Mitomycin C alters corneal stromal wound healing and corneal haze in rabbits after argon-fluoride excimer laser photorefractive keratectomy.

PURPOSE: To investigate the effects of mitomycin C (MMC) on rabbit cornea wound healing after photorefractive keratectomy (PRK). MATERIALS AND METHODS: Rabbit corneas were stained with dichlorotriazinyl aminofluorescein immediately after PRK. MMC was applied to the right eye and phosphate-buffered salt solution (PBS) to the left. Corneal epithelial wound healing rate and corneal haze were examined. Ultrasound pachymetry was performed. Stromal collagen regeneration was evaluated by fluorescent microscopy. We used terminal deoxyribonucleotidyl transferase-mediated D-uridine 5'-triphosphated-digoxigenin nick-end labeling (TUNEL) assay and transmission electron microscopy (TEM) to evaluate keratocyte apoptosis. RESULTS: In eyes treated with MMC, there was no delay to the healing rate of corneal epithelial wound, and less haze 4 weeks after PRK. Ultrasound pachymetry showed thinner corneal thickness in MMC-treated eyes at week 4. Corneal stromal thickness regression was less in MMC-treated eyes observed by fluorescent microscope at week 4. Keratocyte apoptosis was noted in both MMC- and PBS-treated eyes by TUNEL assay and TEM observation. This study discovered the phenomenon that MMC prolongs keratocyte apoptosis. CONCLUSIONS: Applying MMC after PRK is an effective method to decrease haze formation and corneal stromal thickness regression in rabbit corneas. The effect may be related to MMC prolonging keratocyte apoptosis.     

Comparison of Chen Medium and Optisol-GS for human corneal preservation at 4 degrees C: results of transplantation

PURPOSE: To compare results after transplantation of donor corneas stored in Chen Medium (containing beta-hydroxybutyrate without sodium bicarbonate or chondroitin sulfate) to corneas stored in Optisol-GS medium (containing sodium bicarbonate and 2.5% chondroitin sulfate). METHODS: We performed 32 consecutive penetrating keratoplasties with donor corneas stored at 4 degrees C in either Chen Medium or Optisol-GS by random assignment. Corneal thickness measurements were made at 1 day, 1 week, 3 weeks, 2 months, and 1 year postkeratoplasty. Specular microscopic images of the donor endothelium were obtained at the beginning of storage and 2 months and 1 year postkeratoplasty. The percentage of intact epithelium 1 day after keratoplasty and the graft epithelialization time were estimated by the surgeons. Donor rim cultures were performed. RESULTS: No statistically significant differences in corneal thickness or endothelial cell loss between the corneas stored in the two media were found at any time, although differences of less than 12% cell loss or 0.09-mm thickness at 2 months or less than 25% cell loss or 0.10-mm thickness at 1 year could not be excluded with 90% certainty in this small series. The mean percentages of intact graft epithelium on day 1, 64% for Chen Medium and 65% for Optisol-GS, were not significantly different. Endothelial cell density 2 months postkeratoplasty was significantly decreased for corneas stored in both media. Endothelial cell loss at 2 months was directly correlated with storage time in both media. CONCLUSIONS: After keratoplasty, no statistically significant differences in corneal thickness, epithelial survival, and endothelial cell loss were found between corneas stored in Chen Medium and Optisol-GS. Endothelial cell loss at 2 months was significantly correlated with storage time in both media.     

The role of apoptosis in the early corneal wound healing after excimer laser keratectomy in the rat

Background: The potential role of apoptosis in corneal wound healing after excimer laser keratectomy was investigated in a rat model. Methods: Lewis rats underwent laser keratectomy using a 193-nm excimer laser. The central corneas were ablated in three depths: group A, epithelium; group B, superficial stroma; group C, deep stroma. Eyes were collected at 1, 12, 24, and 36 h and 1 week. Cellular markers associated with apoptosis – Fas, Fas ligand (FasL), Bcl-2, and Bax – were examined by immunohistochemistry. Keratocyte depletion and endothelial changes were evaluated histologically. In situ end labeling of double-stranded DNA breaks was used to demonstrate apoptosis in corneal sections. Results: Keratocyte depletion was observed in 6 (50%) of 12 rats (total from groups A, B, and C) at 12 h, 11 (73%) of 15 at 24 h, 3 (20%) of 15 at 36 h, and 2 (15%) of 13 at 1 week after laser surgery. Corneal endothelial edema was observed in the ablation zone. Expression of Fas, FasL, Bcl-2, and Bax in corneal cells showed dynamics similar to that of keratocyte depletion and endothelial changes. There was less expression of apoptotic molecules in newly generated epithelial cells and more in endothelial cells of the stromal ablation groups. Conclusions: Excimer laser keratectomy triggered apoptosis of corneal keratocytes and endothelial cells. More endothelial edema was observed in the stromal ablation than in the epithelial ablation group. The expression of apoptotic molecules coincided with the period of keratocyte depletion and regeneration and of endothelial recovery, suggesting that apoptosis is a dynamic part of corneal wound healing and remodeling after excimer laser keratectomy. Received: 9 November 1999 Revised: 18 May 2000 Accepted: 10 April 2000     

ICAM-1 mediates surface contact between neutrophils and keratocytes following corneal epithelial abrasion in the mouse

Corneal epithelial abrasion elicits an inflammatory response involving neutrophil (PMN) recruitment from the limbal vessels into the corneal stroma. These migrating PMNs make surface contact with collagen and stromal keratocytes. Using mice deficient in PMN integrin CD18, we previously showed that PMN contact with stromal keratocytes is CD18-dependent, while contact with collagen is CD18-independent. In the present study, we wished to extend these observations and determine if ICAM-1, a known ligand for CD18, mediates PMN contact with keratocytes during corneal wound healing. Uninjured and injured right corneas from C57Bl/6 wild type (WT) mice and ICAM-1^−/− mice were processed for transmission electron microscopy and imaged for morphometric analysis. PMN migration, stromal thickness, and ICAM-1 staining were evaluated using light microscopy. Twelve hours after epithelial abrasion, PMN surface contact with paralimbal keratocytes in ICAM-1^−/− corneas was reduced to  ˜ 50% of that observed in WT corneas; PMN surface contact with collagen was not affected. Stromal thickness (edema), keratocyte network surface area and keratocyte shape were similar in ICAM-1^−/− and WT corneas. WT keratocyte ICAM-1 expression was detected at baseline and ICAM-1 staining intensity increased following injury. Since ICAM-1 is readily detected on mouse keratocytes and PMN-keratocyte surface contact in ICAM-1^−/− mice is markedly reduced, the data suggest PMN adhesive interactions with keratocyte-stromal networks is in part regulated by keratocyte ICAM-1 expression.     

Corneal stroma regeneration in felines after supradescemetic keratoprosthesis implantation

PURPOSE: To show corneal regeneration in 3 cats that underwent lamellar keratectomy (90%) depth during supradescemetic keratoprosthetic implantation. METHODS: Three 2-year-old cats that underwent spontaneous keratoprosthesis extrusion between 15 and 150 days after implanting a supradescemetic prosthesis into their right eyes were studied. Corneal structures and stroma thickness were evaluated by slit-lamp photographs, pachymetry, and confocal microscopy. Regenerated corneal epithelial cells, stroma matrix, and keratocyte morphology were studied with histology and transmission electron microscopy. Epithelial and stromal cell immunocharacterization was performed. RESULTS: Corneas progressively regained normal thickness and improved clarity within 40 to 60 days. Slit-lamp photographs and pachymetry showed gains in stromal thickness until 600 microm or more. In vivo confocal microscopy showed the restoration of normal epithelium and stroma in all cats. Corneal nerves were seen in the regenerated stroma of 2 cats. Immunostaining showed absent alpha-smooth muscle actin (SMA) expression and a keratin K3-expressing epithelium. Electron microscopy showed regeneration of normal epithelium with a well-formed basement membrane, organized corneal lamellae, and the presence of normal keratocytes. CONCLUSION: Felines are capable of regenerating corneal structures including epithelium and reinnervated stroma matrix after deep lamellar keratectomy. The use of feline models in corneal keratoprosthesis is therefore questionable.     

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.     

Increased apoptosis and abnormal wound-healing responses in the heterozygous Pax6+/- mouse cornea

PURPOSE: Corneal wound healing involves a cascade of interactions between the epithelium and stroma. Pax6 is upregulated, and early events include epithelial cell migration and apoptosis of superficial keratocytes. The mouse heterozygous Pax6 (Pax6+/-) corneal phenotype mimics human aniridia-related keratopathy (ARK), and some aspects of wound healing have been shown to be abnormal, including matrix metalloproteinase (MMP)-9 expression. The purpose of this study was to test whether the Pax6+/- genotype affects corneal wound-healing responses, including stromal cell apoptosis, epithelial cell migration rate, and MMP secretion in culture. METHOD: Pax6+/- and wild-type (Pax6+/+) mice were killed and their corneas wounded by epithelial debridement. Whole eyes were cultured in organ culture and corneal epithelial healing rates and keratocyte apoptosis were quantified by topical fluorescein staining and TUNEL, respectively. Dissociated corneal epithelial cells from Pax6+/- and wild-type mice were cultured, and the activities of secreted MMP-9 were determined by zymography. RESULTS: Wound-healing rates during the first 6 hours were significantly faster for larger wounds and for Pax6+/- corneas. Compared with wild-type, wounded Pax6+/- eyes showed significantly more stromal cell apoptosis, and cultured Pax6+/- corneal epithelial cells produced lower MMP-9 activity. CONCLUSIONS: The cumulative effect of abnormal wound-healing responses, characterized by increased stromal cell apoptosis and reduced levels of MMP-9 secretion may contribute to the corneal changes in the Pax6+/- mice. Possible contributions of elevated stromal cell apoptosis and other abnormal wound-healing responses to ARK are discussed.     

Keratocyte activation and apoptosis in transplanted human corneas in a xenograft model

PURPOSE: To study keratocyte activation and cellular apoptosis in transplanted human corneas during the early postoperative period. METHODS: Ten human donor corneas preserved for 6 days at 4 degrees C were transplanted into the eyes of 10 adult cats. After confocal and specular microscopy in vivo 1 week after keratoplasty, the cats were killed, and the fixed corneas were examined by TUNEL assay and by scanning (SEM) and transmission electron microscopy (TEM). RESULTS: Abnormal keratocytes, in which portions of cell bodies and processes as well as nuclei were visible, were present in all corneas and occupied the anterior 16 to 562 microm of the stroma. By TEM in the same corneas, these abnormalities represented keratocytes that were activated to a repair phenotype. Only 0% to 1% of all corneal cells were apoptotic by TUNEL assay, except for the donor keratocytes near the wound, where 7% were apoptotic. The midstromal keratocyte density was decreased at 13,936 +/- 5,910 cells/mm(3) (mean +/- SD), and the endothelial cell density was 2,298 +/- 688 cells/mm(2), representing an endothelial cell loss of 7% +/- 16%. CONCLUSIONS: Substantial keratocyte activation and low levels of cellular apoptosis occur 1 week after human corneal transplantation. The human-to-cat xenograft model of corneal transplantation demonstrated endothelial cell loss and other clinical findings similar to human allografts. The model will be useful for preclinical testing of new methods of long-term corneal preservation and of donor endothelial cell augmentation, as well as the study of human corneal wound healing and keratocyte replacement during the early postoperative period.     

Protective role of corneal epithelium against ultraviolet radiation damage

PURPOSE: It is known that the corneal epithelium strongly absorbs ultraviolet radiation (UVR). The aim of the present study was to examine the protective role of corneal epithelium against UVR damage by comparing the biological effect of UVR exposure on whole corneas with that on de-epithelialized corneas. METHODS: Six New Zealand albino rabbit corneas were exposed to UVR centred around 280 nm at a dose that causes biomicroscopically significant keratitis (012 J/cm(2)). Three corneas underwent manual de-epithelialization prior to UVR exposure. A control group of three rabbits underwent only manual de-epithelialization. The animals were killed 76 hours after treatment. The corneas were stained with haematoxylin and evaluated by light microscopy. RESULTS: Corneas that underwent only the exposure to UVR showed a loss of epithelial cells in the treated area. No damage to keratocytes or the stroma was detected. Corneas that underwent manual de-epithelialization showed a loss of epithelial cells, and also keratocytes in the anterior quarter of the corneal stroma. However, corneas that were exposed to UVR after manual de-epithelialization showed very deep stromal damage. The keratocytes disappeared through the entire thickness of the stroma in the UVR-exposed area. CONCLUSION: Exposure to UVR at 280 nm alone does not result in any deep damage to the corneal stroma and keratocytes. Manual de-epithelialization causes the disappearance of anterior keratocytes. However, the stromal damage caused by UVR in the de-epithelialized corneas was very deep. The corneal epithelium serves to protect the deeper corneal structures against UVR damage, probably by absorbing a substantial amount of the UVR energy applied to the eye.     

Keratocyte apoptosis after corneal collagen cross-linking using riboflavin/UVA treatment

PURPOSE: Combined riboflavin/UVA treatment inducing collagen cross-links in the cornea has been shown to increase the biomechanical rigidity of the cornea and has been used successfully in the treatment of progressive keratoconus. The current study was undertaken to investigate the possible cytotoxic effect of combined riboflavin/UVA treatment on corneal keratocytes in vivo. METHODS: Thirty-four New Zealand white rabbits were treated with 0.1% riboflavin solution and surface UVA irradiances ranging from 0.75 to 4 mW/cm2 (1.35- 7.2 J/cm2) for 30 minutes. The animals were euthanized either 4 (n = 6) or 24 (n = 28) hours postoperatively. Four additional control eyes underwent epithelial debridement alone. The corneas of the enucleated eyes were evaluated in routine histologic sections. In addition, the TUNEL technique and transmission electron microscopy were used for the detection of keratocyte apoptosis. RESULTS: In the control eyes with corneal epithelial debridement only, apoptotic keratocytes were found in the anterior 50 microm of the corneal stroma 4 hours postoperatively. However, riboflavin/UVA-induced apoptosis was only visible in the rabbit eyes enucleated 24 hours postoperatively. In these eyes, we found apoptosis of keratocytes down to a variable stromal depth depending on the applied UVA irradiance. A cytotoxic UVA irradiance for keratocytes in the range of 0.5-0.7 mW/cm2 could be deduced. CONCLUSIONS: Riboflavin/UVA treatment leads to a dose-dependent keratocyte damage that can be expected in human corneas down to a depth of 300 microm using a surface UVA dose of 5.4 J/cm2. Future studies should be done to examine the keratocyte repopulation and exclude possible adverse sequelae of keratocyte loss like stromal scarring or thinning.     

Role of keratocyte loss on corneal wound repair after LASIK

PURPOSE: To investigate whether an initial keratocyte loss intensifies central corneal wound repair after LASIK in rabbits. METHODS: New Zealand White rabbits received either conventional LASIK (-8 D, 6-mm diameter) or LASIK combined with a 7-mm diameter, epithelial denudation (LASIK-scrape). Animals were examined during 4 months by slit lamp and in vivo confocal microscopy to monitor changes in central corneal morphology, light backscattering (haze), and sublayer thickness. At various time points, corneas were processed for histology and stained for nuclei; F-actin; ED-A fibronectin; alpha-smooth muscle actin; TGF-beta1, -beta2, and -beta receptor II; and connective tissue growth factor (CTGF). RESULTS: In vivo confocal microscopy identified no major acellular zones or changes in cell morphology or reflectivity after conventional LASIK. By contrast, a complete loss of keratocytes was observed in the anterior 77 +/- 25 microm stroma 1 week after LASIK-scrape. Highly reflective, migratory fibroblasts gradually repopulated the acellular zone, and by week 8, quiescent-appearing keratocytes were observed throughout the stroma. Correspondingly, stromal light backscattering peaked at 2 weeks after LASIK-scrape (2200 +/- 620 U) followed by a decline to approximately 60 U from week 8; comparable to the slightly increased reflectivity (approximately 50 U) observed after conventional LASIK (ns). Stromal thickness appeared stable 8 weeks after both LASIK and LASIK-scrape, after a regrowth of 13 +/- 3 and 20 +/- 11 microm, respectively (ns). In addition, both procedures induced a minor and comparable epithelial hyperplasia of 4 +/- 2 and 7 +/- 5 microm, respectively (ns). No myofibroblast transformation or TGF-beta growth factor expression was observed below the flap after either treatment. CONCLUSIONS: LASIK-scrape induces an anterior keratocyte loss, leading to development of temporary haze during cell repopulation. However, 8 weeks after both LASIK and LASIK-scrape, only a slightly increased reflectivity is noted at the interface. Corneal thickness is stable by week 8, and stromal regrowth and epithelial hyperplasia are comparable after both treatments. Thus, an initial loss of stromal keratocytes does not appear to intensify corneal wound repair after LASIK.     

Caspase inhibitor z-VAD-FMK inhibits keratocyte apoptosis, but promotes keratocyte necrosis, after corneal epithelial scrape

The purpose of this study was to determine whether the caspase inhibitor z-VAD-FMK could be applied topically prior to epithelial scrape injury to inhibit keratocyte apoptosis. Rabbit corneas were treated with z-VAD-FMK or vehicle alone prior to epithelial scrape injury. Cell fate was analysed at 4 hr after epithelial scrape using quantitative TUNEL assay, propidium iodide staining, and transmission electron microscopy. Less stained anterior stromal keratocytes were detected with the quantitative TUNEL assay in corneas pre-treated with z-VAD-FMK than in corneas pretreated with vehicle at 4 hr after epithelial scrape. This difference appeared to be confirmed by propidium iodide staining of keratocyte nuclei. It was observed that fewer nuclei were stained with propidium iodide in the DMSO vehicle treated corneas compared to the z-VAD-FMK treated corneas. Analysis of corneas with transmission electron microscopy, however, indicated that many anterior stromal keratocytes in corneas pretreated with z-VAD-FMK, but not vehicle, had cell morphologic changes more consistent with necrosis. Although pretreatment of corneas with the caspase inhibitor z-VAD-FMK inhibited keratocyte apoptosis detected with the TUNEL assay, transmission electron microscopy revealed that many anterior stromal keratocytes in z-VAD-FMK-treated corneas instead died by necrosis. Thus, z-VAD-FMK is unlikely to be useful to modulate corneal would healing through inhibition of keratocyte apoptosis induced by epithelial injury. The TUNEL assay should not be used to monitor cell fate without confirmation using analyses that also detect necrosis.     

Improved preparation and preservation of human keratoplasty lenticules

PURPOSE: To improve the preparation of lenticules from human cornea and to obtain their preservation without loss of viable keratocytes. METHODS: The epithelium was manually removed after bathing the surface of the cornea with a solution of trypsin and EDTA. Lenticules were prepared by microkeratome resection and viable keratocytes were visualized by staining with thiazolyl blue (MTT). RESULTS: The pretreatment with trypsin-EDTA allowed the removal of the epithelium without damage to the keratocytes and the stroma. When these lenticules were incubated in Optisol-GS for 7 days at 4 degrees C, they showed a limited thickness increase and a preservation of keratocyte viability. CONCLUSION: This procedure allows the preparation of lenticules with viable keratocytes that can be preserved in the cold for at least 1 week.     

准分子激光屈光性角膜切削术后兔角膜基质细胞凋亡及其防治的研究

目的：探讨准分子激光屈光性角膜切削术（photorefractive keratectomy,PRK）后凋亡机制介导的角膜创伤愈合反应对屈光度数回退和角膜雾状混浊（haze）的影响,以及局部应用锌制剂的药物效果。方法：对90只新西兰白兔行双眼PRK,将左眼作为实验眼,手术前、后分别给予A组0．1％地塞米松眼液、B组0．5％硫酸锌眼液和C组0．04％丝裂霉素眼液滴眼;右眼作为对照眼。术后定期裂隙灯下观察haze的程度,测量角膜的厚度,进行光镜和透射电镜观察,采用脱氧三磷酸尿苷缺口末端标记法行凋亡细胞检测,并进行对比性分析。结果：（1）PRK术后角膜前基质细胞出现凋亡,实验眼B组凋亡细胞最少（P＜0．01）。（2）术后角膜厚度增加,前基质细胞增多,实验眼haze和增生程度眼B组凋亡细胞最少（P＜0．01）。（3）术后角膜上皮增生,实验眼B组角膜上皮厚度最小（P＜0．01）。结论：PRK术后屈光度数回退和haze的形成是凋亡机制介导的角膜创伤愈合过程,锌制剂可阻止角膜前基质细胞凋亡,最大限度减轻反应性过度增生,有望成为临床防治haze形成和屈光度数回退的理想药物。     

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。     

Neonatal corneal stromal development in the normal and lumican-deficient mouse

PURPOSE: The purpose of this study was to characterize temporally stromal growth and transparency in lumican-deficient and normal neonatal mice. METHODS: Lumican-deficient mice and CD1 wild-type mice were evaluated by in vivo confocal microscopy through-focusing (CMTF) to quantify stromal and epithelial thickness and corneal light-scattering and by laser scanning CM to determine density of keratocytes from 1 day to 12 weeks after birth. RESULTS: CD1 corneas showed a rapid loss of light-scattering, decreasing by 50% from day 1 to day 12, that paralleled a 60% decrease in density of keratocytes. By contrast, the stroma demonstrated a marked swelling from day 8 to day 12, followed by thinning at day 14. Compared to corneas from CD1 mice, lumican-deficient corneas showed significantly increased (P < 0.05) light-scattering beginning at week 3 that remained elevated above wild-type levels for the duration of the study. Stromal development was also markedly altered, with thinning detected at week 3, followed by no detectable stromal growth for the duration of the study. Density of keratocytes was significantly increased, but the total cell number was similar compared with that in the wild-type cornea, suggesting no effect on keratocyte differentiation. CONCLUSIONS: Development of normal neonatal corneal transparency appears related to changes in density of keratocytes. The stroma, however, undergoes a marked swelling and thinning at the time of eyelid opening (days 8-14). In the lumican-deficient mouse, stromal swelling is abolished, indicating that this critical phase in stromal development is lumican dependent and essential for normal stromal growth and maintenance of stromal transparency.     

圆锥角膜各期的共焦显微镜表现

目的评价共焦显微镜在圆锥角膜临床研究中的应用价值。方法应用共焦显微镜观察圆锥角膜患者32例（48眼）及正常对照组17例（28眼），分别比较早、中、晚期圆锥角膜与正常对照组的图像特点。结果早期圆锥角膜出现激活状态的角膜细胞、浅基质层的细小皱褶、深基质层的暗纹、部分内皮细胞异形性明显，中、晚期圆锥角膜出现角膜上皮细胞拉伸、细胞核皱缩；基质细胞排列紊乱、基质层暗纹；而对照组未发现上述表现。各期圆锥角膜的角膜基质层厚度、不同深度角膜基质细胞密度、内皮细胞密度与对照组之间差异有统计学意义（P〈0．05）。结论共焦显微镜对早期圆锥角膜的发现以及圆锥角膜病理发展的研究具有重要的临床价值。     

Wound healing following anterior keratectomy and lamellar keratoplasty in the pig

PURPOSE: To examine corneal wound healing in an animal model of two types of mechanical lamellar keratectomy. METHODS: One eye from each of 28 pigs was studied. Using a motorized keratome, corneas were subjected to an anterior lamellar keratectomy with removal of anterior stroma and epithelium, or to automated lamellar keratoplasty (ALK) with reapposition of a corneal flap. The exposed stromal surfaces were labeled intraoperatively with a fluorescent dye (DTAF) to assess deposition of stromal components during subsequent wound healing. Examination before surgery and enucleation included measurement of corneal curvature and intraocular pressure, and assessment of corneal haze. Eyes were prepared for histological examination, fluorescence microscopy, and for fibronectin immunohistochemistry. RESULTS: Both keratectomy procedures produced flattening of corneas by up to 3.80 diopters, 28 days after surgery. Corneal haze was more pronounced in eyes from which epithelium was removed (anterior lamellar keratectomy group). The increased haze in this group was associated histologically with appearance of many reactive keratocytes and inflammatory cells, deposition of new stromal material, and more widespread appearance of fibronectin immunoreactivity. In the lamellar keratoplasty group, only the edges of the corneal wound showed significant reactivity, and included keratocyte activation and epithelial ingrowth. CONCLUSIONS: The pig provides a useful model for studies of refractive surgical techniques using procedures and instruments designed for use in humans. Mechanized keratectomy procedures that minimize disruption of the epithelium and Bowman's layer produce a less reactive corneal wound than procedures in which an expanse of epithelium and anterior stroma are removed.     

Early keratocyte apoptosis after epithelial scrape injury in the human cornea

Animal studies in mice, rats, rabbits, pigs and hens demonstrated that anterior keratocytes undergo programmed cell death or apoptosis after corneal epithelial injury. Many other wound healing changes subsequently follow the keratocyte apoptosis response. This study evaluated early keratocyte apoptosis after corneal epithelial scrape injury in human eyes scheduled for enucleation for malignancy. Two eyes had corneal epithelial scrape 1 h prior to the enucleation and another eye served as a control and had no corneal scrape prior to enucleation. One additional eye was enucleated, washed with balanced salt solution, and then had the corneal epithelium scraped 1 h prior to processing for analysis. Apoptosis was identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and confirmed by transmission electron microscopy (TEM). Anterior keratocyte apoptosis was detected in the three corneas that had epithelial scrape injury, but not in the control unwounded cornea. This study confirmed that keratocyte apoptosis is also an early response to corneal epithelial injury in humans and showed that tears are not essential for keratocyte apoptosis to occur in response to epithelial injury.     

Confocal microscopy in vivo in corneas of long-term contact lens wearers

PURPOSE: To compare keratocyte density, stromal backscatter, epithelial thickness, and corneal sensitivity between corneas of long-term contact lens wearers and those of non-contact lens wearers. METHODS: Twenty corneas of 20 daily contact lens wearers (>10 years' duration) and 20 corneas of 20 age-matched (+/-5 years) control subjects who had never worn contact lenses, were examined by confocal microscopy in vivo. The contact lens wearers removed their lenses 12 to 24 hours before the examination. Full-thickness images were recorded from the central and temporal cornea, and bright objects (keratocyte nuclei) in images were manually counted to calculate keratocyte density. Stromal intensity (backscatter) was measured by calculating the mean grayscale value (corrected for camera and light source variations) from the center of stromal images. Epithelial thickness was determined from the distance between images of the surface epithelium and subbasal nerve plexus. Central corneal sensitivity was measured by Cochet-Bonnet esthesiometry and correlated with the number of nerve fiber bundles in the subbasal nerve plexus. RESULTS: Full-thickness central and temporal keratocyte densities in contact lens wearers were 22,122 +/- 2,676 cells/mm(3) (mean +/- SD) and 20,731 +/- 2,627 cells/mm(3), respectively, and were not significantly different from central and temporal keratocyte densities in control subjects (P = 0.29). The minimum detectable difference in cell density was 11% (2346 cells/mm(3) and 2235 cells/mm(3) in central and temporal stroma, respectively). Temporal epithelial thickness was 46.3 +/- 4.7 microm in contact lens wearers and 50.9 +/- 4.7 microm in control subjects (P = 0.02). Central epithelial thickness and stromal backscatter did not differ between contact lens wearers and control subjects (P > 0.05). Corneal sensitivity was lower in contact lens wearers than it was in control subjects (P = 0.05) and did not correlate with the number of nerve fiber bundles in the subbasal nerve plexus. CONCLUSIONS: Long-term daily contact lens wear and its associated stromal hypoxia and acidosis have no demonstrable effect on keratocyte density. The temporal epithelium is thinner in corneas of long-term contact lens wearers than in control subjects. Decreased corneal sensitivity in contact lens wearers is not accompanied by decreased nerve fiber bundle density.