圆锥角膜基质细胞端粒长度与衰老相关标记物的变化

目的 探讨圆锥角膜基质细胞内端粒长度的变化和圆锥角膜基质内衰老相关β-半乳糖苷酶与衰老标记蛋白30(SMP-30)的表达,以及其在圆锥角膜发牛发展中的临床意义.方法 实验研究.收集2006年1月至12月间于山东省眼科研究所青岛眼科医院接受角膜移植治疗的圆锥角膜患者(32例)的病变角膜37份、来源于眼库的正常角膜20份.所收集圆锥角膜患者年龄范围13～34岁,平均(19±5)岁;正常角膜供体年龄范围9～25岁,平均(19±4)岁.采用Southern印迹杂交检测圆锥角膜和正常角膜基质细胞的端粒长度.以5-溴4-氯-3-吲哚-β-D-半乳糖苷原位染色法染色圆锥角膜和正常角膜基质中的衰老相关β-半乳糖苷酶,应用逆转录PCR分别检测圆锥角膜和正常角膜基质中的SMP-30.同时对圆锥角膜和正常角膜基质进行组织病理学观察.应用t检验进行统计处理.结果 圆锥角膜基质细胞的端粒长度为10.29～14.12 kb,平均端粒长度为(11.54±1.41)kb;正常角膜基质细胞的端粒长度为12.64～15.32 kb,平均端粒长度为(13.45±0.99)kb;统计学分析显示两者比较差异有统计学意义(t=4.753,P＜0.05).组织化学染色显示圆锥角膜基质内X-Gal染色町见散在分布蓝色阳性着色,表明存在衰老相关β-半乳糖苷酶的表达;而正常角膜基质中X-Gal染色阴性,未见衰老相关-β-半乳糖苷酶表达.RT-PCR检测结果 显示圆锥角膜与正常角膜中均无SMP-30蛋白的表达.组织学病理学观察显示正常角膜基质纤维排列规则紧密,角膜细胞规则地分布在基质中.而圆锥角膜基质胶原纤维排列呈现疏松不规则,细胞分布较前者散乱且数量减少.结论 与正常角膜基质相比,圆锥角膜基质细胞的端粒长度有所缩短,基质中衰老相关β-半乳糖苷酶表达增加.圆锥角膜可能是一种与组织异常老化有关的疾病.     

圆锥角膜的光镜及透射电镜研究

目的观察圆锥角膜在光镜及透射电镜下的超微结构,试图了解圆锥角膜的发病机制.方法应用常规病理方法将正常角膜片与圆锥角膜片制成石腊切片,光镜下观察角膜中央区角膜基质细胞的密度,结果应用t检验;将正常角膜片与圆锥角膜标本制成超薄切片,在透射电镜下观察正常角膜与圆锥角膜基质细胞超微结构及胶原的变化.结果光镜观察结果,圆锥角膜中央基质区的角膜细胞密度与正常角膜相似,二者间差异无显著性(P＞0.05).电镜观察结果,圆锥角膜前基质层胶原纤维排列欠规则.角膜细胞附近,实质板层之间可见微丝状的沉积物.同正常角膜比,圆锥角膜基质细胞细胞核缩小,核内异染色质减少,线粒体增多、肿胀,粗面内质网增多、扩张,高尔基体正常结构消失.结论圆锥角膜的发病与基质细胞数量无关;圆锥角膜基质细胞超微结构的异常可能是圆锥角膜发病的根源.     

圆锥角膜组织中EGF和bFGF变化免疫组织化学的研究

目的 观察正常人及圆锥角膜中表皮生长因子(EGF)和碱性成纤维细胞生长因子（bFGF)的表达，探讨EGF和hFGF在圆锥角膜中的变化。方法 采用免疫组织化学SABC法检测角膜组织中EGF、bFGF的表达，结果 在圆锥角膜上皮层、Bowman层和内皮层有EGF表达，EGF比正常角膜的表达强；在圆锥角膜的上皮层、实质层和内皮层可见bFGF的阳性表达，圆锥角膜的基质瘢痕区bFGF有较强的阳性表达。结论 圆锥角膜组织中EGF水平增高，促进上皮细胞和基质细胞的DNA合成，从而使组织愈合。bFGF表达增强可以促进角膜上皮、基质细胞和内皮细胞的增殖。     

圆锥角膜的光密度分析

目的 使用Pentacam对圆锥角膜和健康角膜的光密度进行测量,比较健康角膜与不同等级圆锥角膜的透明度.方法 使用Pentacam眼前节分析系统测量对照组(30例30眼正常角膜)以及圆锥角膜组(36例36眼圆锥角膜)的光密度,以角膜顶点为中心,分别测量0～2 mm、2～6 mm、6～ 10 mm、10 ～ 12 mm直径范围的前层(120μm)、后层(60 μm)以及中间层的角膜光密度.使用Amsler-Krumeich分级方法对圆锥角膜进行分级,对比不同等级光密度差异.分析圆锥角膜光密度与厚度、陡峭K值的相关性.结果 总直径的全层角膜光密度圆锥角膜组(17.96±3.23)和对照组(17.39±1.95)之间差异无统计学意义(P =0.124);但是在0～2 mm直径范围内的前层、中层、全层,2～6mm直径范围内的前层以及总直径的前层中,圆锥角膜组的光密度均显著高于对照组(均为P＜0.05).按照圆锥角膜等级分组,对照组角膜光密度与轻度圆锥角膜组各层比较,差异均无统计学意义(均为P＞0.05),但对照组角膜光密度与中度圆锥角膜组0～2mm直径范围内的前层比较差异有统计学意义(P＜0.05).重度圆锥角膜在0～2 mm直径范围内各层光密度较对照组、轻度圆锥角膜组、中度圆锥角膜组均显著增高(均为P＜0.05),在2～6 mim直径范围和总直径的前层、中层和全层与其余三组相比均显著增加(均为P＜0.05).圆锥角膜组的陡峭K值与0～2 mm直径范围的总角膜光密度显著相关(P＜0.05).圆锥角膜组的角膜顶点以及角膜最薄点的厚度与光密度也存在明显的相关性(均为P＜0.05).结论 相比正常角膜,轻度圆锥角膜光密度不发生改变,中度圆锥角膜光密度增高主要发生于角膜前层0～2 mm直径范围内,重度圆锥角膜光密度增高主要发生于角膜前层0～6 mm直径范围内和角膜中层2 mm直径范围内,而且圆锥角膜光密度随疾病发展而增高.角膜光密度是圆锥角膜的一个重要参考指标,可以用来检测圆锥角膜发病进程以及治疗效果.     

Ⅲ型和Ⅵ型膠原在正常角膜和圆锥角膜中的表达

目的 观察正常人及圆锥角膜中胶原Ⅲ、Ⅵ型胶原的表达,探讨Ⅲ、Ⅵ型胶原在圆锥角膜病变中的变化。方法 采用免疫组织化学-SABC法检测角膜组织中Ⅲ、Ⅵ型胶原的表达。结果 Ⅲ型胶原在正常角膜和圆锥角膜的上皮层、实质层及内皮层均有阳性表达,但圆锥角膜比正常角膜的表达弱,而圆锥角膜基质斑块状瘢痕区可见Ⅲ型胶原呈强阳性表达;正常角膜和圆锥角膜组织中在Bowman's层、Descemet's层和基质层均可检测到Ⅵ型胶原表达,但圆锥角膜中Ⅵ型胶原表达减低,特别在圆锥角膜的疤痕区内,Ⅵ型胶原明显降低。结论 Ⅲ、Ⅵ型胶原的异常表达会导致角膜基质纤维间隙的稳定性降低,改变结缔组织中胶原束的组成,最终影响角膜的正常形态。     

I型和II 型胶原在正常角膜和圆锥角膜中的表达

目的　观察正常人及圆锥角膜中 I、II型的表达 ,探讨胶原 I、II型在圆锥角膜病变中的变化。方法　采用免疫组织化学 - SA BC法检测角膜组织中胶原 I、II型胶原的表达。结果　胶原 I型在正常角膜和圆锥角膜的上皮层、实质层及内皮层均有阳性表达 ,但圆锥角膜比正常角膜的表达弱 ;而胶原 II型在正常角膜和圆锥角膜组织的基质层和 Bow m an层均可检测到 ,但圆锥角膜中 II型胶原表达减低 ,在圆锥角膜基质斑块状瘢痕区 II型胶原呈强阳性表达。结论　 I、II型胶原的减少会导致角膜的稳定性降低 ,使角膜的机械抵抗力减弱 ,并使角膜前凸变薄。     

人类转化生长因子诱导的细胞外基质黏附蛋白βig-h3在圆锥角膜和正常角膜中的表达

目的：观察圆锥角膜患者角膜组织及正常人角膜组织中人类转化生长因子诱导的细胞外基质黏附蛋白βig-h3的表达，探讨细胞外基质结构成分在圆锥角膜病变中的作用。方法：采用原位杂交技术，以BIGH3基因的cDNA探针对圆锥角膜患者的角膜组织及正常人角膜组织中βig-h3表达主要在基质层；圆锥角膜患者角膜基质层中βig-h3的表达随角膜病变的程度加深而减弱，严重者无βig-h3阳性表达。在圆锥角膜病变区与正常角膜交界处，βig-h3表达呈强阳性。结论：细胞外基质结构成分βig-h3在角膜纤维间质有序化的发育中起细胞黏附作用；圆锥角膜病变的发生过程可能与βig-h3的水平下降有关。     

机械法准分子激光上皮瓣下角膜磨镶术与去瓣Epi-LASIK术后兔角膜基质细胞凋亡的研究

目的观察机械法准分子激光上皮瓣下角膜磨镶术（Epi-LASIK）与去瓣Epi-LASIK术后不同时间点角膜基质细胞的凋亡,探讨2种手术方式对角膜创伤修复过程的影响。方法 42只新西兰大白兔随机编号,其中40只兔各取一侧眼行Epi-LASIK,对侧眼行去瓣Epi-LASIK,剩余2只兔4只眼作为正常对照组。分别于术后4h,1、3、7d共4个时间点取出角膜组织,透射电镜下观察各组兔角膜组织的超微结构改变,采用TUNEL法检测2组兔手术后不同时间点角膜基质细胞的凋亡情况,应用免疫组织化学法检测2组兔手术后不同时间点白细胞介素-1β（IL-1β）在角膜基质细胞中的表达。结果 Epi-LASIK组术后1d及3d角膜基质细胞凋亡细胞数明显高于去瓣Epi-LASIK组,差异均有统计学意义（t=2.42,P〈0.05;t=4.04,P〈0.05）;Epi-LASI组术后1、3、7d角膜基质中IL-1β表达的阳性细胞数明显高于去瓣Epi-LASIK组,差异均有统计学意义（t=2.13,P〈0.05;t=3.71,P〈0.05;t=3.06,P〈0.05）。Epi-LASIK组术后透射电镜下可见兔角膜基质细胞核固缩,染色质边集并可见凋亡小体。免疫组织化学染色显示Epi-LASIK组术后角膜基质中IL-1β的表达强于去瓣Epi-LASIK组。结论去瓣Epi-LASIK对角膜基质细胞的损害较Epi-LASIK轻,提示去瓣Epi-LASIK术后早期角膜创伤愈合反应的程度较轻,有利于角膜组织的快速修复。     

丝裂霉素C诱导PRK后角膜基质细胞凋亡的实验研究

目的 探讨丝裂霉素C(MMC)对准分子激光角膜切削术(PRK)后角膜基质细胞凋亡的影响.方法 30只兔随机取1只眼行PRK并术中使用0.02%MMC为PRK MMC组,另1只眼仅行PRK设为PRK组,2只兔设为正常对照组.观察角膜上皮下混浊(haze)程度;采用苏木精-伊红染色法、TUNEL法和免疫组织化学法观察角膜细胞.结果 术后1周,1、2、3个月角膜haze差异均有统计学意义(P＜0.05).术后1周,1、2、3个月PRK MMC组与PRK组TUNEL染色、α-SMA阳性细胞数相比,差异均有统计学意义(P＜0.05).结论 术中眼局部应用0.02%MMC溶液可促进活化的角膜基质细胞凋亡,减轻PRK术后haze.     

准分子激光原位角膜磨镶术后角膜细胞凋亡的研究

目的 了解准分子激光原位角膜磨镶术（laser in situ keratomileusis,LASIK）后早期角膜基质细胞凋亡的特点并探索LASIK术后角膜愈合反应程度与角膜刀的机械损伤、激光能量的相关性。方法 20只新西兰白兔,其中10只兔左眼为LASIK-4.0D组、右眼为LASIK-8.0D组、另10只兔左眼为单纯角膜瓣组、右眼作为空白对照组,在术后4小时处死兔取角膜制作病理切片,行透射电镜检查,用脱氧核糖核苷酸末端转移酶介导的缺口末端标法（terminal-deoxynucleotidyl transferase medi-ated dUTP nick end labeling,TUNEL）原位显示凋亡细胞,激光扫描共聚焦显微镜观察凋亡细胞形态,定量统计比较凋亡水平差别。结果 三个手术组中角膜基质中均出现凋亡细胞,主要分布于角膜瓣和基质床交接面两侧50μm范围内的基质层中;瓣缘的凋亡水平高于中央部。而空白对照组中,凋亡细胞仅出现在表层上皮,角膜基质细胞未见凋亡。三个手术组的TUNEL阳性细胞计数进行方差分析,三组之间差异无显著意义（F=1.008,P〉0.05）;每组不同个体的TUNEL阳性细胞计数存在明显的差异。结论 正常角膜基质细胞不发生凋亡。角膜基质细胞凋亡与角膜损伤有关。LASIK术后早期即可出现角膜基质细胞的凋亡表达,该表达与角膜瓣的制作有关,与激光切削的深度、能量无明显的相关性。     

Keratocyte apoptosis associated with keratoconus.

Keratoconus is an ectatic corneal dystrophy associated with stromal thinning and disruption of Bowman's layer. The purpose of this study was to explore a possible association between keratocyte apoptosis and keratoconus. Keratocyte apoptosis was evaluated in corneas of patients with keratoconus, corneas of patients with stromal dystrophies, and normal donor corneas using the transferase-mediated dUTP-digoxigenin nick and labeling (TUNEL) assay. Keratocyte apoptosis was also studied in keratoconus and normal corneas using transmission electron microscopy. TUNEL-stained keratocytes were detected in 60% of corneas with keratoconus, but only 35% of corneas with stromal dystrophies (P =0.03). The number of TUNEL-positive keratocytes detected in the keratoconus, stromal dystrophy, and normal corneas was 7+/-1 (mean+/-standard error, range 0-20), 2+/-0. 8 (range 0-9), and 0+/-0 (range 0-0) TUNEL-positive cells per section, respectively. The differences between the keratoconus and the stromal dystrophy (P =0.0097) or the normal cornea (P =0.01) groups were statistically significant. The difference between the stromal dystrophy and normal cornea groups was not statistically significant (P =0.45). The stromal dystrophy group was included to account for surgery-associated keratocyte apoptosis. No TUNEL-stained keratocytes were detected in normal corneas. Cell morphologic changes consistent with apoptosis were detected by transmission electron microscopy (TEM) in keratocytes of keratoconus corneas, but not in keratocytes in normal corneas. Chronic keratocyte apoptosis associated with ongoing epithelial injury may link risk factors associated with keratoconus such as chronic eye rubbing, contact lens wear, or atopic eye disease. Similarly, increases that have been detected in several different degradative enzymes in keratoconus corneas could be associated with chronic keratocyte apoptosis and less than perfect control of release of intracellular contents.     

Evidence of apoptotic cell death in keratoconus

PURPOSE: To determine the potential role of apoptosis in the noninflammatory degeneration characteristic of keratoconus. METHODS: Four normal corneas and 16 keratoconus corneas were obtained as archival specimens. Tissues were examined histopathologically for TUNEL immunoreactivity to detect the presence of DNA fragmentation. Tissues were also subjected to single-stranded DNA (ssDNA) analysis, a more apoptosis-specific stain. RESULTS: Normal corneas exhibited fewer than five TUNEL-positive epithelial cells per section, these being very lightly stained. All 16 keratoconus corneas demonstrated extensive, intense TUNEL staining in at least one layer. Fifteen of 16 exhibited staining in the epithelial layer, 11 of 16 in the stromal layer, and 13 of 16 in the endothelial layer, whereas 10 of 16 keratoconus cases demonstrated TUNEL immunoreactivity in all three layers. The ssDNA stain was also positive and evident in all three layers of the cornea, although to a lesser degree than the TUNEL assay. CONCLUSIONS: The noninflammatory nature of keratoconus, coupled with the TUNEL in situ results, suggests apoptosis as a mode of cell death in this degenerative disease.     

20%乙醇处理兔角膜后上皮增生和细胞凋亡的研究

目的探讨准分子激光角膜上皮瓣下磨镶术(LASEK)中采用20%乙醇浸润兔角膜40s后角膜上皮增生和角膜细胞凋亡情况与机械刮除角膜上皮后的异同。方法实验组42只新西兰大白兔,用直径为8mm的LASEK专用角膜上皮刀切割角膜上皮,20%的乙醇浸润单眼40s,机械刮除对侧眼中央8mm直径的角膜上皮,随机分7组,于术后0、4h,1、3、5、8、30d取材;6只兔眼为空白对照。角膜冰冻切片,行Ki67免疫组化检查和TUNEL检测,计数角膜中央前基质细胞。结果乙醇浸润后5d中央角膜上皮增生达峰值,术后1d周边角膜上皮增生达峰值;术后4h上皮刀口下方局限的角膜基质细胞TUNEL染色阳性,数量最多;各组角膜中央前基质细胞计数和空白对照比差异无统计学意义(P=0.68)。机械刮除角膜上皮后3d周边角膜上皮增生达峰值,其高于乙醇浸润后角膜上皮的增生峰值;术后4h可见大量中央前基质细胞TUNEL阳性;术后1d中央前基质细胞数量最少(P<0.05)。结论与机械刮除角膜上皮相比,20%乙醇浸润40s对角膜损伤轻,恢复快,乙醇浸润后的角膜上皮对基质细胞有保护作用。     

Reduced expression of the gap junction protein Connexin 43 in keratoconus

PURPOSE: The purpose of the present study was to examine whether keratoconus, which is a bilateral noninflammatory corneal ectasia with multifactorial aetiology, shows altered expression of Connexin (Cx43). Cx43 is an important gap junction protein that contributes crucially to epithelial and stromal integrity of cornea. METHODS: Eight keratoconic human corneal buttons were examined with immunohistochemistry and Western blotting and compared with eight normal human corneal buttons, to unravel changes in Cx43 expression. RESULTS: All normal corneas exhibited similar epithelial Cx43 expression patterns, with the protein located in the basal epithelial layer. In contrast, some keratoconic corneas showed an altered pattern of immunostaining and Western blotting confirmed a decreased expression of Cx43 in keratoconic corneas. CONCLUSIONS: Our results indicate that a decrease in Cx43 amount together with functional alteration of the protein is associated with keratoconus pathophysiology However, these changes apply only to some of the corneas examined and may not generally account for the development of keratoconus.     

Repeated UVR exposures cause keratocyte resistance to apoptosis and hyaluronan accumulation in the rabbit cornea.

PURPOSE: To evaluate hyaluronan (HA) production and level of apoptosis of corneal cells after repeated UVR exposures. METHODS: Fifteen albino rabbit corneas were exposed to 310 nm UVR at a dose that causes biomicroscopically significant keratitis (0.47 J/cm2). Nine rabbits received a single dose of UVR. Six rabbits were irradiated 3 times at 7-day intervals. Rabbits exposed to the single dose of UVR, were sacrificed 24 hours, 7 and 14 days after irradiation. Rabbits exposed to the repeated doses of UVR, were sacrificed 24 hours and 14 days after the last irradiation. The corneal tissue specimens were processed for histological analysis using specific staining for HA, and the TdT-dUTP terminal nick-end labeling (TUNEL) assay. RESULTS: Corneas exposed to a single UVR dose showed extensive positive TUNEL staining 24 hours after exposure. Almost all basal epithelial cells, keratocytes throughout the entire thickness of the stroma, and endothelial cells were TUNEL-positive. No HA was found 24 hours after exposure. Extracellular HA staining of high intensity was found at day 7 throughout the entire central stroma, except the anterior one-fourth. At day 14 only a faint HA staining was detected in the posterior stroma, close to Descemet's membrane. Corneas exposed to repeated UVR doses showed at 24 hours positive TUNEL staining only in epithelial cells and in very few stromal cells. The majority of stromal cells and endothelial cells were unaffected. At the same time HA staining of very high intensity was found both at 24 hours and day 14, and it was evenly distributed throughout the entire thickness of the stroma. CONCLUSION: Repeated UVR exposures lead to increased production and accumulation of HA in the corneal stroma. The repopulated keratocytes are much more resistant to apoptosis than the native ones. HA accumulation may be a sign of long-term changes in the cornea.     

Apoptosis in human non-necrotizing stromal herpes simplex keratitis

BACKGROUND: Herpes simplex virus (HSV-1) stromal keratitis is an immune-mediated disease. Recently, it has been shown that infection of cells with HSV-1 induces apoptosis. In this study we investigated the presence of apoptotic cell death in keratectomy specimens from patients with HSV-1 stromal keratitis. PATIENTS AND METHODS: Keratectomy specimens from six patients with HSV-1 non-necrotizing stromal keratitis were chosen and compared to healthy corneas. Paraffin sections were analyzed histologically and cryosections were studied by the immunoperoxidase technique for the presence of HSV-1, Fas and FasLigand (FasL) antigens. Apoptosis was assessed by TUNEL assay (TdT-mediated dUTP nick-end labeling). RESULTS: In healthy corneas, Fas was detected in the epithelium, keratocytes and endothelium; FasL was present in the epithelium and endothelium; TUNEL-positive cells were only found in the superficial epithelial cells. In contrast, inflammatory cells and scars were found in all HSV-diseased corneas; HSV-1 antigen was detected in only one specimen. Cells within inflammatory infiltrations and epithelium were apoptotic. Fas was detected in all corneal cell layers and inflammatory cells. FasL was restricted to areas of inflammation. CONCLUSIONS: The data suggest that apoptosis plays a role in the pathogenesis of HSV keratitis. The Fas-FasL system appears to be involved in the induction of apoptosis. Apoptosis could be involved in the depletion of inflammatory cells in the cornea and may limit the extension of viral replication to the eye.     

Expression of type XII collagen and hemidesmosome-associated proteins in keratoconus corneas

PURPOSE: Keratoconus is a disease characterized by thinning of the central and paracentral cornea and scarring in advanced cases. This study was performed to examine the expression of type XII collagen, proteins associated with hemidesmosomes, and beta1 integrin in keratoconus corneas. METHODS: Corneal buttons were collected from normal subjects and patients with keratoconus and other corneal diseases. Immunofluorescence staining was performed on frozen sections for type XII collagen, bullous pemphigoid antigen (BP180), and integrin subunits alpha6, beta4, and beta1. RESULTS: To varying degrees, all proteins examined were expressed in normal human corneas. The staining intensity of type XII collagen was diminished in keratoconus corneas in the epithelial basement membrane zone and the stromal matrix. No significant variation was found in either the staining patterns or intensities for BP180, or integrins alpha6, beta4, and beta1. CONCLUSIONS: The level of type XII collagen was reduced in the epithelial basement membrane zone and stromal matrices in keratoconus corneas. These alterations may affect critical interactions of the corneal epithelium with the under-lying basement membrane, and cell-matrix interactions and matrix organization in the stroma.     

Delay of corneal epithelial wound healing and induction of keratocyte apoptosis by platelet-activating factor

PURPOSE: To examine the role of the lipid mediator platelet-activating factor (PAF) in epithelial wound healing. METHODS: A 7-mm central de-epithelializing wound was produced in rabbit corneas, and the tissue was incubated with 125 nM carbamyl PAF (cPAF), an analogue of PAF. Rabbit corneal epithelial and stromal cells were also cultured in the presence of cPAF. Cell adhesion, proliferation, and migration assays were conducted. Apoptosis was assayed by TUNEL staining on preparations of corneal tissue sections and in cells in culture. RESULTS: Twenty-four hours after injury, 50% of the wounded area was covered by new epithelium, whereas only 30% was covered in the presence of cPAF. At 48 hours, the epithelium completely closed the wound, but only 45% of the original wound was covered in corneas treated with cPAF. Similar inhibition of epithelial wound closure was found with human corneas incubated with PAF in organ culture. Moreover, addition of several growth factors involved in corneal wound healing, such as epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, could not overcome the inhibitory action of PAF in wound closure. Three PAF antagonists, BN50727, BN50730, and BN50739, abolished the effect of PAF. A significant increase in TUNEL-positive staining occurred in corneal stromal cells (keratocytes), which was inhibited by preincubating the corneas with PAF antagonists. However, no TUNEL-positive staining was found in epithelial cells. TUNEL-staining results in cultured stromal cells (keratocytes) and epithelial cells in first-passage cell culture were similar to those in organ-cultured corneas. In addition, PAF caused 35% to 56% inhibition of adhesion of epithelial cells to proteins of the extracellular matrix: collagen I and IV, fibronectin, and laminin. There were no significant changes in proliferation or migration of epithelial cells induced by the lipid mediator. CONCLUSIONS: The results suggest PAF plays an important role in preventing corneal wound healing by affecting adhesion of epithelial cells and increasing apoptosis in stromal cells. PAF antagonists could be of therapeutic importance during prolonged ocular inflammation, helping to avoid loss of corneal transparency and visual acuity.     

Second-harmonic imaging microscopy of normal human and keratoconus cornea

PURPOSE: The purpose of this study was to evaluate the ability of second-harmonic imaging to identify differences in corneal stromal collagen organization between normal human and keratoconus corneas. METHODS: Six normal corneas from eye bank donors and 13 corneas of patients with keratoconus, obtained after penetrating keratoplasty were examined. A femtosecond titanium-sapphire laser with 800-nm output was used to generate second-harmonic signals collected at 400 nm from central and paracentral corneal tissue blocks. Three-dimensional (3-D) data sets were collected and reconstructed to evaluate the location and orientation of stromal collagen lamellae. RESULTS: Imaging of second-harmonic signals combined with 3-D reconstruction of the normal cornea identified a high degree of lamellar interweaving, particularly in the anterior cornea. Of note was the detection of lamellae that inserted into Bowman's layer and were oriented transverse to the corneal surface, penetrating posteriorly approximately 120 mum. In keratoconus corneas, imaging second-harmonic signals identified less lamellar interweaving and a marked reduction or loss of lamellae inserting into Bowman's layer in 12 of 13 cases, particularly in regions associated with cone development without breaks in Bowman's layer or scarring. CONCLUSIONS: Compared with normal adult corneas, marked abnormalities were detected in the organization of the anterior corneal collagen lamellae of keratoconus corneas by second harmonic imaging. These structural abnormalities are consistent with the known changes in collagen organization and biomechanical strength of keratoconus.     

Apoptosis in the endothelium of human corneas for transplantation

PURPOSE: To determine whether endothelial cell loss of human corneas stored in organ culture before transplantation is due to apoptosis. METHODS: The corneal endothelium of human corneas, stored in organ culture at 34 degrees C for varying periods of time, were analyzed for the presence of apoptotic cells using the TdT-mediated dUTP nick-end labeling (TUNEL) technique. Corneal endothelial cell apoptosis was confirmed by Hoechst staining and immunolabeling with anti-caspase 3 active antibody. RESULTS: Apoptotic cells were identified in the corneal endothelium of human organ cultured corneas: their number and distribution demonstrated a close correlation with corneal folding and overall quality of the corneal endothelium. TUNEL-positive labeling of cells was confirmed as apoptotic by the presence of morphologic nuclear alterations identified by Hoechst staining and the presence of immunostaining for caspase-3 activity. Corneal endothelial cell apoptosis was independent of cause of donor death, death to enucleation time, and death to culture times. CONCLUSIONS: Corneal endothelial cell apoptosis appears to determine the suitability of a cornea for transplantation.