人体角膜移植内皮排斥反应的共焦显微镜研究

目的：应用共焦显微镜探讨活体角膜内皮排斥反应的早期形态学特征和变化。方法：19例(19只眼)角膜移植术后发生内皮排斥反应的人群根据角膜内皮排斥反应轻重的不同分为轻、中、重三组。应用共焦显微镜(Confoscan2．0)对角膜植片进行检查，并分析其相关因素。结果：活体角膜内皮排斥反应特有的共焦显微镜改变主要包括：①早期发现在KP及其附近的内皮细胞面有少量的免疫细胞附着，紧靠KP处的角膜基质细胞出现轻度水肿，细胞排列较混乱，胞浆中出现少量空泡状暗区。②中期发现在内皮细胞表面可见较多的免疫细胞聚集并被完全激活，基质细胞明显水肿，细胞核不清楚，基质中有免疫细胞进入；可见由高反光的免疫细胞和不明成份的组织碎片混合组成内皮排斥线。③在晚期排斥的患者中可见大量“激活”的免疫细胞在角膜内皮细胞面聚集，内皮细胞几乎完全破坏；基质细胞明显水肿，部分被“激活”且伴有大量免疫细胞聚集。结论：①内皮排斥反应的早期能够发现角膜内皮面有免疫细胞沉积；②角膜内皮面的免疫细胞沉积与角膜内皮排斥反应的轻重成正相关；③角膜内皮排斥反应实际上是一个混合排斥反应的过程；④共焦显微镜对角膜内皮排斥反应具有实时、无创、敏感、准确以及可莺蒽、可比较、可活体观察等临床价值。     

生物工程角膜移植术后排斥反应的临床及免疫病理观察:附5例报告

目的：对生物工程角膜移植术后发生免疫排斥反应患者进行激光共焦显微镜观察和免疫病理研究,初步探究生物工程角膜移植排斥反应的发生机制。

方法：回顾性病例研究。纳入2018-09/2020-12我院收治的感染性角膜炎行生物工程角膜移植术后发生免疫排斥患者5例5眼。裂隙灯检查角膜透明度、角膜新生血管; 激光共焦显微镜对角膜进行动态观察,记录免疫排斥反应后角膜上皮下朗格罕氏细胞和基质炎症细胞密度,并以对侧眼作为对照进行统计分析。行同种异体角膜移植时取下生物工程角膜,免疫组织化学染色后对CD4⁺细胞和CD8⁺细胞及炎症细胞的浸润和分布进行观察。

结果：激光共焦显微镜显示,术眼角膜上皮下可见大量活化郎格罕氏细胞,其活化比例与对侧眼比较有差异(χ²=38.29,P<0.001)。基质层大量炎症细胞,与对侧眼比较具有差异(t=32.5,P<0.05)。对生物工程角膜组织进行免疫组织化学染色和HE染色,角膜基质层均可见CD4⁺细胞和CD8⁺细胞,仅1眼角膜基质层可见大量嗜酸性粒细胞。

结论：生物工程角膜移植术后免疫排斥反应经免疫病理证实以T细胞介导的细胞免疫在发病机制中具有重要作用,对异种抗原的超敏反应可能参与其中。     

活体共聚焦显微镜对角膜移植后排斥反应的观察

目的 应用活体共聚焦显微镜(CMTF)观察角膜移植术后排斥反应。 方法 对穿透角膜移植患者术后定期行CMTF检查。 结果 通过连续共聚焦扫描及焦点分析,得到角膜X、Y轴和Z轴多层精确的、可重复的图像及时间记录四维显示。角膜移植术后角膜可出现多种反应,如角膜上皮反应、基质反应。角膜移植排斥反应主要表现为移植片周围大量圆形细胞浸润、大的异常上皮细胞、基质水肿、基质细胞变为成纤维细胞及细胞排列紊乱。 结论 应用CMTF可直接观测活体角膜各层细胞形态、结构、病理转归及角膜移植免疫排斥的早期反应,以及反应过程的发展,为较早地诊断角膜移植排斥反应提供依据。这是裂隙灯检查所不及的。     

角膜移植术后角膜在共焦显微镜下的形态学改变

目的：研究角膜移植术后角膜组织在共焦显微镜(Confocal microscopy)下的形态学改变。方法：应用Conoscan2．0共焦显微镜对板层角膜移植术后3～7d患者12例(12只眼)，术后1a患者8例(8只眼)，穿透性角膜移植术后3～7d患者10例(10只眼)，术后1a患者11例(11只眼)进行扫描检查，记录各层角膜图像。结果：板层角膜移植术后3～7d，植片中基质细胞较小，可见裂隙状暗纹和细小神经，层间为大面积高反光区，有点状颗粒沉积，植床水肿，细胞成像不清。移植术后1a，植片中未见神经，层间反光明显减弱，但仍有点状高反光颗粒沉积，植床中出现粗大裂隙状暗纹，内皮细胞密度正常。穿透角膜移植术后3～7d，植片中基质细胞“激活”，可见神经和后基质层的粗大暗纹，内皮细胞密度正常，细胞间可镶嵌有高反光点。术后1a，植片中基质细胞仍较小，未见神经，后基质层仍有裂隙状暗纹，内皮细胞体积增大，密度减少，高反光点消失。结论：Confoscan 2．0共焦显微镜可活体检查角膜移植术后角膜组织结构和细胞的病理改变，这对评估手术效果和临床观察以及跟踪随访具有重大意义。     

角膜共焦显微镜早期诊断兔高危角膜移植排斥反应的价值

目的:角膜共焦显微镜检查对兔碱烧伤角膜移植排斥反应进行研究,找寻排斥反应早期诊断的客观指标。方法:制作兔角膜碱烧伤模型,36d后行穿透性角膜移植,于角膜移植术后4,9,14,21~28d诊断排斥反应时,角膜共焦显微镜检查角膜。结果:排斥反应时角膜共焦显微镜检查见角膜植片炎性细胞浸润,角膜细胞丢失,新生血管生长。结论:角膜共焦检查有助于早期诊断排斥反应。     

板层角膜移植术后全层角膜组织改变的共焦显微镜观察

目的研究活体扳层角膜移植术后角膜各层组织的共焦显微镜(confocal microscopy)形态学改变，方法应用Confoscan 2.0共焦显微镜对板层角膜移植术后3月～3年，角膜植片稳定的患者共17例17眼，进行扫描检查，记录与分析各层角膜图像。结果共焦显微镜上能够清楚地观察到稳定角膜的上皮细胞、基底层细胞、前弹力层，它们同正常角膜的相应细胞在图像上没有显著差别。但植片与植床的基质细胞在共焦显微镜图像上有着明显的差异。板层角膜移植术后稳定角膜植片的基质细胞，细胞核较小呈中等反光，排列较植床的基质细胞紊乱。而角膜植床的基质细胞，细胞核较大也呈中等反光，排列较植片的基质细胞稍整齐，结论1、Confoscan 2.0共焦显微镜可活体检查，板层角膜移植术后角膜组织结构和细胞的病理改变。2、板层角膜移植术后可成功的重建眼表。3、活体板层角膜移植术后稳定角膜上有自体角膜基质细胞与异体的角膜基质细胞同时共存，角膜内发细胞的形态结构正常。4、角膜板层间的不平滑与植片基质细胞结构紊乱可能是导致板层角膜移植术后视力不佳的重要原因。     

激光扫描共焦显微镜观察穿透角膜移植术后角膜的形态学特征

目的研究活体穿透角膜移植术后各层角膜组织的激光扫描共焦显微镜(Laser Scan-ning Confocal Microscope,LSCM)的形态学特征。方法应用海德堡视网膜激光断层扫描仪Ⅱ/Rostock角膜模块(HRTⅡ)对20例20眼穿透角膜移植术后40天～2年的患者进行检查,记录并分析其各层角膜图像。结果穿透角膜移植术后早期角膜上皮细胞排列欠规则;Bowman膜和Descemet膜在激光共焦显微镜下无细胞结构;所有患者浅基质层细胞均较深基质层密集;稳定角膜植片其内皮细胞排列紧密,形态为均一六边形结构;所有患者均未发现上皮下神经丛及基质神经;术后植片排斥者有其特殊形态学表现。结论激光扫描共焦显微镜可活体检查穿透角膜移植术后角膜各层组织结构和细胞的病理改变,为临床诊断及用药提供有价值的客观依据,并在跟踪随访方面具有重大意义。     

共聚焦显微镜对角膜移植术后内皮型免疫排斥反应观察

目的 应用激光共聚焦显微镜(Confocal LaserScaning Microscope,CLSM)观察角膜移植术后内皮型免疫排斥反应的特征及抗排斥治疗后的转归.方法 应用CLSM对16例角膜移植术后内皮型免疫排斥反应的病例,分别在抗排斥治疗前、治疗3～7d和1月后,对其病灶的特定位点进行检查,分析角膜上皮细胞、基质细胞、内皮细胞及免疫细胞的数量及形态学变化.结果 (1)16例患者中,8例可见典型的内皮排斥线,CLSM下:内皮排斥线是由体积较小反光明亮的炎症细胞和体积较大边界不清的异常内皮细胞构成.排斥线经过的区域上皮细胞体积增大,上皮基底层大量树突状Langerhans细胞,基质层水肿增厚,呈网格状改变.内皮细胞体积增大,边界模糊,六边形结构消失,可见多核细胞.基质水肿较重的病例,内皮层面见大量强反光团,内皮细胞无法成像.排斥线以上角膜植片相对透明的部分则各层细胞排列基本规则,内皮面少量免疫细胞附着.另8例表现为植片弥漫水肿混浊,CLSM下表现与内皮排斥线经过区域类同.(2)药物治疗3～7d后,12例患者明显好转.CLSM下见上皮基底层下树突状Langerhans细胞明显减少;基质细胞核清晰可见;内皮细胞恢复六边形结构,密度增加,其表面免疫细胞数量减少.4例患者基质层水肿减轻,中深基质层细胞有变性纤维化现象,内皮细胞层强反光团明显减少,但内皮细胞仍不能清晰成像.此4例患者用药1月后基底膜下仍有局限性树突Langerhans细胞聚集,中深基质层变性纤维化,内皮细胞排列不规则,有变圆或拉长现象.结论 应用CLSM可在细胞学水平对角膜移植术后内皮型免疫排斥反应的过程与转归进行活体的动态观察,对指导抗排斥治疗,有效提高角膜移植排斥反应的诊治水平,具有重要的临床意义.

Abstract：

Objective To demonstrate the features and the outcomes after treatment of endothelial rejection in human corneal allograft rejection by in vivo confocal microscopy. Methods Sixteen patients with endothelium rejection ofallogratt after penetrating keratoplasty were examined by confocal microscopy, to analyze the quantity and morphological changes of corneal epithelial cells, stroma cells, endothelium cells and immune cells before treatment and 3-7days and 1 month after treatment. Results Among the 16 patients, typically endothelial rejection line were seen in 8 cases, another 8 cases' keratic precipitates was diffusely scattered.Confocal microscope images revealed that the endothelial rejection lines were formed by cellular aggregate of small inflammatory cells and damaged larger endothelial cells with pyknotic highly reflective nuclei. With the progression of endothelial endothelia line, epithelium cells were large in size and lose distinct boundaries, numerous Langerhans cells (LCs) were seen in the basement epithelial cell region, keratocytes were altered in appearance with increased visibility of the cytoplasm, damaged endothelial cells decreased in number, increased in size with loss of normal polygonal shape and sticked by highly reflective immune cells which scattered or gathered. In the severely edematous area, the endothelial cells were not visualized with confocal microscopy.After 3 or 7 days of treatment, 12 cases were cured. Confocal microscopy examination revealed that the density of LCs reduced gradually and normal keratocytes were detected, the endothelial cells restored to hexagon structure and the density of immune cells decreased. Endothelial cells of 4 cases still were not visualized with confocai microscopy because of the degeneration fibrosis of deep stromal layer. LCs in the basement epithelial cell region still could be found after I month of treatment, and their endothelial cells appeared elongated or rounded with loss of their normal cell junctions. Conclusions In vivo confocal microscopy can provide us with detailed histopathology proofs and appear dynamically the transformation after treatment of endothelial rejection at cell level. It plays an important role in improving the diagnostic level and directing anti-rejection medication in our clinical practicing works.     

人体角膜内皮细胞的共焦显微镜研究

目的应用共焦显微镜观察活体角膜内皮细胞的形态学特征。方法应用共焦显微镜观察正常人（20例）、角膜炎（12例）、角膜移植术后（19例）、葡萄膜炎（5例）、大疱性角膜病变（3例）、角膜白斑（6例）、角膜营养不良（7例）、青光眼（10例）、圆锥角膜（17例）等患者的角膜内皮,分析所得活体角膜内皮图象,总结其形态学特征。结果活体共焦显微镜下角膜内皮的图象主要有8种：①完全正常角膜内皮。②水肿角膜内皮。③失代偿角膜内皮。④“激活”角膜内皮。⑤排斥角膜内皮。⑥有KP角膜内皮。⑦有“疣状物”的角膜内皮。⑧“双核”角膜内皮细胞。结论共焦显微镜能对活体角膜内皮进行实时、无创、准确以及可重复、可比较的观察。     

Sjgren综合征患者的角膜激光共焦显微镜活体观察

目的应用激光共焦显微镜观察Sjgren综合征(SS)患者角膜各层的形态改变。方法应用激光共焦显微镜对15例(30眼)SS患者和15例(30眼)同龄正常对照组的角膜进行检查,获取角膜全层图像,并进行比较、分析。结果与对照组相比,SS组角膜上皮各层细胞密度均明显下降,并出现炎性细胞和树突状细胞浸润。角膜基质细胞呈多突起星状,细胞体积增大,胞核反光减弱,胞质反光增强,浅基质出现炎性细胞浸润。SS组较对照组角膜上皮下神经纤维变细,弯曲度变大,走行方向明显改变,部分神经纤维断裂。结论SS患者不仅角膜上皮、浅基质细胞、上皮下神经出现明显改变,且角膜基质细胞形态与角膜免疫状态均出现异常。     

Alpha-1--antitrypsin in aqueous humour from patients with corneal allograft rejection

PURPOSE: To investigate the presence and concentration of alpha1-antitrypsin in aqueous humour at the time of corneal rejection and to compare results obtained from patients with reversible and irreversible rejection. METHODS: Samples of aqueous humour were obtained from 17 patients with acute corneal endothelial allograft rejection. The presence of alpha1-antitrypsin in aqueous humour was confirmed by immunoblotting and measured employing a sandwich ELISA. Total protein concentrations in aqueous humour were measured using Bradford's method. The outcome of corneal rejection episodes was determined 1 month after diagnosing corneal rejection and described as reversible or irreversible rejection. RESULTS: alpha1-antitrypsin was detected in aqueous humour. Patients with reversible rejection had significantly higher alpha1-antitrypsin concentration than patients with irreversible rejection (p = 0.044). There was no significant difference in total protein concentrations (p = 0.745), and no correlation was found between alpha1-antitrypsin and total protein concentrations (p = 0.368). CONCLUSIONS: alpha1-antitrypsin in aqueous humour seems to signal a favourable outcome of corneal rejection. The possible mechanism is discussed.     

联合自-异体穿透性角膜移植减少排斥反应研究

目的：观察联合自－异体穿透性角膜移植术后植片排斥反应的发生率和疗效。方法：对25例角膜病灶靠近边缘的患者,采用周边部新月形自体植片及中央部异体植片作穿透性移植术,另以植床靠边的旁中央穿透性移植术28例为对照组,比较其术后排斥反应的发生率和角膜散光。结果：两组植片的上皮排斥反应发生率分别为8．0％和32．1％,实质层和内皮排斥反应为28．0％及64．3％,角膜散光为3．82D和6．82D。结论：联合自－异体穿透性角膜移植术可降低术后植片排斥反应发生率及减少术后角膜散光。     

角膜移植术后可溶性白细胞介素-2受体水平的动态变化

目的:观察角膜移植术后血清可溶性白细胞介素-2受体(sIL-2R)水平与排斥反应的相关性,将其发展成为角膜移植排斥反应早期诊断和指导临床用药的辅助性检查。方法:在Lewis大鼠建立同种异体穿透性角膜移植模型,使用放射免疫法动态测定了移植术后血清sIL-2R的变化。结果:同基因移植术后sIL-2R无显著变化。同种异体移植组术后sIL-2R第6天开始增高,第9天达高峰,第11天进入平台期。其显著变化出现于临床表现之前。结论:sIL-2R水平的消长与角膜移植免疫排斥反应的发生存在显著相关性,移植术后检测sIL-2R有助于排斥反应的早期诊断。眼科学服 1996;12;51—53。     

The impact of corneal allograft rejection on the long-term outcome of corneal transplantation

PURPOSE: To examine the influence of corneal allograft rejection on the survival of penetrating corneal transplantation, to review the status of conventional therapies to improve graft survival, and to consider prospects for alternative approaches to reduce the impact of rejection. DESIGN: Perspective, including prospective, observational cohort study. METHODS: An examination of the literature on human corneal graft rejection and data from the Australian Corneal Graft Registry, reviewed in the context of clinical experience. RESULTS: Corneal graft outcome is not improving with era. The sequelae of inflammation, whether occurring before corneal transplantation or subsequently, exert a profound influence by predisposing the graft to rejection. Of the developments that have been instrumental in reducing rejection in vascularized organ transplantation, living-related donation is not an option for corneal transplantation. However, HLA matching may be beneficial and requires reassessment. The evidence base to support the use of systemic immunosuppressive agents in corneal transplantation is thin, and topical glucocorticosteroids remain the drugs of choice to prevent or reverse rejection episodes. Experimental approaches to local allospecific immunosuppression, including the use of antibody-based reagents and gene therapy, are being developed but may be difficult to translate from the laboratory bench to the clinic. CONCLUSIONS: Corneal allograft rejection remains a major cause of graft failure. High-level evidence to vindicate the use of a particular approach or treatment to prevent or treat corneal graft rejection is lacking. In the absence of extensive data from randomized, controlled clinical trials, corneal graft registers and extrapolation from experimental models provide some clinically useful information.     

Influence of donor storage time on corneal allograft survival

PURPOSE: To investigate the influence of graft storage time on corneal allograft survival in high-risk and low-risk patients. DESIGN: Comparative retrospective nonrandomized clinical trial. PARTICIPANTS: Overall, 193 patients with 210 corneal allografts were classified as high risk or low risk for corneal allograft rejection on the basis of recipient corneal neovascularization and number of ipsilateral transplants. METHODS: Data from 3 groups were evaluated. The first group received fresh (no storage in culture medium) corneas, the second received corneas of donor storage time less than 7 days in minimum essential medium (MEM) at 37 degrees C, and the final group received corneas stored in MEM longer than 7 days. Recipients were analyzed for development of immune rejection according to storage time of the corneal tissue. Corneal allograft survival rate was determined with Kaplan-Meier survival analysis. The log-rank test was used to determine statistical significance. MAIN OUTCOME MEASURES: Risk of reversible and irreversible allograft rejection in corneas stored at various intervals in high-corneal and low-corneal transplant patients. RESULTS: High-risk corneal allograft recipients had a significantly prolonged allograft survival when the tissue was stored for 7 days or greater, compared with recipients receiving fresh tissues. Patients at low risk of corneal allograft rejection also showed a tendency for prolonged survival, although not statistically significant (P = 0.06). CONCLUSIONS: Storage of corneal tissue may reduce the frequency of allograft rejection, especially in high-risk patients.     

Tacrolimus immunosuppression in high-risk corneal grafts

BACKGROUND: Unlike the immune privilege enjoyed by low-risk corneal grafts, high-risk corneal grafts experience rejection rates comparable to liver and kidney transplants. Systemic immunosuppression reduces the risk of rejection in high-risk corneal grafts. METHODS: Systemic tacrolimus, a specific T cell inhibitor, was used at a mean daily dose of 2.5 mg to immunosuppress 43 patients undergoing high-risk corneal transplantation. Immunosuppression was continued for a period of 18-24 months after the high-risk corneal graft. RESULTS: During a mean follow-up period of 33.7 months, clarity of the graft was maintained in 65% of patients. Eight patients experienced rejection episodes while on tacrolimus, and this led to graft failure in five patients. CONCLUSION: Tacrolimus is relatively safe and effective in reducing rejection and prolonging graft survival in patients with high-risk keratoplasty compared with other series where similar immunosuppression was not used.     

细胞间粘附分子在角膜疾病中的表达

目的：探讨细胞间粘附分子（Intercellular Adhesion Molecule-1,ICAM-1）在感染性角膜疾病和角膜移植免疫排斥中的表达及作用。方法：应用抗细胞间粘附分子ICAM-1的单克隆抗体，对42例角膜移植所取下的病变角膜进行免疫组织化学研究。角膜组织连续冰冻切片，进行SABC免疫组化及HE染色，研究细胞间粘附分子的表达与炎性细胞分布以及炎症程度的关系。结果：各种感染性角膜疾病、角膜变性以及角膜移植排斥过程中，ICAM-1在角膜上皮，尤其是基底细胞、角膜细胞、内皮细胞和血管化的基质层新生血管内皮细胞中均有表达，在炎性细胞包括单核细胞、巨噬细胞及淋巴细胞浸润部位表达增强；ICAM-1表达强度与炎症程度相平行。结论：ICAM-1在感染性、变性角膜病及角膜移植排斥中均有表达，在炎性细胞浸润部位表达增强；I-CAM-1对白细胞募集于炎症部位、炎症的发生、发展具有重要的中介作用。     

DCD角膜移植术后角膜内皮细胞与排斥反应的相关性分析

目的：探讨心脏死亡供体器官捐献(donation after cardiac death,DCD)来源角膜植片术后排斥反应与角膜内皮细胞的相关性。

方法：用角膜内皮显微镜对心脏死亡供体来源角膜植片行穿透性角膜移植术后发生排斥反应的28例28眼角膜植片分别于术后<1、2～3、4～6、7～12mo行角膜内皮镜检查。

结果： 28例患者术后<1、2～3、4～6、7～12mo的角膜内皮细胞变异系数分别为38.23%、49.56%、57.18%、65.04%; 角膜内皮细胞密度分别为2071.15±311.47、1771.33±348.18、1626.59±353.92、1553.14±307.31个/mm²; 角膜内皮细胞变异系数与排斥反应呈正相关关系(r=0.95,P<0.05); 术后角膜内皮细胞密度与排斥反应呈负相关关系(r=-0.93,P<0.05)。

结论：DCD穿透性角膜移植术后发生排斥反应时有角膜内皮细胞变异系数逐步增高,角膜内皮细胞密度逐步降低的趋势; 角膜内皮细胞变异系数、角膜内皮细胞密度可作为早期检测术后排斥反应的指标。     

In vivo confocal microscopy of subepithelial infiltrates in human corneal transplant rejection

PURPOSE: Corneal allograft rejection is the leading cause of penetrating keratoplasty failure in the first year after surgery. We report 2 cases of subepithelial infiltrates in corneal transplant rejection imaged by in vivo confocal microscopy. METHODS: Case report and review of relevant literature. RESULTS: Two subjects with subepithelial infiltrates in previously clear penetrating corneal transplants were assessed. In vivo confocal microscopy revealed focal accumulations of hyperreflective dendritic-like particles, postulated to represent Langerhans cells, at the level of the basal epithelium and Bowman membrane. Altered keratocytes with visible cytoplasmic processes were observed posterior to these foci. CONCLUSIONS: To our knowledge, these are the first reported cases of in vivo confocal microscopy appearance of corneal allograft rejection in humans. In vivo confocal microscopy may provide a valuable clinical tool to aid in the diagnosis of early corneal transplant rejection and in the differential diagnosis of other inflammatory conditions of the cornea.