Rabbit corneal endothelial cell membrane potential

Membrane potential of rabbit corneal endothelial cells measured using microelectrodes was − 29.3 ± 0.8 mV, n = 45. (mean ± SEV1). Histological location of Lucifer Yellow dye iontophoresed out of the microelectrode confirmed that the microelectrode was located intracellularly, The Lucifer Yellow diffused five to six cell diameters away from the impaled cell indicating endothelial cell coupling. Depolarization by ouabain (10⁻⁴ M) and high extracellular potassium (potassium for sodium substitution) showed the cells to be responsive to changes in the bathing solution whilst impaled, that the cell membrane is more permeable to potassium than sodium and that membrane bound Na⁺-K⁺ -ATPase activity generates the transmembrane electrolyte gradients.     

The corneal endothelial cell interface

The apical edges of corneal endothelial cells have been separated along lines forming a crisscross pattern, by the help of a special method of preparation. The visualization by SEM of the lateral faces of the cells is thereby made possible. The separation leads to the formation of thin strands spanning the gap between the apical edges of adjoining cells. They are formed by stretching of the normal apical flaps. This stretching demonstrates that the main intercellular adherence is located at the tips of the apical flaps. The basal edges of the endothelial cells can be inspected, and it can be seen that the basal face of the cells has and irregular form. Threadlike processes extend from the basal cell edges. They ascend in the intercellular space and make contact with the lateral face of the neighbouring cell at varying levels. These processes are presumed to mediate intercellular transfer.     

Rabbit corneal endothelial cells in vitro: effects of EGF

Rabbit corneal endothelial cells were grown in tissue culture. Epidermal growth factor (EGF) increased the mitotic rate during the growth phase by 70% over control without affecting the plating efficiency. Within 48 hr of exposure to EGF, the endothelial cells became spindle-shaped. This morphological change was quantitated by morphometry; cells treated with EGF had a major axis 1.5 X larger than that of non-EGF treated cells. The spindle-shaped morphological change did not occur in response to other growth factors, was not related to cell density, and was reversible within 24 hr after removal of EGF from the media or subculture in the absence of EGF. The addition of 5-fluorouracil blocked cell division but did not affect the EGF-induced morphological change. The appearance of the endothelial cells following EGF stimulation is similar to migrating cells closing a wound in vivo.     

Cultured human corneal endothelial cell transplantation

Researchers have demonstrated the feasibility of transplanting human cultured corneal endothelial cells (HCEC) in various animal models. This review provides an overview of recent advances in our understanding of cultured corneal endothelial cell transplantation. We propose HCEC transplantation with a collagen sheet as the substitute carrier of HCEC. We also propose a novel strategy for corneal endothelial cell deficiency with the injection of adult human corneal endothelial precursors (HCEP). Using white rabbits or nude rats as keratopathy models, cultured HCEC were seeded on a collagen sheet. Descemetorhexis was performed on rabbit eyes. The HCEC collagen sheet was brought into the anterior chamber and fixed to the posterior stroma (HCEC group). Rabbit corneas with collagen sheet transplantation after descemetorhexis(collagen group) and with only descemetorhexis(no transplantation group) were the controls, respectively. As for HCEP transplantation, HCEP, isolated from rabbit corneal endothelial cells by sphere-forming assay, were injected into the anterior chamber and a face-down position was maintained for 24 hours in the rabbits (HCEP group). Pump function parameters of the HCEC sheets were 76-95% of those of human donor corneas. Mean corneal thickness in the HCEC group was significantly less than in the collagen and no transplantation groups 1, 3, 7, 14, 21, and 28 days (p< 0.05) after surgery. Cells were spread over the rear corneal surface in the HCEC group. In HE staining, marked stromal edema was present in the collagen and in the no transplantation groups, but not in the HCEC group with collagen sheets bearing monolayer cells. In the HCEP group, injected spheres were spread over the rear surface of the cornea and corneal edema was markedly suppressed. Our findings indicate that transplantation of cultured HCEC from adult human donor cornea by means of a collagen sheet can maintain the function of corneal dehydration. This suggests the feasibility of transplantation using cultured HCEC with a collagen sheet for corneal endothelial cell dysfunction. Additionally, adult precursor injection therapy can be also an effective strategy for corneal endothelial cell deficiency in place of conventional full-thickness corneal transplantation.     

角膜内皮细胞治疗研究进展

人角膜内皮细胞(HCECs)是一种有丝分裂后的单层内皮细胞,因此,其在体内和体外的增殖能力十分有限。HCECs在严重受损的情况下会发生内皮失代偿,极易引起失明。目前,唯一有效的治疗方法是使用含健康角膜内皮的供体植片进行角膜移植。因此,世界范围内供体材料的严重短缺推动了对角膜内皮替代来源的研究。随着HCECs的细胞培养研究的不断开展,细胞治疗为角膜内皮失代偿提供了希望。本文对角膜内皮细胞治疗方面的最新研究进展进行综述。     

The inheritance of corneal endothelial cell density

Background: Although it is known that some corneal diseases and degenerations have a significant heritable background, heritability on corneal endothelial cell density (ECD) has never been clearly determined. Our aim was to determine the heritability of corneal ECD.

Material and methods: Corneal ECD of 114 eyes (66 eyes of 33 monozygotic and 48 eyes of 24 dizygotic pairs; mean age 49.0 ± 15.5 years) was investigated by Konan Noncon Robo NSP-9900 specular microscopy. Structural equation modeling (ACE model) was applied.

Results: Endothelial corneal cell density was highly heritable (82.0%, 95%CI, 70.0–92.0%), whereas the unique environmental contribution was 18.0% (95%CI, 8.0–29.0%). Shared environmental factors had no influence on the endothelial corneal cell density.

Discussion: In this twin study, we established first that the density of the corneal endothelial cells is strongly heritable, which should stimulate future genetic studies to identify genes and pathways that are involved in determining ECD which might in turn lead to future treatments to prevent EC loss.     

角膜葡萄膜炎患者角膜内皮细胞相关性分析

目的:探讨角膜葡萄膜炎患者角膜内皮细胞在治疗前后相关参数的变异和临床意义。方法:对我院眼科2012-10/2013-12收治的角膜葡萄膜炎患者52例52眼,在治疗前后分别应用非接触型角膜内皮细胞仪测量角膜内皮细胞的相关参数,并进行统计学分析。结果:与正常组比较,患病组内皮细胞水肿明显,变异大;治疗时间越短,内皮细胞恢复越好,细胞丢失越少;反之,愈合时间越长,正常六边形细胞比率越小,变异系数较大。治疗前后各参数比较,差异有统计学意义(P<0.05)。结论:患者病程直接影响角膜内皮细胞功能的恢复。     

白内障超声乳化手术与角膜内皮细胞损伤

白内障是目前眼科最常见的致盲性眼病,超声乳化手术以切口小、反应轻、术后恢复快已被世界公认为最先进而成熟的主流手术方式。现代白内障手术从复明手术逐渐向以改善视功能为目标的屈光性手术发展。角膜作为屈光系统中最重要的组织,其透明度的维持很大程度上取决于角膜内皮细胞的功能,白内障手术不可避免地在一定程度上损伤角膜内皮细胞,由此引起的角膜水肿、混浊甚至失代偿等并发症严重的影响了白内障患者术后视力的改善,本文对白内障超声乳化手术及其角膜内皮细胞损伤因素进行综述。     

小梁切除手术前后角膜内皮细胞观察

目的:观察小梁切除术对角膜内皮细胞有无影响.方法:采用非接触型角膜内皮显微镜,对40例56眼行小梁切除术的患者,做术前术后角膜内皮细胞密度和细胞形态学的检测.结果:行青光眼小梁切除术的患者40例56眼,除了4眼有2度浅前房的患者外,其余52眼术前角膜内皮细胞密度均值为2 580.90±323.20个/mm2,术后1 wk均值为2 558.28±341.83/mm2,细胞形态学方面,最大细胞面积、最小细胞面积、平均细胞面积、细胞面积标准差、细胞面积变异系数、六角形细胞百分数术前术后无显著性差异(P＞0.05).结论:在通常情况下,小梁切除术不会对角膜内皮细胞产生不良影响.     

Rabbit corneal endothelial permeability in the presence and absence of adenosine and glutathione

Rabbit corneal endothelial fluxes of sodium, bicarbonate, inulin, and dextran have been measured in ambient solutions containing either 0.5 mM adenosine alone (A), 0.3 mM glutathione alone (G), both adenosine and glutathione, or neither adenosine nor glutathione. Addition of A alone to a solution lacking both additives caused a decrease in both Jstrendo and Jendostr sodium fluxes coupled with an increase in Jstrendo net. Addition of G alone caused no effect, whereas addition of A and G together caused a decrease in both Jstrendo and Jendostr sodium fluxes and a large increase in Jstrendo net. Addition of A or G caused an increase in Jstrendo bicarbonate flux, but no change in Jendostr flux. Addition of A and G caused an increase in both Jstrendo and Jendostr bicarbonate flux and a decrease in Jstrendo net. None of the solution variations caused an alteration in either inulin or dextran permeability. Since both A and G influence Jstrendo net bicarbonate and sodium transport, this may provide an explanation of the beneficial effects of these compounds on the endothelium to both enhance and prolong corneal thickness and hydration regulation.     

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。     

Quantification of allospecific and nonspecific corneal endothelial cell damage after corneal transplantation

Purpose

To investigate the effect of host immunity (allospecific) and surgical manipulation (non-allospecific) on corneal endothelial cells (CECs) in corneal transplantation.

Methods

Draining lymph nodes and grafted C57BL/6 corneas were harvested from syngeneic recipients, allograft acceptors, and allograft rejectors (BALB/c) 1, 3, and 8 weeks after transplantation. We analyzed CEC apoptosis using an ex vivo cornea-in-the-cup assay, and visualized cell-to-cell junctions using immunohistochemical staining (ZO-1). Automatic cell analysis using Confoscan software was used to measure CEC density as well as changes in CEC morphology by quantifying the coefficient of variation in cell size (polymegethism) and shape (pleomorphism).

Results

The cornea-in-the-cup assay showed that allogeneic acceptor T cells and to an even greater extent rejector T cells (but not syngeneic T cells) induced CEC apoptosis. CEC density after corneal transplantation was significantly reduced in allogeneic acceptors compared with syngeneic grafts (P<0.001), and CEC density was even further reduced in the allo-rejector group compared with the allo-acceptor group. Allogeneic grafts showed a greater increase in the coefficient of variation in cell size (polymegethism) when compared with syngeneic grafts 1 week after transplantation (P=P<0.001). However, pleomorphism was not significantly different between syngeneic and allo-acceptor grafts, indicating that polymegethism (but not pleomorphism or cell density) is a sensitive indicator of the effect of alloimmunity on CECs.

Conclusions

Our data demonstrate that host alloimmunity rather than surgical manipulation alone is the major cause of CEC damage in corneal transplantation, and such morphologic changes of CECs can be detected before the clinically visible onset of allograft rejection.     

Latent endothelial cell damage after experimental corneal cryopreservation

Ninety porcine corneas were evaluated by vital staining with alizarin red S and trypan blue in a three-step experiment. Central cell densities were counted (a) on freshly dissected corneas (n = 30), (b) on cryopreserved corneas directly after thawing (n = 30), and (c) after a postthawing organ culture interval of 24 h (n = 30). Two freezing methods were used: (a) minimum essential medium — containing 20% fetal calf serum and (b) the same but containing additionally 2% chondroitin sulfate. Directly after thawing neither method showed significant cell loss (3.9% and 3%) compared to fresh tissue. After postthawing organ culture, however, tissue that had been frozen without chondroitin sulfate displayed a cell loss of 73.5% compared to corneas of the same freezing protocol directly after thawing. Corneas in chondroitin sulfate containing medium showed a cell loss of only 33.2%. We conclude that reliable morphologic evaluation should not be obtained from cryopreserved corneas examined directly after thawing.     

Two-year corneal endothelial cell assessment following INTACS implantation

PURPOSE: To evaluate the 2-year effects of intrastromal corneal ring segments (INTACS) on the corneal endothelium. METHODS: Non-contact specular microscopy was performed as a subgroup test in a Phase III clinical trial. Endothelial cell images were collected before surgery and at 6, 12, and 24 months after surgery at the central and peripheral (6 and 10 o'clock) regions. Images were recorded and analyzed later by a central reading center. Cell density, coefficient of variation, and percent hexagonal cells were determined. RESULTS: There were no clinically significant changes in the endothelial cell structure at 6, 12, and 24 months (102 eyes). There was a gain of 5 cells/mm2 (6 months) and 3 cells/mm2 (12 months) at the central region of the cornea and a loss of 28 cells/mm2 at 24 months. At the 6 o'clock region of the cornea, there was a loss of 0, 24, and 92 cells/mm2 at 6, 12, and 24 months. At the 10 o'clock region of the cornea, there was a loss of 14, 30, and 94 cells/mm2 at 6, 12, and 24 months. INTACS did not statistically affect the central cell density at 6 and 12 months, however, there was a slight loss centrally at 24 months. At 24 months, all corneal regions had a slight decrease in cell density. In all eyes, mean central and peripheral endothelial cell counts remained above 2495 cells/mm2. Coefficient of variation improved and percent hexagonal cells remained unchanged. CONCLUSION: Endothelial cell density changes at 2 years after INTACS implantation were not clinically significant and endothelial cell remodeling was present.     

脂质体介导兔角膜内皮细胞基因转染的观察

目的观察阳离子脂质体Lipofectamine2000能否介导目的基因转移至兔角膜内皮细胞内及脂质体潜在的毒副作用。方法RT-PCR检测特异性胶原Ⅷ进行细胞鉴定,通过体外细胞转染实验优化阳离子脂质体与质粒DNA的浓度和比例,选择Lipo-fectamine^TM 2000/pEGFP-N1比值3∶1,脂质体体积分别为0μL、6μL、9μL、12μL转染兔角膜内皮细胞,荧光显微镜观察目的基因的表达,透射电镜观察细胞超微结构。结果RT-PCR扩增得到单一产物,与预期的扩增序列大小完全一致,角膜内皮细胞内可见脂质体介导的绿色荧光蛋白表达,Lipofectamine^TM 2000浓度能显著影响其对兔角膜内皮细胞的转染效率,存在最佳浓度,在35mm培养皿中,3μg DNA与9μL脂质体转染效率最高,透射电镜显示12μL脂质体可导致细胞器出现肿胀、崩解现象。结论阳离子脂质体可有效介导目的基因转移至角膜内皮细胞内,在35mm培养皿中,12μL脂质体对角膜内皮细胞有一定的毒副作用。     

前葡萄膜炎患者角膜内皮细胞的计算机图像分析

目的探讨前葡萄膜炎患者角膜内皮细胞相关参数的变化及其临床意义。方法选择我院收治的前葡萄膜炎患者115例(115眼),分为首次发病组(45例)、首次复发组(35例)、多次复发组(35例),并将首次发病组按病程分为2～4周组、4～8周组、8～12周组,另设正常对照组35例(35眼)。应用非接触型角膜内皮细胞仪测量角膜内皮细胞的相关参数,并对结果进行统计学分析。结果首次发病组六边形细胞百分比与细胞面积变异系数分别为(53.8±5.4)%、(33.8±5.4)%,与正常对照组(55.7±5.7)%、(31.6±2.9)%比较,差异均有统计学意义(均为P<0.05);首次复发组最大细胞面积、细胞面积变异系数、六边形细胞百分比分别为(769.8±103.0)μm2、(34.3±6.1)%、(53.2±6.9)%,与正常对照组比较,差异均有统计学意义(均为P<0.05);多次复发组各参数与正常对照组比较,差异均有统计学意义(均为P<0.05)。8～12周组细胞面积变异系数与2～4周组、4～8周组比较,差异均有统计学意义(均为P<0.05);8～12周组六边形细胞百分比与2～4周组比较,差异有统计学意义(P<0.05);各组间其余参量比较,差异均无统计学意义(均为P>0.05)。结论前葡萄膜炎对角膜内皮有明显影响,角膜内皮细胞损害与前葡萄膜炎发病频数呈正相关,与病程及疾病严重程度有直接关系。     

Low endothelial cell count and clear corneal grafts

Specular microscopic study on clear corneal grafts indicates that at times surprisingly low endothelial cell density can maintain the grafted cornea in a relatively dehydrated state. The critical limit of the endothelial cell count for corneal decompensation is thought to be 700 cells/mm2. This communication reports 13 cases of clear corneal graft with endothelial cell count below 700 cells/mm2.