复杂性角膜穿孔伤角膜移植手术对植片内皮细胞的观察研究

目的探讨复杂性角膜外伤后穿透性角膜移植手术角膜植片内皮细胞的变化。方法用角膜内皮显微镜对穿透性角膜移植联合手术12例（12眼）的术后移植片行内皮细胞摄像分析,并用超声角膜测厚仪测量术前供体角膜及术后移植片厚度。结果穿透角膜移植术后远期角膜内皮丧失率为34．98％,平均细胞面积明显增大,变异系数也明显增大,六角形细胞比例下降,但植片的厚度在正常范围内。其中11例移植片保持透明,表明多数未发生内皮细胞功能失代偿。结论穿透性角膜移植在严格掌握适应证的情况下是一种安全的方法,虽然移植片内皮细胞各种指标变化较大,但其厚度正常,透明成功率较高,所以穿透角膜移植联合手术仍不失为治疗复杂性角膜穿孔伤的良好办法。     

Ex vivo transfer of Smad7 decreases damage to the corneal endothelium after penetrating keratoplasty

PURPOSE: To determine whether transient gene transfer and expression of the intracellular antagonist of transforming growth factor beta (TGF-beta), Smad7, to corneal endothelial cells decreases corneal endothelial cell damage after penetrating keratoplasty in a rabbit model. METHODS: Rabbit corneas were transfected ex vivo with replication-deficient adenoviruses encoding Flagtagged Smad7, Flag-tagged Smad3, or LacZ (termed AdCMV-Smad7, AdCMV-Smad3, AdCMV-LacZ) and then transplanted to normal rabbits. Expression of the exogenous Smads and phosphorylation of endogenous Smad2 in the transplanted corneal endothelium were examined by immunoblotting and immunohistochemistry with anti-Flag or anti-phosphorylated Smad2 antibodies. Cellular density and morphological changes in the corneal endothelium of the transplanted cornea were evaluated by scanning electron microscopy after transplantation of the Smad-transfected corneas. RESULTS: Transplanted AdCMV-Smad7-transfected corneas significantly inhibited the decrease in cellular density and accelerated wound healing at the host-graft junction when compared with transplanted AdCMV-LacZ-transfected corneas. Transplanted AdCMV-Smad3-transfected corneas showed decreased cellular density and delayed wound healing at the host-graft junction. CONCLUSIONS: Ex vivo gene transfer of Smad7 to corneal endothelial cells inhibits the decrease in cellular density and accelerates wound healing after penetrating keratoplasty in rabbits. Thus, modulation of Smad7 expression in corneal endothelial cells may decrease corneal endothelial cell damage after penetrating keratoplasty.     

不同保存方法对角膜形态结构及朗格罕斯细胞的影响

角膜的正常结构及内皮细胞功能的稳定对于角膜的透明极为重要，术前供体角膜材料的处理和保存方法直接影响穿透角膜移植术后的成功与失败。良好的中长期角膜保存为适时进行穿透角膜移植手术提供了便利的条件。综述了短期、中期和长期保存的供体角膜材料在保存过程中以及使用该材料行穿透角膜移植术后角膜的厚度、内皮细胞密度及角膜缘的HLA—DR阳性朗格罕斯细胞的变化情况。     

Outcomes of penetrating keratoplasty with imported donor corneas

PURPOSE: To analyze factors influencing the surgical success of penetrating keratoplasty and long-term graft survival when using imported donor corneas. METHODS: Sixty-three donor corneas imported to Taipei from the Cincinnati Eye Bank from July 1992-June 1993 were used for penetrating keratoplasty. The corneal endothelium was examined using specular microscopy on arrival in Taiwan. The endothelial morphology and endothelial cell density (ECD) were compared with the photograph of the same cornea taken in the United States. The relationships of the surgical success rate with donor age, death to enucleation time, death to surgery time, and ECD were analyzed. The long-term graft survival and ECD of clear grafts were analyzed 4 years after surgery. RESULTS: On specular microscopic examination. the imported corneas showed diminished endothelial reflection, blurred cellular borders, and increased dark areas, which were markedly different from the pictures of the corneal endothelium taken in the United States. The average ECD before transportation was 2,525+/-267/mm2 and decreased to 1,934+/-250/mm2 after transportation (p < 0.001), with an average endothelial cell loss of 590+/-247/mm2. The overall surgical success rate was 89% and did not correlate with any of the donor factors tested except death to surgery time. The surgical success rate decreased when the time from death to surgery was >7 days (p = 0.05), mainly because of poor reepithelialization. Four years after surgery, 24 grafts remained clear. The ECD had decreased by 72+/-5% in the clear grafts. CONCLUSIONS: Our findings show that endothelial changes in imported donor corneas do occur after transportation, but the surgical success rate may not be influenced significantly if the penetrating keratoplasty is performed within 7 days after donor death. However, the ECD in the clear grafts 4 years after surgery is low.     

Autoradiographic study of retrocorneal membranes.

Fibrous membranes in the posterior cornea occur in failed transplants. The purpose of these studies was to investigate the role of corneal endothelium in the formation of these membranes. The corneal cellular components of rabbit corneas were labeled with tritiated thymidine. Labeled rabbit corneal stroma was replaced with unlabeled tissue (pre-Descemet's lamellar graft), leaving only labeled endothelium. Weeks later small unlabeled penetrating grafts, without endothelium, were placed inside this lamellar graft. The surrounding labeled host endothelial cells were found in a fine retrograft membrane and in the scar of grafts. The experiment demonstrated that host endothelial cells participate in the formation of retrograft membranes, but it does not exclude other origin of fibroblasts.     

Ultrastructural changes in the posterior layers of the cornea in Schnyder's crystalline dystrophy

A case of central crystalline dystrophy of Schnyder is presented in which ultrastructural studies were performed on the corneal specimen after penetrating keratoplasty. Electron microscopic changes were documented not only in the anterior but in the posterior stroma and in the corneal endothelium. In the posterior stroma and at the interface between the stroma and Descemet's membrane, numerous ovoid, electron-lucent spaces were present that most likely represented foci of lipid deposition. In addition, rare, focal areas of endothelial cell degeneration were observed that produced minute discontinuities in the endothelial cell covering of Descemet's membrane. Changes in the posterior layers of the cornea in Schnyder's crystalline dystrophy may contribute to the comparatively poorer surgical results obtained with lamellar rather than full-thickness grafts in these cases.     

穿透性角膜移植术后慢性失功移植片的超微结构观察

目的探讨穿透性角膜移植术后慢性失功能移植片的超微结构改变及发生机制。方法角膜植片慢性失功（CCAD）组：穿透性角膜移植（PK）术后因CCAD导致移植失败的12只（12例患者）角膜植片;12例患者两次PK手术的平均间隔时间为69个月,10例患者初次PK术后曾发生1次或以上免疫排斥反应,2例患者未发现免疫排斥反应;正常对照组：由山东省眼库提供的5只角膜植片作为正常供体。对两组角膜片行组织病理学、透射电镜及扫描电镜检查,结合患者的病史进行综合分析。结果透射电镜观察发现CCAD组较正常对照组角膜上皮层变薄,可见大空泡形成,角膜基质层纤维排列紊乱,无明显炎性细胞浸润;后弹力层与角膜内皮细胞之间可见异常的间隙及纤维增生;角膜内皮层萎缩变薄,细胞变形、核染色质浓缩,偶见炎性细胞与角膜内皮细胞黏附。扫描电镜观察发现CCAD组较正常对照组角膜上皮细胞微绒毛数量明显减少,暗细胞增多;角膜内皮细胞数量减少,存在缺失区,内皮细胞可见凋亡小体。结论CCAD植片特征性超微结构改变是内皮细胞的萎缩性改变和非炎性细胞成分的纤维增生。慢性亚临床的抗原依赖与非抗原依赖因素可能共同参与了CCAD的发生,免疫排斥反应可能诱导和促进了CCAD的发生、发展。     

角膜后弹力层剥除联合自动角膜刀取材内皮移植术的临床研究

目的 探讨角膜后弹力层剥除联合自动角膜刀取材内皮移植术的术后疗效、并发症、处理及适应证的选择.方法 临床病例系列研究.2007年9至12月期间,北京大学第三医院、北京大学眼科中心选择9例角膜内皮失代偿的患者行角膜后弹力层剥除自动角膜刀取材及角膜内皮移植手术,术后观察视力、角膜透明性的恢复、植片的脱位率、角膜厚度、角膜曲率及角膜内皮细胞数,随访时间3～7个月.结果 手术中1例虹膜角膜内皮综合征患者的角膜内皮植片植入失败,改行穿透性角膜移植术;其余8例患者术后植片明显脱位1例,再处理后复位.术后8例手术成功患者视力全部提高,植片透明,角膜厚度为(775±30)μm;角膜曲率为(44.19±2.28)D;角膜散光度数为(2.20±0.83)D;角膜内皮细胞数为(1439±296)个/mm~2.结论 角膜后弹力层剥除联合自动角膜刀取材内皮移植术有可能成为一种治疗角膜内皮失代偿的重要术式.     

Endothelium microscopy studies following keratoplasty

The quality of the endothelium in 15 patients with transplants after penetrating keratoplasty was analyzed. Donor age was not taken into account (average age of donors 65.8 years). At the time of the investigation the mean age of the grafts was comparatively high (oldest donor 92 years). There were only a few morphological changes in the endothelium and visual acuity was satisfactory. Even with low cell count (625/mm2) the graft had normal clarity. A number of pathologic changes in corneal endothelial cells after keratoplasty are described.     

Effect of 1% sodium hyaluronate (Healon) on a nonregenerating (feline) corneal endothelium

A series of experiments were performed to investigate the effect of 1% sodium hyaluronate (Healon) on the nonregenerating corneal endothelium of the cat. Aqueous humor replacement with 1% sodium hyaluronate resulted in mild, transient elevations of intraocular pressure compared to eyes that were injected with balanced salt solution. Sodium hyaluronate 1% protected the feline endothelium against cell loss incurred by contact with hyaluronate-coated intraocular lenses compared to endothelial contact with lenses that were not coated with sodium hyaluronate. The use of intraoperative 1% sodium hyaluronate, however, did not protect against endothelial cell loss incurred by penetrating keratoplasty or prevent subsequent skin graft-induced corneal homograft rejections. Homograft rejections were milder, however, in some eyes that received grafts coated with 1% sodium hyaluronate. Image analysis of photographs of trypan blue- and alizarin red-stained corneal buttons after trephining, stretching of Descemet's membrane, rubbing against iris-lens preparations, or immediately after penetrating keratoplasty demonstrated that the stretching of the posterior cornea is an important cause of endothelial damage that would not be protected against by a viscoelastic coating.     

Fate of lyophilized xenogeneic corneal lenticules in intrastromal implantation and epikeratophakia

The antigenicity of intrastromal and epikeratophakia xenografts of lyophilized corneal tissue was evaluated in nonimmune and immune recipients. Lyophilized feline lenticules were implanted into intrastromal pockets in unsensitized rabbits and rabbits sensitized to the donor cat. In both cases, the grafts remained clear. Sensitized rabbits with clear intrastromal grafts received fresh tissue penetrating keratoplasty grafts from the same donor cat, placed adjacent to the intrastromal grafts. The fresh tissue penetrating keratoplasty grafts were rapidly rejected, while the lyophilized intrastromal grafts remained clear. Cats sensitized to rabbits received lyophilized and rehydrated epikeratophakia grafts shaped from rabbit cornea; these lyophilized grafts also remained clear for the 3-month period of the study. The results indicate that lyophilized and rehydrated corneal stroma, which is devoid of living cells, is not antigenic and is not subjected to immunologic attack, even in cases where the donor and host are of different species and the host has been previously immunized to the donor.     

Corneal endothelium and postoperative outcomes 15 years after penetrating keratoplasty

PURPOSE: To determine changes in the central endothelium and thickness of grafted corneas and the cumulative probability of developing glaucoma, of graft rejection, and of graft failure 15 years after penetrating keratoplasty. DESIGN: Longitudinal cohort study of 500 consecutive penetrating keratoplasties by one surgeon. METHODS: Regrafted eyes, fellow eyes of bilateral cases, and patients not granting research authorization were excluded, leaving 388 grafts for analysis. At intervals after surgery, we photographed the endothelium and measured corneal thickness using specular microscopy. The presence of glaucoma, graft rejection, and graft failure were recorded. RESULTS: The 67 patients examined at 15 years represented 30% of the available clear grafts. Endothelial cell loss from preoperative donor levels was 71 +/- 12% (mean +/- standard deviation, n = 67), endothelial cell density was 872 +/- 348 cells/mm(2), and corneal thickness was 0.59 +/- 0.06 mm. Endothelial cell density was unchanged between 10 and 15 years, whereas corneal thickness increased (P = .001, n = 55). The mean annual rate of endothelial cell loss from 10 to 15 years after surgery was 0.2 +/- 5.7% (n = 54). The cumulative probability of developing glaucoma, graft rejection, or graft failure was 20%, 23%, and 28%, respectively, and 6 of the 8 graft failures after 10 years resulted from late endothelial failure. CONCLUSIONS: From 10 to 15 years after penetrating keratoplasty, the annual rate of endothelial cell loss was similar to that of normal corneas, corneal thickness increased, and late endothelial failure was the major cause of graft failure.     

Endothelium and pachymetry of clear corneal grafts 15 to 33 years after penetrating keratoplasty.

PURPOSE: To evaluate long-term endothelial cell count and thickness of clear corneal grafts after penetrating keratoplasty. METHODS: Specular microscopy and ultrasonic pachymetry were performed in 20 eyes (14 eyes that were keratoconus, three aphakic/pseudophakic bullous keratopathy, one Fuchs dystrophy, one had herpetic keratitis, and one avascular scar after injury) of 18 patients (mean age +/- SD 58+/-15 years; range, 34 to 82 years) with a mean follow-up of 22+/-6 years (range, 15 to 33 years). RESULTS: Mean endothelial cell count was 808+/-194 cells per mm2 (range, 575 to 1243 cells/mm2), and thickness was 608+/-75 microm (range, 430 to 751 microm). Endothelial cell count was neither correlated with thickness (P = .25, r2 = .08) nor with follow-up interval (P = .31, r2 = .028). We observed predominantly enlarged endothelial cells and mild polymegethism. No graft rejections were recorded. CONCLUSION: Despite a reduced cell density, the dehydration function of the endothelium may still be sufficient in corneal grafts up to 33 years after penetrating keratoplasty.     

组织工程角膜三维构建的实验研究

目的以人胚胎干细胞（hESC）诱导细胞为种子细胞,以脱细胞猪角膜基质（APCM）为支架三维构建生物工程角膜,以期用于穿透性角膜移植,解决角膜供体极度匮乏的难题。方法实验研究。无菌条件下将新鲜猪角膜组织置于0.5% SDS溶液中4 ℃脱细胞 24 h,获取APCM。将hESCs与人角膜基质细胞通过Transwell共培养5 d,获取眼周间充质干细胞（POMPs）,再于人晶状体上皮细胞源性条件培养基继续培养14 d获取角膜内皮样细胞并进行鉴定和筛选纯化。将纯化后扩增的角膜内皮样细胞接种于APCM构建角膜内皮植片,并移植入角膜内皮功能失代偿动物模型进行泵功能评估;采用人角膜缘干细胞（LSCs）来源的条件培养基培养hESCs 12 d,诱导其分化人角膜上皮样细胞并筛选鉴定,将其与APCM构建的角膜上皮植片移植于LSC失代偿动物模型的角膜缘,观察其眼表修复能力。结果诱导的人角膜内皮样细胞表达内皮细胞相关标记物vimentin、N-cadherin、Na+/K+ATP酶和ZO-1。构建的角膜内皮植片能够促使角膜内皮功能失代偿动物的角膜逐渐恢复透明。构建的角膜上皮细胞植片具有4～5层细胞复层结构,类似于正常角膜上皮,且能够一定程度上修复LSC失代偿动物模型眼表。结论采用hESCs诱导分化来源的细胞与APCM构建的人角膜内皮植片和人角膜上皮植片具有类似于正常角膜的功能,为全层生物角膜的构建提供了良好的实验和理论基础,具有良好的临床应用前景。     

The triple procedure. Penetrating keratoplasty, extracapsular cataract extraction and posterior chamber lens implantation. A clinical and specular microscopic study

The combination of penetrating keratoplasty, cataract extraction and intraocular lens implantation, the triple procedure, is the surgical choice for the patients with corneal disease and cataract. The success of the corneal graft depends chiefly on the endothelium of the transplanted cornea. Preservation, donor age, cadaver time, histocompatibility and operation itself affect endothelial cells. We present clinical results including endothelial evaluation of 20 patients who have undergone the triple procedure during the years 1984-1986. Mean age was 73 years. The follow-up time varied from 1 to 30 months. The visual recovery was very good. Every patient had better visual acuity post-operatively than pre-operatively. We had no operative complications. Three patients had rejection episodes which all were cured with cortisone. No re-transplantations were needed. The mean endothelial cell density was 1723 cells/mm2. The possible factors affecting the outcome are discussed.     

Pseudophakic bullous keratopathy. Relationship to preoperative corneal endothelial status

Pseudophakic bullous keratopathy is one of the complications of intraocular lens implantation. A knowledge of the preoperative status of corneal endothelium may help to minimize the incidence of this complication. The preoperative corneal endothelial status of 118 eyes of 102 patients who received Worst-Medallion intraocular lenses more than five years ago was analyzed retrospectively. This data was then correlated with the postoperative clinical status of the cornea. Twelve eyes (10%) underwent penetrating keratoplasty for irreversible corneal edema, and 28 of the remaining eyes (22%) had clinical evidence of peripheral corneal edema. No correlation was found between the preoperative endothelial cell density or the degree of postoperative cell loss and the development of corneal edema. Significant correlation was found between variation in cell size (pleomorphism) and the development of postoperative corneal edema. Greater density of precipitates on endothelium and abnormality in cell shape postoperatively were also frequently seen in corneas that developed edema subsequently.     

Evaluation of donor corneas by means of a computerized image analysis

The authors compare two groups of patients and specify characteristics of possible cornea donors for penetrating keratoplasty. Donors are selected by examining several parameters of the corneal endothelium: cellular density, average cell area, form factor, polymegetism quotient. These parameters are studied using an endothelial microscope and an image analysis system. The data thus obtained are then used for statistical analysis.     

Corneal endothelial cell damage in penetrating keratoplasty.

Fifty-eight corneal grafts were examined by specular microscopy to determine the corneal endothelial cell damage following penetrating keratoplasty. The average postoperative follow-up period was 10.1 months. The cell density decreased continuously during the observation period, and the endothelial cell loss of the central area of the cornea in all the cases averaged 10.4% at two weeks after the surgery, 16.0% at one month, 33.0% at three months, 39.4% at six months, and 48.2% at one year. However, the cell loss in the keratoconus group averaged -1.9, 1.2, 9.9, 30.6 and 33.4% for the corresponding postoperative periods. The cell loss in the bullous keratopathy eyes showed higher values compared to those in the keratoconus and the corneal leukoma eyes. It was concluded that cell loss in penetrating keratoplasty during the first postoperative year depends on the host diseases.     

Further studies on the functional decompensation of corneal endothelial cells

30 cases of corneal endothelial decompensation occurred in 241 transparent grafts of penetrating keratoplasty during a 5-year follow-up with the specular photomicroscope. The study indicated that the critical cell density for corneal endothelial decompensation was 786/mm2; the critical cell dimension as represented by the sum of mean maximal and minimal diagonal was 35.43 microns; and the critical difference between the mean maximal and minimal diagonal was 14.70 microns. When these three criteria all approximated the critical values, the endothelium was regarded as in a predecompensation state that heralded impending endothelial decompensation. Therefore, these values may be regarded as a reference standard for diagnosing early stage endothelial decompensation.     

The iridocorneal endothelial syndrome

The iridocorneal endothelial syndrome represents a unique group of ocular pathologies (Chandler syndrome, progressive iris atrophy, and Cogan-Reese syndrome) characterized by the proliferation of corneal endothelial cells that migrate toward the iridocorneal angle and iris surface causing, to a degree varying according to the subtype, corneal edema and decompensation and secondary glaucoma, whether by obstructing the angle or producing peripheral anterior synechiae by contraction of the basement membrane of the migrating cells over the surface of the iris. A triggering factor, possibly viral, induces the corneal endothelial cells to proliferate and behave like epithelial cells. Diagnosis is made based on typical ocular findings on the cornea and iris. Iridocorneal endothelial syndrome is more frequent in young women, with unilateral involvement in most cases. In vivo confocal microscopy is an excellent diagnostic tool, especially in borderline presentations like early cases of Chandler syndrome, which affects the cornea predominantly. Typical clinical management consists of treating the corneal edema and decompensation, where endothelial keratoplasty techniques have replaced in many cases the need for a penetrating keratoplasty and treating the secondary glaucoma, which usually requires surgical intervention.