真菌性角膜炎的共焦显微镜影像学特点分析

目的探讨应用共焦显微镜观察真菌性角膜炎患者角膜各层的活体形态学特点。方法对65例（65只眼）真菌性角膜炎患者的病灶进行活体共焦显微镜检查,应用NAVIS软件测量、分析角膜各层菌丝、孢子形态、直径、密度。结果通过连续共焦扫描及焦点分析：65例中共焦显微镜显示：①10例显示上皮层下高反射直径2μm～4μm的树枝状菌丝;②27例显示浅、中基质层杂乱分布的直、长线状菌丝,直径约3μm～6μm,长度150μm～300μm。③25例显示浅、中基质层弥漫分布的高反射短、段状菌丝,直径3μm～6μm,长度40μm～60μm。④3例显示：直径12μm～15μm的高亮度圆形、椭圆形实心球体孢子。菌丝侵入分布密度与炎性细胞分布密度成负相关（r=-0.019;p=0.026）。结论共焦显微镜下真菌性角膜炎菌丝有不同的影像学特点,随着菌丝侵入角膜深度不同,菌丝形态亦不相同。明确角膜基质深层菌丝形态在共焦显微镜检查中尤显重要。     

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。     

共焦显微镜观察慢性角膜水肿患者角膜各层形态特点

目的：应用活体共焦显微镜(IVCM)观察慢性角膜水肿患者角膜各层形态特点。

方法：使用IVCM观察不同病因的慢性角膜水肿的患者21例21眼,并与5例拟行白内障手术患者的正常角膜进行对照。

结果：IVCM观察到所有慢性角膜水肿患者角膜上皮层均可见大泡,表现为黑色、圆形、边缘清晰。18眼(86%)上皮细胞出现高反射的无细胞结构的片状区域和瘢痕。12眼(57%)患者中央区角膜上皮下未发现神经纤维,9眼(43%)患者中央区角膜上皮下神经平均密度显著降低。所有患者Bowman膜(BZ)表现为明显的异常,除了瘢痕外,BZ呈分支的、细的、黑线状。13眼(62%)患者前基质表现为细颗粒或粗颗粒,且反光不同。所有患者角膜基质细胞密度降低。所有患者角膜内皮表现为正常六边形结构消失,细胞边界不清。对照组角膜正常未见上述改变。

结论：IVCM可以用来观察慢性角膜水肿角膜各层微观结构上的变化,包括上皮瘢痕形成、上皮下神经纤维及基质细胞的减少。随着角膜内皮移植术的日益普及,本研究支持IVCM在定量评估术前、术后角膜水肿的作用。     

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。     

镰孢菌属真菌性角膜炎的临床及共焦显微镜特征分析

目的 回顾性分析镰孢菌感染所致的真菌性角膜炎共焦显微镜影像特征及疗效.方法 将43例(43眼)临床确诊的镰孢菌感染所致的真菌性角膜炎进行裂隙灯显微镜及共焦显微镜检查,并详细记录.结果 43例镰孢菌感染所致的真菌性角膜炎裂隙灯显微镜观察:23例(92%)可见菌丝苔被,20例(80%)可见羽毛状浸润边缘,8例(18.6%)观察到前房积脓;共焦显微镜下显示:19例(44.2%)上皮层及浅基质层检查到长度50～200 μm,直径2～5 μm的树枝状菌丝;16例(37.2%)显示浅中基质层的杂乱分布的直、长线状菌丝,直径3～7 μm,长度150～300 μm.结论 菌丝苔被、羽毛状浸润边缘是镰孢菌属感染所致的真菌性角膜炎的主要临床特点.不同菌种镰孢菌属感染所致的真菌性角膜炎的共焦显微镜影像表现较为一致,可见典型的菌丝形态.     

颗粒状角膜营养不良活体共焦显微镜形态学研究

目的研究颗粒状角膜营养不良角膜各层组织的共焦显微镜形态改变。方法应用Confoscan2.0共焦显微镜对13例(26眼)颗粒状角膜营养不良患者的角膜进行扫描检查,记录与分析各层角膜图像。结果所有患眼前基质细胞及16/26眼后基质细胞结构不清,排列紊乱,并可见短棒状多形性强反光;6/26眼前弹力层不规则并增厚,神经纤维密度明显下降;6/26眼角膜上皮基底细胞层可见不定型的强反光;2/26眼角膜上皮细胞边界不清,排列呈疏松的蜂窝状,并出现不透明的强反光;所有患者角膜内皮细胞形态基本正常。视力0.3以下的患眼角膜上皮细胞层、上皮基底细胞层、前弹力层、后基质层发生形态异常的比例高于0.3以上的患眼(P<0.05)。结论1.共焦显微镜可活体检查颗粒状角膜营养不良角膜组织各层结构,起到类似病理组织切片的作用。2.前基质层形态异常可能是颗粒状角膜营养不良最基本的共焦显微镜形态特征,病情越重,前基质层以外的其它层次发生形态异常的可能性越大,但内皮细胞层一般不受累。3.共焦显微镜检查对颗粒状角膜营养不良手术方式的选择具有一定的参考价值。     

准分子激光原位角膜磨镶术后弥漫性板层角膜炎的共焦显微镜观察

目的探讨准分子激光原位角膜磨镶术（LASIK）后弥漫性板层角膜炎（DLK）的临床病理学特征及发病机制。方法LASIK术后DLK患者30例（39只眼）（Ⅰ～Ⅳ期）,术后1、3、5、7d及1个月进行裂隙灯显微镜检查,术后3、7d及1个月进行共焦显微镜检查。结果Ⅰ期及Ⅱ期DLK的典型表现出现在术后3d,共焦显微镜观察所见：角膜板层切口前基质及层间可见大量直径12～20μm的圆形或卵圆形细胞,反光较强,散在分布或排列成行,细胞内可见偏心的高反光的核和低反光的细胞内结构。层间还可见大量直径8～12μm的圆形细胞,强反光,多聚集成簇或排列成行,细胞核形态不规则。术后7d上述细胞几乎消失。Ⅲ期DLK出现于术后3～5d,表现为前基质中的细胞浸润更浓密,层间无定形的高反光物质较明显。Ⅳ期DLK在术后5～7d出现明显的前基质结构模糊,高反光,角膜瓣全层皱褶,晚期形成大量高反光的瘢痕组织。结论LASIK术后弥漫性板层角膜炎是角膜瓣层间的炎性反应,主要病理学特征为角膜瓣层间的炎性细胞浸润,其发病是多种因素通过内源性途径和外源性途径共同作用的结果。     

Diagnosis and treatment of pseudomonas aeruginosa infected keratitis caused by wearing orthokeratology lenses: a case report北大核心CSCD

患儿男性,11岁,配戴角膜塑形镜矫正近视1年,戴镜后出现右眼疼、睁眼困难,当地医院诊断为右眼角膜炎,给予妥布霉素滴眼液及小牛血去蛋白提取物眼用凝胶4次/d点眼治疗,2 d后症状持续加重,遂于河南省立眼科医院就诊。患儿既往体健;视力右眼为指数/20 cm,左眼为0.8;右眼睑水肿,睑裂区有黄绿色黏液状分泌物,结膜水肿,混合充血(+++),角膜水肿,中央鼻上方见4 mm×4 mm白色溃疡病灶(图1A);右眼角膜刮片Giemsa染色镜检发现少量杆菌;角膜活体共聚焦显微镜检查发现右眼角膜病灶及周围大量炎性细胞浸润及大量树突状细胞,可见高反光物沉积,角膜内皮隐见较多炎性细胞附着,未发现典型真菌菌丝、孢子及阿米巴包囊(图1B)。初步诊断:右眼细菌性角膜溃疡。     

正常人角膜神经的共焦显微镜观察

角膜内感觉神经的保护作用十分重要，交感神经纤维可调节角膜上皮完整性、上皮的增殖及伤1：3的愈合等。以往对角膜神经的研究方法多为离体检查，共焦显微镜作为一种新型的光学显微镜，可在活体上对角膜从四维(三维空间和时间)水平进行实时扫描成像。我们对37例(42只眼)正常人进行了共焦显微镜检查，以了解正常角膜内神经的形态，为各种病理状态下角膜神经形态的研究提供参考。     

超声生物显微镜测量正常人眼前节结构的初步研究

目的了解正常人活体状态下的眼前节组织结构。方法通过超声生物显微镜（ｕｌｔｒａ－ｓｏｕｎｄｂｉｏｍｉｃｒｏｓｃｏｐｙ，ＵＢＭ）的超高频（５０～１００ｍＨｚ）换能器，对活体眼球的眼前节结构进行测量。结果ＵＢＭ能清晰观察角膜、巩膜、虹膜、晶体、睫状体、前房、后房和眼外肌等结构。结论ＵＢＭ是一种无创伤、且在活体状态下观察眼前节结构的新方法，它弥补了既往不能在活体组织上动态观察眼前节结构的不足。因换能器频率高，探查深度仅４～５ｍｍ，只适用于观察眼部的表浅结构。     

A Study of the Characteristics of Candida with In Vitro and In Vivo Confocal Microscopy

Objective: To use in vivo laser confocal microscopy (IVCM) to observe the in vitro culture and living characteristics of candida spores and pseudohyphaes, and to provide abasis for the early diagnosis of candida keratitis. Methods: The data of 26 patients (26 eyes) who were diagnosed with candida keratitis in the Eye Hospital of Shandong First Medical University from January 2016 to December 2019 wascollected. At the same time, candida albicans and candida parapsilosis were taken for laboratory fungal culture, and the typical morphology of candida spores and pseudohyphae in vitro and in vivo were observed by IVCM. Results: Candidaspores in vitro and in vivo presented uniformly in the shape of round spots with a diameter of 2-5 μm and a clear boundary. Pseudohyphae have a highly reflective linear structure. The reflection of spores was significantly enhanced in vivo. Eighteen eyes showed a scattered arrangement of spot-like, strongly reflective structure, and 11 eyes showed a dense fog structure. The morphology of pseudomycelia varied greatly. The mycelia in 2 eyes were slender, the spore structure was present in 5 eyes, and branches were visible. In 1 eye, an irregular thin and long rod shape with high contrast was observed, and in another eye, an irregular thin and long rod shape and spore structure could be observed simultaneously. Candida albicans pseudohyphae were relatively slender, with a length of 20-150 μm and a diameter of 2-10 μm. Pseudohyphae of candida parapsilosis were short and thick, with a length of about 30-100 μm and a diameter of 2-5 μm. Part of the branch spore structure was highlighted. Conclusions: Candida keratitis showstypical spore and pseudohyphae structures with IVCM. The morphology of candida keratitis spores in vitro and in vivo are consistent with observations under IVCM, but the morphology of pseudohyphaes are relatively different. IVCM can be used for the early diagnosis of candida keratitis.     

丝状角膜炎的共聚焦显微镜观察

目的：应用活体激光共聚焦显微镜（ IVCM）观察丝状角膜炎患者丝状物的组成结构及位置特点,分析角膜丝状物的形成机制。方法收集深圳市眼科医院2014年6月至2016年1月收治的丝状角膜炎患者48例（60只眼）,详细记录患者的病史和相关检查。采用共焦显微镜观察角膜丝状物的组成结构和角膜丝状物附着处的角膜结构。结果活体激光共聚焦显微镜检查显示,丝状物起自前弹力层,由上皮细胞、炎性细胞、黏液、螺旋状条形核心构成,核心结构中亦含有上皮细胞、炎性细胞及纤维状组织。结论丝状角膜炎丝状物的共聚焦表现特点可以为临床治疗方法的选择提供影像的参考依据。     

Contribution of In Vivo Confocal Microscopy to the Diagnosis and Management of Infectious Keratitis

Infectious keratitis can lead to irreversible complications and even blindness. Identifying the infectious agent in this condition is a challenge for the ophthalmologist. Corneal cultures are considered to be the gold standard diagnostic tool for this condition. Nevertheless, routine culture and viral investigations may yield positive results in only half the cases. In vivo confocal microscopy (IVCM) is a noninvasive imaging technique that provides high-resolution images of all the corneal layers. Since accurate and rapid diagnosis is important for the management and outcome of infectious keratitis, this disease constitutes one of the most important clinical uses of IVCM. However, in this review, the efficacy of IVCM for infectious keratitis remains inconclusive. Although the value of IVCM has been demonstrated in the diagnosis and management of Acanthamoeba and filamentous fungal keratitis, the current resolution of this imaging technique limits its use in cases of bacterial and viral keratitis. By providing in vivo, repeated, and noninvasive analyses of the cornea, IVCM may also be used to evaluate the effectiveness of treatment and the healing of the cornea in the follow-up of infectious keratitis patients.     

In vivo confocal microscopy of the inflamed anterior segment: A review of clinical and research applications

In vivo confocal microscopy (IVCM) allows non‐invasive imaging of the living human cornea, specifically enabling the detection of immune cells in the healthy and diseased ocular anterior segment. Studies using IVCM have provided insight into the effects of contact lens wear on corneal Langerhans cell density and morphology, and the effects of eye drops on conjunctiva‐associated lymphoid tissue. IVCM has also been shown to be a useful adjunctive diagnostic tool in distinguishing infective and non‐infective uveitis and in diagnosing atypical infective keratitis. In the research setting, this technology has enhanced our understanding of the role of inflammatory cells in corneal neuropathy and angiogenesis. In vivo‐ex vivo correlation using animal models has helped overcome some of the difficulties in identifying cell type on IVCM images. As highlighted in this review, currently there are multiple established, and emerging, clinical and research applications for IVCM in the inflamed anterior segment.     

青光眼睫状体炎患者角膜共聚焦显微镜表现

目的：应用活体共聚焦显微镜（IVCM）观察青光眼睫状体炎（又称Posner-Schlossman综合征,PPS）患者角膜组织形态学改变。方法：回顾性研究。对2014 年5 月至2017 年5 月于北京大学第三医院眼科就诊的56 例PPS患者（56 眼）的病历资料进行归纳整理,记录患者的视力、眼压、裂隙灯显微镜检查结果及共聚焦显微镜下表现,包括角膜基质细胞有无活化、内皮细胞形态、有无角膜后沉着物（KP）及KP的形态、有无朗格罕氏细胞（LCs）及其形态特点。采用卡方检验进行数据分析。结果：56 例PSS患者LogMAR视力为0.3±0.2,眼压为（29.7±13.4）mmHg,使用青光眼药物的种类为（2.6±1.0）种。全部患者受累眼角膜内皮细胞呈六角形。50 例患者（89%）的患眼可以观察到角膜基质细胞活化;32 例患者（57%）的患眼可以观察到内皮面LCs;44 例患者（79%）的患眼可观察到KP,共6 种类型。以上与对侧眼相比,差异均具有统计学意义（分别为χ2=69.14,41.29,60.89,P<0.001）。结论：IVCM下可见PSS患者的角膜基质细胞活化,内皮面出现LCs及多种类型的KP。     

Activation of Conjunctiva-Associated Lymphoid Tissue in Patients With Infectious Keratitis Using In Vivo Confocal Microscopy

PurposeWe aimed to evaluate activation of conjunctiva-associated lymphoid tissue (CALT) in patients with keratitis using in vivo confocal microscopy (IVCM) and conjunctival impression cytology (CIC).MethodsIn addition to anterior segment photography and corneal fluorescein staining, IVCM revealed the palpebral conjunctiva in all subjects, and CIC and immunofluorescence staining were performed.ResultsDiffuse lymphoid tissue cell density in the eyes of patients with keratitis was significantly greater compared with healthy volunteers (P < 0.001). Similar trends were found in perifollicular lymphocyte density (P < 0.001), follicular density (P = 0.029), follicular center reflection intensity (P = 0.011), and follicular area (P < 0.001). Immunofluorescence staining showed that the proportions of CD4⁺ (61.7% ± 8.0% vs. 17.3% ± 10.2%, respectively, P < 0.001) and CD8⁺ (46.9% ± 10.0% vs. 19.6% ± 11.5%, respectively, P < 0.001) cells in patients with keratitis was greater compared with healthy volunteers. Interestingly, we also observed changes in the contralateral eye in subjects with keratitis.ConclusionsOur research suggests that CALT, as an ocular immune structure, is activated and plays an important role in the pathogenesis of keratitis. This has been overlooked previously. CALT is also active in the contralateral eye of subjects with keratitis.     

Fungal Keratitis – Improving Diagnostics by Confocal Microscopy

Purpose

Introducing a simple image grading system to support the interpretation of in vivo confocal microscopy (IVCM) images in filamentous fungal keratitis.

Setting

Clinical and confocal studies took place at the Department of Ophthalmology, Aarhus University Hospital, Denmark. Histopathological analysis was performed at the Eye Pathology Institute, Department of Neuroscience and Pharmacology, University of Copenhagen, Denmark.

Methods

A recent series of consecutive patients with filamentous fungal keratitis is presented to demonstrate the results from in-house IVCM. Based upon our experience with IVCM and previously published images, we composed a grading system for interpreting IVCM images of filamentous fungal keratitis.

Results

A recent case series of filamentous fungal keratitis from 2011 to 2012 was examined. There were 3 male and 3 female patients. Mean age was 44.5 years (range 12–69), 6 out of 17 (35%) cultures were positive and a total of 6/7 (86%) IVCM scans were positive. Three different categories of IVCM results for the grading of diagnostic certainty were formed.

Conclusion

IVCM is a valuable tool for diagnosing filamentous fungal keratitis. In order to improve the reliability of IVCM, we suggest implementing a simple and clinically applicable grading system for aiding the interpretation of IVCM images of filamentous fungal keratitis.Key words: In vivo confocal microscopy, Fungal keratitis, Diagnostics     

29例念珠菌性角膜炎的临床特点和治疗预后

目的：分析念珠菌性角膜炎的发病特征和临床表现,并评估其治疗预后。方法：回顾性系列病例研究。 收集2013年1月至2019年12月于山东第一医科大学附属眼科医院明确诊断为念珠菌性角膜炎的患者29例（29眼）,其中男18例,女11例,均为单眼发病;1例患者为7月大女婴,其余28例患者年龄为 29～91（59.0±13.5）岁;发病时间为2～90 d,中位数12 d。分析其发病趋势、感染前相关因素、临床表现、角膜激光共聚焦显微镜图像特点、治疗方法和疗效。结果：角膜念珠菌培养阳性共29例（其 中白念珠菌10例,近平滑念珠菌6例,其他菌种13例）,占同期真菌性角膜炎患者2%。感染前患者最常见的情况是眼部手术史26例（90%）,其次是局部糖皮质激素使用史24例（83%）、全身疾病9例 （31%）和眼部外伤2例（7%）,25例（86%）患者合并2个或更多上述因素。念珠菌性角膜炎结膜充血一般较轻,不伴有典型的伪足、苔被、免疫环等体征。4例早期患者角膜病变表现为单个或多个孤立圆点状病灶,角膜病灶切除联合药物治疗治愈。病变进展后病灶表现多样,最常见为散在多灶性浸润灶（10例,占35%）。最终23例（80%）患者需行手术治疗。结论：念珠菌性角膜炎患者的眼部手术史和局部激素使用史是感染前常见的情况,发病早期特征性表现为孤立圆点状病灶,病灶清除联合药物治疗预后好,晚期病灶表现多样,多数患者需要行角膜移植手术治疗。     

Corneal nerves: structure,contents and function

This review provides a comprehensive analysis of the structure, neurochemical content, and functions of corneal nerves, with special emphasis on human corneal nerves. A revised interpretation of human corneal nerve architecture is presented based on recent observations obtained by in vivo confocal microscopy (IVCM), immunohistochemistry, and ultrastructural analyses of serial-sectioned human corneas. Current data on the neurotransmitter and neuropeptide contents of corneal nerves are discussed, as are the mechanisms by which corneal neurochemicals and associated neurotrophins modulate corneal physiology, homeostasis and wound healing. The results of recent clinical studies of topically applied neuropeptides and neurotrophins to treat neurotrophic keratitis are reviewed. Recommendations for using IVCM to evaluate corneal nerves in health and disease are presented.     

In Vivo Confocal Microscopy of Corneal Nerves in Health and Disease

In vivo confocal microscopy (IVCM) is becoming an indispensable tool for studying corneal physiology and disease. Enabling the dissection of corneal architecture at a cellular level, this technique offers fast and noninvasive in vivo imaging of the cornea with images comparable to those of ex vivo histochemical techniques. Corneal nerves bear substantial relevance to clinicians and scientists alike, given their pivotal roles in regulation of corneal sensation, maintenance of epithelial integrity, as well as proliferation and promotion of wound healing. Thus, IVCM offers a unique method to study corneal nerve alterations in a myriad of conditions, such as ocular and systemic diseases and following corneal surgery, without altering the tissue microenvironment. Of particular interest has been the correlation of corneal subbasal nerves to their function, which has been studied in normal eyes, contact lens wearers, and patients with keratoconus, infectious keratitis, corneal dystrophies, and neurotrophic keratopathy. Longitudinal studies have applied IVCM to investigate the effects of corneal surgery on nerves, demonstrating their regenerative capacity. IVCM is increasingly important in the diagnosis and management of systemic conditions such as peripheral diabetic neuropathy and, more recently, in ocular diseases. In this review, we outline the principles and applications of IVCM in the study of corneal nerves in various ocular and systemic diseases.