明胶酶在兔真菌性角膜炎病理改变中的作用研究

目的探讨明胶酶包括基质金属蛋白酶（MMP）2与MMP-9在兔真菌性角膜炎病理改变中的作用。方法80只新西兰白兔随机分为4组,每组20只。其中3组为实验组,兔右眼分别注入100μl茄病镰刀菌、烟曲霉菌及白色念珠菌的悬液;另1组为对照组,兔右眼注入等量生理盐水。免疫组织化学方法观察MMP-2与MMP-9的来源,明胶酶谱法检测其活性。组织病理学方法观察炎性细胞的浸润、角膜细胞外基质（ECMs）的降解以及真菌菌丝在角膜内的生长方式与入侵深度。结果MMP-2主要由角膜基质细胞产生,真菌感染后5d检测出活性,8d活性升高。MMP-9主要来源于嗜中性粒细胞,接种后1d即检测到活性,3d活性升高,之后逐渐下降。茄病镰刀菌感染后3d,角膜内散在嗜中性粒细胞,浅层ECMs被降解,菌丝平行于角膜基质纤维生长。烟曲霉菌和白色念珠菌感染后3d,角膜内可见大量嗜中性粒细胞,周围ECMs降解明显,菌丝表现为垂直生长。接种后8d,茄病镰刀菌和白色念珠菌感染的角膜内炎性细胞和菌丝明显减少,而烟曲霉菌感染的角膜变化不明显。结论茄病镰刀菌、烟曲霉菌及白色念珠菌感染兔角膜后,产生的明胶酶活性明显不同;明胶酶对降解角膜ECMs发挥了重要作用;随着ECMs降解程度的不同,菌丝在角膜内的生长方式、入侵深度等病理改变出现差异。     

改良角膜表面镜片术法建立兔曲霉菌性角膜炎动物模型

背景真菌性角膜感染动物模型是研究真菌性角膜炎发病机制的工具,目前的制作方法主要有划痕法、基质注射法和角膜表面镜片术法,但均有其不足之处。目的探讨一种简便、易操作的改良兔曲霉菌性角膜炎动物模型制作方法。方法成年新西兰白兔18只,采用烟曲霉菌孢子附贴滤纸片的改良角膜表面镜片术法制作真菌性角膜炎动物模型。将浸有10^8孢子／ml（10^8孢子／ml组,6只）或10^6孢子／ml（106孢子／ml组,6只）真菌混悬液的滤纸贴敷于去上皮的角膜基质并用角膜接触镜覆盖,将浸有生理盐水的滤纸贴敷于另6只兔眼角膜作为对照组。分别于造模后3、7、14d裂隙灯下观察眼前节症状,参照Dong的标准进行症状评分。制备角膜刮片并用质量分数10％KOH和荧光白染色在荧光显微镜下检测真菌菌丝,角膜组织切片分别行苏木精-伊红和过碘酸希夫染色,光学显微镜下观察角膜形态学改变和菌丝生长情况。对感染组织进行真菌培养以验证模型的质量。结果10^8孢子／ml组和10^6孢子／ml组真菌性角膜炎模型成功者分别为6眼和4眼,裂隙灯检查表明造模3d后10^8孢子／ml组眼前节症状明显重于10^6孢子／ml组,且随着时间的延长,炎性损伤逐渐转向增生期。造模后3d和7d,2组感染的兔眼症状评分明显高于对照组,差异均有统计学意义（P〈0．01）,10^8孢子／ml组兔眼的症状评分均明显高于10^6孢子／ml组,差异有统计学意义（P〈0．01）,造模后14d,10^8孢子／ml组兔眼的症状评分明显高于对照组,差异有统计学意义（P〈0．05）。造模后3d和7d,2组兔眼角膜刮片中均可见真菌菌丝。角膜组织病理学检查显示,造模3d和7d后10^8孢子／ml组可见炎性细胞浸润和角膜基质细胞坏死,并可见真菌菌丝,造模后14d可见新生血管长入。10^6孢子／ml组炎症轻于10^8孢子／ml组。真菌培养结果表明,造模后3d和7d时10^8孢子／ml组均见菌丝生长,而10^6孢子／ml组仅在造模3d时可见菌丝生长。结论改良角膜表面镜片术法可成功制备兔曲霉菌性角膜炎动物模型,是一种简便、易于操作的真菌性角膜炎动物模型制作方法。     

丝状真菌所致真菌性角膜炎的临床研究及相关分析

目的 探讨丝状真菌所致真菌性角膜炎的临床研究方法及指导临床治疗的意义。方法 对110例真菌性角膜炎进行10％KOH湿片镜检、大体培养、鉴定培养、药敏试验、病理检查和动物模型实验以指导临床分型及治疗。结果 本组110例中10％KOH湿片镜检可见大分生孢子59例，小分生孢子51例，有隔菌丝101例，无隔菌丝6例，关节状菌丝3例；鉴定培养镰刀菌59株，曲霉菌31株，青霉菌7株，无孢菌5株，头孢子菌2株，链互隔菌2株，附球菌2株，枝顶孢霉菌2株；药敏试验提示氟康唑对丝状菌均呈现耐药性，其他药物对不同的菌属呈现不同的敏感性和耐药性；病理检查可见病变角膜内大量炎细胞及菌丝浸润；15只实验兔角膜划痕接种的动物模型中4例出现角膜真菌感染。结论进行丝状真菌所致真菌性角膜炎的临床研究，对确定真菌在角膜内的生长情况和指导临床分型及选择个性化治疗、减低医疗成本有较大意义。     

豚鼠真菌性角膜炎模型的建立及病理观察

目的 探讨理想的真菌性角膜炎动物模型的制作方法,并观察其病理学改变.方法 豚鼠25只(50眼)制作真茵性角膜炎动物模型.分别于接种后2 d、4 d、7 d、14 d过量乙醚麻醉处死豚鼠,取角膜,光镜下观察组织学改变及真菌孢子和茵丝.结果 接种真茵2 d后,可见角膜全层水肿,溃疡形成,干燥致密,粗糙不平,稍隆起.HE染色显示角膜上皮层可见散在孢子及茵丝,且茵丝向基质层长入.随病情进展,溃疡部位角膜上皮及浅基质修复,基质内较多成纤维细胞,细胞走行与基质纤维平行,茵丝平行或斜行于角膜板层生长,有分隔,圆形孢子散在生长,部分标本前房可见茵丝和孢子.结论 利用豚鼠采用剖除角膜上皮,眼睑缝合,茵液滴眼的方法可以成功建立真茵性角膜炎的动物模型.     

吉林地区135例真菌性角膜炎病原菌的调查分析

目的 调查吉林地区真菌性角膜炎的病原菌种类及分布情况.方法 取2004～2008年间135例疑似真菌性角膜溃疡患者行KOH涂片镜检,发现真菌菌丝或孢子者,取角膜刮片进行病原菌的分离培养及鉴定.结果 真菌培养阳性率为70.37%(95/135),其中混合感染为14.74%(14/95).培养阳性的真菌菌株中镰刀菌最多(45.87%),其次为念珠菌(20.18%)和曲霉(15.60%).结论 吉林地区真菌性角膜炎的优势病原菌为镰刀菌.     

仿LASEK法建立烟曲霉菌角膜炎动物模型的研究

目的 探讨建立真菌性角膜炎动物模型的新方法.方法 38只新西兰大耳白兔随机分为3组,右眼为实验眼.A组15只兔,采用准分子激光角膜上皮磨镶术(LASEK)制作直径7.0 mm、厚度50μm的角膜上皮瓣,瓣下接种25μL标准烟曲霉菌株(105孢子);B组15只兔,接种前7 d 0.1%地塞米松滴眼液点眼,每日4次,其余步骤同A组;对照组8只兔,以25μL生理盐水代替真菌菌液进行接种.术后采用裂隙灯显微镜、病理组织学检查、角膜真菌培养等方法观察评价角膜真菌感染情况.结果 A、B两组均见典型的真菌性角膜溃疡发生,经真菌培养鉴定为烟曲霉菌.对照组未见有角膜感染.A、B两组接种成功率分别为93.33%和100%,差异无统计学意义(χ2=1.345,P>0.05);B组角膜炎症较A组重、病程长,两组间临床评分差异有统计学意义(接种后3、7、15 d P<0.05,接种后30 d P<0.01).结论 采用LASEK制作角膜上皮瓣能够较理想地建立真菌性角膜炎动物模型.     

离体及活体念珠菌在共聚焦显微镜下的特征

目的：应用活体激光共聚焦显微镜（IVCM）观察离体培养及活体状态下念珠菌孢子及假菌丝的典型 特征，为念珠菌性角膜炎的早期诊断提供依据。方法：比对观察研究。收集2016年1月到2019年12月 在山东第一医科大学附属眼科医院被诊断为念珠菌性角膜炎的26例（26眼）患者资料，同时取白色 念珠菌和近平滑念珠菌进行实验室真菌培养，应用IVCM分别观察离体及活体状态下念珠菌孢子及 假菌丝的典型形态。结果：离体与活体状态下念珠菌孢子均呈排列均匀的点状结构，形状类圆形， 直径约2～5 μm，边界清晰；假菌丝呈高反光线状结构。活体状态下孢子反光明显增强，18眼呈分散 排列点状强反光结构，11眼呈团雾状中强反光结构；假菌丝形态变异较大，2眼菌丝形体细长，5眼 呈芽孢结构，可见分支，1眼呈高对比度的不规则细长短棒状，1眼可同时观察到不规则细长短棒状 和芽孢结构。白色念珠菌假菌丝结构较细长，长度约20～150 μm，直径约2～10 μm，与母细胞连接 处收缩呈缢痕；近平滑念珠菌假菌丝短粗，长度约30～100 μm，直径2～5 μm，部分可见高亮的分支 芽孢结构。结论：念珠菌性角膜炎在IVCM下呈现典型的孢子和假菌丝结构，离体和活体状态下孢 子形态较为一致，而假菌丝形态变异较大，IVCM可用于念珠菌性角膜炎的早期诊断。     

应用激光共焦显微镜诊断真菌性角膜炎的临床研究

目的:应用激光共焦显微镜观察真菌性角膜炎患者图像特点及菌丝和孢子检出率,探讨激光共焦显微镜检查在真菌性角膜炎临床诊断中的意义。方法:对41例41眼经门诊确诊为真菌性角膜炎的患者行角膜激光共焦显微镜检查,观察不同治疗期真菌性角膜炎患者活体角膜各层图像特点。结果:共焦显微镜下真菌性角膜炎患者图像有如下特点:(1)病变部位角膜各层形态结构破坏明显;角膜上皮至基质不同程度水肿;炎细胞浸润;神经结构破坏;基质层结构紊乱,透过度降低;(2)真菌菌丝是本病的特异性诊断依据,不同菌种感染在镜下菌丝有不同的影像学特点;(3)不同病变时期和治疗阶段,图像有很大差异,真菌菌丝的检出并不是诊断本病的唯一依据。结论:激光共焦显微镜检查具有无创、及时等优点,在临床诊断真菌性角膜炎中有重要参考意义,尤其是指导临床早期诊断、合理治疗及评价预后。     

共焦显微镜鉴别诊断真菌性角膜炎的实验研究

目的研究利用眼科用共焦显微镜鉴别诊断不同真菌性角膜炎的可行性。方法18只新西兰白兔均分为3组,右眼分别接种白色念珠菌、烟曲霉菌和腐皮镰刀菌。接种时,去除角膜中央区5mm直径的上皮,滴新鲜培养的1×107个细胞／ml的白色念珠菌悬浮液,或涂上新鲜培养的烟曲霉菌或腐皮镰刀菌孢子,盖上软性接触镜,行睑裂缝合。在角膜感染后的第5,10和15天时,行共焦显微镜检查、真菌涂片检查和病理切片检查。结果感染早期(1～5天)炎症反应明显,中期(5～10天)炎症逐渐减轻,10～15天角膜形成疤痕而自愈。烟曲霉菌和腐皮镰刀菌的病程较迁延,反应较白色念珠菌重。共焦显微镜下,感染的白色念珠菌早期主要呈现芽孢状,成高亮度结构,以后发展成具有大量分支的树枝状结构,再发展成链状结构。感染10天后未见任何真菌结构。烟曲霉菌的菌丝在早期则呈较白色念珠菌菌丝粗但分支相对较少的明亮的树枝状结构。中期以后其分支减少并变长,它在角膜组织中保留的时间较白色念珠菌长。在感染早期,腐皮镰刀菌则表现为蚯蚓状结构,以后发展为分支很少的粗短树枝状结构,其分支在3种真菌中最少但直径最粗。真菌涂片和组织学检查的结果与共焦显微镜检查相符。结论共焦显微镜检查对真菌性角膜炎的鉴别诊断有重要的参考价值。     

单纯疱疹病毒性角膜炎的免疫学

单纯疱疹病毒性角膜炎 (herpes simplex keratitis,HSK)是世界范围内角膜盲的最常见原因。单纯疱疹病毒 (herpes simplexvirus,HSV)不仅可直接导致角膜的感染性破坏 ,而且还可通过免疫病理机制诱导某些难以处理的疾病 ,如基质型角膜炎。1　免疫学机制1 .1　动物模型　目前 ,已有数种 HSK的动物模型 ,比较常用的是兔模型和小鼠模型。兔模型比较理想 ,因为兔眼球大小与人类相似易于观察 ;另外 ,存在有自发性病毒释放和疾病复发现象。而小鼠模型则缺乏病毒的自发性释放和疾病的复发 ,但在感染后经过一段时间才发生角膜的炎症反应 ,该特征类…     

Differences in virulence between two Candida albicans strains in experimental keratitis

PURPOSE: To study the differences in disease caused by two wild-type strains of Candida albicans in a model of contact lens-facilitated keratitis in rabbits. METHODS: Two strains, SC5314 and VE175, were examined. Standardized inocula were placed on the debrided corneal surface of one eye in Dutch belted rabbits and covered with a contact lens. A temporary tarsorrhaphy was opened after 24 hours with removal of the contact lens. Six days later, corneas were photographed and animals killed. Corneas were bisected with one half for quantitative isolate recovery and the other for stromal penetration by hyphae. RESULTS: Strain SC5314 was significantly more virulent. The mean hyphal penetration into the cornea was 24.4% +/- 8.5% of the corneal thickness, and in three of six corneas hyphae penetrated through the entire cornea. In contrast, for VE175, the mean hyphal penetration was 2.6% +/- 1.2%. The difference between these two strains was statistically significant (P = 0.0297). Hyphae did not penetrate into the deep layers of the cornea in any of the six rabbits infected with VE175. The grading of clinical disease was consistent with histology, in that strain SC5314 caused more severe infection than VE175 and the difference was statistically significant (P = 0.0048). There was no difference in isolate recovery. CONCLUSIONS: Wild-type strains of C. albicans can differ significantly in virulence as measured by depth of fungal invasion into corneas and clinical evaluation of infection. Further characterization of the intrinsic genetic differences between such strains may help identify factors responsible for fungal virulence.     

Effect of corticosteroids in antimycotic therapy of Candida keratitis

The effect of corticosteroid treatment in addition to antimycotic therapy was studied on the basis of a newly developed keratomycosis model. Forty-eight hours after intracorneal injection of a defined strain of Candida albicans, Amphotericin B drops were administered at one-hour intervals ten times a day. To improve penetration of the drops abrasion of the corneal epithelium was performed every three days. In addition, 4 mg of dexamethasone phosphate was injected subconjunctivally into one eye every two days. The results showed that in this low dosage dexamethasone did not worsen the course of the infection in a single case. On the contrary, there was significantly less neovascularization (p less than 0.05) than in the group not treated with dexamethasone. It therefore appears that combination thereby is the best form of treatment for keratomycosis; this is also supported by clinical observations.     

Animal experiment model of keratomycosis

A model of experimental keratomycosis in the rabbit eye is described. Candida albicans strain DSM 70010 (10 microliters; 2.5 X 10(5) cells) is injected intracorneally without employing immunosuppressive measures. This strain is characterized by marked germ-tube formation, which apparently is a major cause of its virulence. All 17 eyes developed an infiltration of the cornea two days after injection. On Day 6 (mean value; standard error +/- 2.34 days) this infiltration developed synchronously to a severe corneal ulcer with hypopyon. The infection remained active for about two weeks and was either complicated by a descemetocele or perforation or led to a reparatory stage with extensive vascularization and successive leukoma. The model presented is reproducible for the first time and is therefore recommended for experimental evaluation of new therapeutic concepts.     

Polyphosphate kinase 1 and the ocular virulence of Pseudomonas aeruginosa

PURPOSE: To determine the role of polyphosphate kinase 1 (PPK1) in the ocular virulence of Pseudomonas aeruginosa. METHODS: Using a mouse model of infection, P. aeruginosa strains PAO1, PAOM5 (an isogenic mutant of PAO1 deficient in PPK1), and PAOM5+PPK1 (the mutant complemented with PPK1 on plasmid pHEPAK11) were compared for ocular virulence. These strains were also characterized with respect to traits associated with survival and pathogenicity in an ocular environment. RESULTS: The PPK1-deficient strain PAOM5 was significantly less virulent than either wild-type PAO1 or the complemented mutant (P <0.016). Loss of virulence was not associated with serum sensitivity or diminished adherence to the cornea. However, PAOM5 has an increased susceptibility to oxidative stress and was cleared from corneal tissue significantly better (P <0.006) than either the wild-type or restored strain. Furthermore, the PPK1-deficient mutant produced significantly less (P <0.022) pyocyanin. CONCLUSIONS: PPK1 is essential for a successful ocular infection by P. aeruginosa. The loss of ocular virulence is probably due to the dysregulation of multiple genes, including those responsible for stress response.     

Candida albicans strain-dependent virulence and Rim13p-mediated filamentation in experimental keratomycosis

PURPOSE: To compare the virulence of wild-type Candida albicans strains in a murine model of corneal candidiasis and to investigate the role of fungal filamentation in disease progression. METHODS: Scarified corneas of immunocompetent or cyclophosphamide-treated BALB/c mice were topically inoculated with one of three human isolates of C. albicans, a homozygous mutant of the pH-dependent filamentation gene rim13 or a mutant reference strain control. Mock-inoculated eyes served as negative controls. Corneal disease was categorized daily for 8 days with quantitative fungal culturing of eyes at 6 hours, 1 day, 4 days, and 8 days after infection and histopathologic examination at 1 day and 4 days after infection. RESULTS: Corneal disease severity differed significantly among wild-type strains (P < or = 0.02). The rim13(-/-) mutant Tn7-rim13 was fully attenuated, whereas the mutant control DAY286 was fully virulent. Pretreatment of mice with cyclophosphamide increased susceptibility to wild-type C. albicans and partially rescued the attenuated phenotype of the genetically deficient rim13(-/-) fungal mutant. All strains replicated with similar kinetics in vitro, and wild-type strains had similar clearance from infected eyes. Histopathologic findings correlated with disease severity. CONCLUSIONS: Wild-type strains of C. albicans that differ significantly in ocular pathogenicity correlate with the ability of yeast to produce pseudohyphae and hyphae and to invade corneal tissue. Full attenuation of the fungal rim13(-/-) mutant is the first direct demonstration of a hyphal morphogenesis-related gene as a specific virulence factor for C. albicans during corneal infection.     

Experimental studies of local therapy of Candida keratomycosis with amphotericin B

The efficiency of Amphotericin B drops was studied using a newly developed keratomycosis model (defined strain Candida albicans DSM 70010, which leads reproducibly to a corneal infection with descemetocele without prior local or systemic immunosuppression in the rabbit). Penetration of the drug (administered ten times a day) into the cornea and aqueous humor was only demonstrated after abrasion of the corneal epithelium. Three groups were studied: (I) therapy with abrasion, (II) therapy without abrasion, and (III) a control group. Both clinically (descemetocele or perforation, hypopyon) and with regard to microbiology (reculture of Candida) the results obtained in Group I were significantly better than those obtained in Group II (p less than 0.001). Repeated corneal abrasion is therefore recommended for treatment of Candida keratitis with Amphotericin B.     

Clinical characteristics and outcome of Candida keratitis

PURPOSE: To characterize the clinical features and therapeutic outcome of Candida keratitis. DESIGN: Retrospective, observational case series. METHODS: We reviewed 26 patients treated for Candida keratitis, including two with recurrent keratitis and one with bilateral infection. RESULTS: Of 29 keratitis episodes resulting from Candida albicans (n = 20) or Candida parapsilosis (n = 9), 16 (55%) complicated chronic ocular surface disease, and nine (31%) followed previous keratoplasty. Only two were clinically suspected to have keratomycosis at initial presentation, and 21 (72%) used antibacterial therapy before corneal scrapings. Reconstructive keratoplasty occurred more often in previously grafted eyes (P = .03). Visual outcome was 20/60 or better in six (100%) medically treated eyes with good presenting visual acuity but in only five eyes (24%) with worse initial vision (P = .002). CONCLUSIONS: Candida keratitis is an opportunistic infection of a compromised cornea that often is misdiagnosed initially and, despite antifungal therapy, occasionally requires corneal grafting.     

Corneal biopsy in the diagnosis of keratomycosis

In two patients, a 55-year-old man and a 49-year-old man, who had fungal keratitis initially undiagnosed by corneal scrapings the condition was successfully diagnosed by corneal biopsy. We compared corneal biopsy specimens and corneal scraping in the diagnosis of keratomycosis in rabbits with experimental bilateral fungal keratitis caused by Fusarium solani, Aspergillus fumigatus, and Candida albicans. Corneal scrapings disclosed three specimens (30%) positive for Candida, five (50%) for Fusarium, and six (60%) for Aspergillus keratitis, whereas corneal biopsy specimens showed fungal elements of Fusarium, Aspergillus, and Candida in all inoculated eyes.     

Differences in response in vivo to amphotericin B among Candida albicans strains

A group of ten Candida albicans strains previously determined to be resistant or susceptible to topical amphotericin B in vivo and in vitro were exposed to treatment with different concentrations of the drug in a quantitative model of candidal keratitis in Dutch-belted rabbits. After 5 days of topical treatment with amphotericin B eye drops in concentrations of 0.3%, 0.03%, or 0.003%, quantitative isolate recovery in treated animals was compared with that of untreated controls. A dose response was observed for all five susceptible strains. The two strains that were most sensitive to amphotericin B in vitro also were the most susceptible in vivo. At each dose level there was a two- to eightfold reduction in isolate recovery among highly susceptible strains compared with less susceptible strains (P less than 0.05). The five resistant strains remained so even when the 0.3% concentration was used. Among strains of C. albicans susceptible to amphotericin B, there appeared to be a variation in degree of susceptibility in vivo that correlated with the minimum inhibitory concentration.