角膜移植排斥反应的共焦显微镜研究

目的：探讨角膜移植术后免疫排斥反应的共焦显微镜特征,在细胞水平为该症的基础和临床研究提供活体、三维与实时的科学依据。方法：应用NIDEK公司提供的共焦显微镜,对11例（11眼）发生角膜移植免疫排斥反应的患眼角膜在不同时期行三维实时扫描成像,并用计算机作定性与定量分析。结果：角膜移植免疫排斥反应的共焦显微镜呈现如下特征：①上皮排斥线是由较小的炎症细胞与受损的较大的上皮细胞混合形成的结构;②上皮下浸润表现为体积较小、折射率高的炎症细胞的聚集,上皮下神经纤维水肿呈串珠状改变,浅基层细胞水肿;③基质排斥反应可见角膜基质细胞水肿,胞体变形,数量减少,并见炎症细胞浸润,后者于缝线周围较明显;④内皮排斥线则是由较小的明亮的炎症细胞与核固缩胞体形态异常的内皮细胞混合而成,随着内皮排斥线的发展,内皮 细胞数量减少,体积变大呈伪足状;⑤以上排斥反应均见新生血管长入植片,并与血管壁可见游动的炎症细胞。结论：应用共焦显微镜可在活体上为角膜移植术后免疫排斥反应提供早期诊断与鉴别诊断的科学依据,并在细胞水平对排斥反应的触发与转归进行活体的动态观察。     

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。     

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.     

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。     

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。     

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。     

应用共焦显微镜观察Fuch角膜内皮营养不良患者的病变形态学特征

目的探讨应用共焦显微镜观察我国Fuch角膜内皮营养不良患者角膜各层的活体形态学特征。方法对19例(38只眼)Fuch角膜内皮营养不良患者的中央部角膜进行活体共焦显微镜检查,分为有症状组(19只眼)和无症状组(19只眼),并选取30只眼作为正常对照组,应用NAVIS软件测量、分析角膜各层组织细胞形态和密度,以及滴状赘疣和角膜神经的直径。结果 (1)有症状组:19只眼的角膜内皮层均见到滴状赘疣,直径20-60 μm,内皮细胞密度与正常对照组比较差异有显著意义(t=18.74,P<0.01);9只眼后弹力膜增厚;14只眼角膜后基质层有长条形暗区结构;19只眼角膜基质反光普遍增强;17只眼Bowman膜有局灶性高反光区域;19只眼基底上皮细胞形态大致正常;10只眼显示正常的角膜神经结构;后、前基质细胞密度,与正常对照组比较差异无显著意义(t=0.854、1.173,P=0.38、0.24)。(2)无症状组:19只眼的角膜内皮层均见到滴状赘疣,数目较有症状组者少,直径15-40μm;内皮细胞密度,与正常对照组比较,差异无显著意义(t=1.998,P=0.053);角膜其余各层未见异常。有症状组与无症状组的内皮细胞密度计数比较,差异有非常显著意义(t=8.352,P<0.01)。结论活体共焦显微镜检查有助于Fuch角膜内皮营养不良患者的诊断,特别适用于角膜水肿、角膜内皮镜无法成像的患者。(     

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。     

利用共焦显微镜对穿透角膜移植术后散光的研究

目的 利用共焦显微镜在活体上对造成角膜移植术后散光的因素进行分析。方法 ６只有色素兔分为两组接受同种异体部分穿透角膜移植术。一组的供受体角膜边缘钻切整齐,缝线均匀,另一组刻意造成角膜切缘不整齐,缝线深浅不一致。在术后２个月内的不同时间,利用共焦显微镜对角膜切口的愈合及缝线对角膜组织的影响进行观察分析。结果 角膜切口整齐对合良好的部位疤痕形成小。角膜切口愈合后再拆除缝线,局部角膜组织的形状仍会发生较大改变。结论 首次从活体显微水平上直接形象地显示了角膜切口和缝线对角膜移植后散光的影响,提出临床型共焦显微镜对分析穿透角膜移植术后散光变化的研究有着重要参考意义。     

共焦显微镜在不典型虹膜角膜内皮综合征诊断中应用

目的 探讨共焦显微镜检查在虹膜角膜内皮综合征临床诊断中的应用价值.方法 对8例(其中6例单眼患病,2例双眼患病)常规检查无法确诊的疑似虹膜角膜内皮综合征病例进行共焦显微镜检查.观察角膜内皮层病变结构.结果 活体共焦显微镜检查发现患眼角膜内皮细胞均偏大而且形状不规则,细胞核反光明显增高,可见散在双核、多核、偏位核及分叶核等改变,7例细胞边界模糊,部分病例有局灶内皮细胞缺失、胞内环形暗区、分裂状细胞等改变.此外,3例单眼患病病例的对侧眼在共焦显微镜下发现内皮细胞密度下降、大小不均等改变.最后确诊为原发性进行性虹膜萎缩型2例、Chandler综合征4例、Cogan-Reese综合征2例.结论 共焦显微镜可以在细胞水平上无创、高分辨率地观察到虹膜角膜内皮综合征患眼角膜内皮层特征性的显微结构改变,且不受角膜水肿的影响,大大提高了虹膜角膜内皮综合征诊断的准确性,并有助于早期诊断,具有很高的应用价值.     

Immunology of corneal allograft rejection: HLA-DR antigens on human corneal cells

Human stromal and endothelial cultures were established from corneas obtained from the Maryland Eye Bank. Cultures were incubated in the presence of human gamma interferon for 4 days, trypsinized, washed, and then stained with a monoclonal reagent specific for human Class II (HLA-DR) antigens. Under these conditions, 100% of human corneal endothelial cells and 40 to 70% human corneal stromal cells expressed HLA-DR antigens.     

Immunology of corneal allograft rejection. Associated human leucocyte populations

Human serum samples and peripheral blood mononuclear leucocytes (PBML) were collected from ten corneal transplant recipients over a two-year period at intervals before and after surgery, and during and after episodes of immunologic (rejection) reaction. Serum was monitored for the development of antibodies to histocompatibility antigens and PBML were stained with a panel of monoclonal reagents specific for T cell and for monocyte populations using indirect immunofluorescence. Patients who demonstrated an elevation in the Leu 2a+ (suppressor/cytotoxic) T cell population following an immunologic reaction ultimately accepted their grafts, whereas patients whose Leu 2a+ population was unaffected developed irreversible graft clouding. Additionally, a direct positive correlation between the development of elevated levels of lymphocytotoxic antibodies and helper T cells was noted in some patients during immunologic reactions. Neither DR-positive nor monocyte population changes demonstrated any consistent correlation with rejection episodes or final graft outcome.     

Immune mechanisms of corneal allograft rejection

Penetrating keratoplasty has been successfully performed on humans for over 100 years and remains the most common form of solid tissue transplantation. Although corneal allografts enjoy a remarkable degree of immune privilege, immune rejection remains the leading cause of keratoplasty failure. The immunologic basis for corneal allograft rejection was established in animal studies over 50 years ago, yet large gaps remain in our knowledge regarding the cellular and molecular mechanisms of corneal allograft rejection. The enormous redundancy in the mammalian immune system creates a condition that favors the development of multiple independent immune mechanisms that can produce corneal allograft rejection. Although there are few absolute principles, it is certain that the immune rejection of corneal allografts is (1) T cell-dependent, (1) heavily dependent upon CD4(+) T cells, (3) not restricted to either Th1 or Th2 T cell populations, and (4) dependent upon an intact repertoire of resident antigen presenting cells.     

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.     

同种异体角膜移植后角膜内皮细胞的特征表现及生物学意义

同种异体角膜移植目前仍是治疗角膜疾病最为有效的方法,但术后发生的免疫排斥所引起的角膜炎症反应和血管化,最终导致植片水肿,甚至功能丧失仍是造成手术失败的最主要原因.角膜内皮细胞作为免疫赦免及角膜正常生理功能维系的首要屏障,在眼部系统中占据着至关重要的位置.本文就同种异体角膜移植术后免疫的识别,应答和最终反应的各阶段角膜内皮所呈现的特征改变及生物学意义进行阐述.

Abstract：

Allograft corneal transplantation is currently the most effective and optimum method to treat corneal disease, however it is the immune rejection that the main reason for graft edema caused by vascularization and inflammation of the cornea and surgical failure after transplantation. Corneal endothelial cells occupy a crucial position as immune privilege and the primary barriers for maintaining normal physiological function of eye. In this paper, the various stages (recognition, response and reaction of immune system) of corneal endothelial changes in the morphological and biological characteristics had been presented after allograft corneal transplantation.     

Cyclosporine-containing collagen shields suppress corneal allograft rejection

Thirty-seven rabbit eyes with penetrating keratoplasty grafts placed in vascularized beds to enhance the possibility of graft rejection were treated with cyclosporine delivered in collagen shields or drops of olive oil. Treatment was begun either immediately after grafting or at the first sign of immune graft reaction. Mean survival time of the grafts in the collagen shield treated eyes was significantly longer than in the eyes treated with drops. In the eyes treated at the first sign of graft reaction, cyclosporine in collagen shields halted the rejection process; seven of these eyes survived the 120-day observation period, compared to one of the eyes treated with drops. These results indicate that the collagen shield is an effective delivery system for cyclosporine and the topically administered cyclosporine is effective in suppressing the initiation of graft rejection and in reversing a graft reaction in progress.     

角膜移植术后免疫排斥反应的防治

目的 为控制角膜移植术后发生排斥反应，探讨防治排斥反应的有效途径。方法 根据不同角膜病变排斥反应发生的规律给予不同期的药物联合治疗，降低和控制排斥反应。结果 129眼中47眼(36．43％)发生免疫排斥反应，其中高危角膜病变40眼，非高危角膜病变7眼。有角膜新生血管者占89．36％。排斥反应发生时间为术后3周—3年。经药物联合治疗，角膜排斥反应得到明显的抑制，有效率达63．83％。结论 免疫排斥反应的发生是多因素参与的复杂过程，尤其高危角膜病变，术后出现排斥反应的机率明显增加，发生的时间相对较早，时间跨度较长。因此应根据不同的角膜病变及手术情况，尽早应用免疫抑制剂可明显降低排斥反应的发生率。