Mitomycin C-induced reduction of keratocytes and fibroblasts after photorefractive keratectomy

PURPOSE: To investigate the effects of mitomycin C (MMC) on the number of keratocytes and the proliferation of fibroblasts after photorefractive keratectomy (PRK) and exposure to ultraviolet B (UV-B) irradiation. METHODS: The right eyes of New Zealand White rabbits in Groups 1, 2, and 3 (n = 18 each) underwent PRK to correct -10 diopters with 5 mm optical zone. Sponges soaked with 0.02% MMC were applied to the right eyes of Group 1 rabbits for 2 minutes. Antibiotic ointment was applied daily to all rabbits until the epithelium healed completely, after which 0.02% MMC eye drops were applied twice daily to the right eyes in Group 2 until 4 weeks after PRK. Three weeks after PRK, the right eyes of all the remaining rabbits were exposed to 100 mJ/cm2 C UV-B radiation. Corneal haziness was assessed biomicroscopically using the Fantes scale every 3 weeks. Six eyes of each group were each enucleated 3, 6, and 12 weeks after PRK, and tissue specimens were stained with hematoxylin and eosin and with TUNEL stain. The tissues were evaluated immunohistochemically with antibody to alpha-smooth muscle actin (SMA). Cellular changes in the anterior stroma and epithelial basement membrane were evaluated by electron microscopy. RESULTS: Corneal haze was observed after PRK and was aggravated by UV-B irradiation. A single intraoperative application of MMC immediately after PRK induced opacity and apoptosis of keratocytes. Twelve weeks after PRK, MMC significantly reduced corneal haze, the number of keratocytes, apoptotic cells, and fibroblasts, even after UV-B irradiation. Relatively large numbers of apoptotic and SMA-positive cells were found only in PRK-treated, non-MMC treated rabbits (Group 3), even after 12 weeks. Three weeks after PRK, dying stromal cells showed cell shrinkage, and chromatin condensation was observed in all treated groups by electron microscopy. Twelve weeks after PRK, fewer keratocytes and inflammatory cells were observed just beneath the epithelial layer in Group 1 than in any of the other groups. CONCLUSIONS: MMC is a potent inhibitor of corneal haze induced by PRK. MMC reduced the number of keratocytes and fibroblasts after PRK and UV-B irradiation. Although MMC would improve the clinical results of PRK, it has significant toxicity on corneal keratocytes, which did not disappear until 3 months after PRK.     

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.     

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。     

Mitomycin C reduces haze formation in rabbits after excimer laser photorefractive keratectomy

PURPOSE: To investigate the effects of mitomycin C on haze after photorefractive keratectomy (PRK). METHODS: Twenty of 24 rabbits underwent bilateral 193-nm excimer laser PRK to correct -10.00 D of myopia; the remaining four rabbits were not operated (no PRK group). The right eyes of the 20 rabbits were treated with 0.02% mitomycin C during surgery (PRK+MMC group) and the left eyes did not receive 0.02% mitomycin C (PRK alone group). Clinical and histopathologic examinations were performed. RESULTS: The most severe haze in the PRK alone group after PRK reached grade 3; the PRK+MMC group did not exceed grade 1 haze. Statistically significant differences were found between the PRK+MMC and PRK alone groups from week 2 to week 26 after treatment (P<.01). Epithelial thickening appeared for 26 weeks in both PRK groups; no statistically significant differences were found between the two PRK groups (P>.05). A marked reduction of keratocytes in the anterior stroma of the PRK+MMC group was observed. At week 1, 2, and 4 after PRK, keratocytes of the PRK+MMC group were only 3.1+/-2.6, 6.8+/-4.7, and 12.4+/-5.7 keratocytes x 10(4)/microm2, respectively, while those of the PRK alone group were 41.2+/-80, 42.3+/-7.8, and 40.0+/-3.3 keratocytes x 10(4)/microm2, respectively. There were statistically significant differences between the two groups (P<.001). CONCLUSION: A single intraoperative application of topical mitomycin C during PRK in rabbits reduced corneal haze by inhibiting the proliferation of keratocytes.     

LASEK and photorefractive keratectomy for myopia: clinical and confocal microscopy comparison

PURPOSE: To compare postoperative visual acuity and corneal morphology after laser epithelial keratomileusis (LASEK) versus photorefractive keratectomy (PRK) in the correction of low to moderate myopia. METHODS: In a double-blind, randomized clinical trial, 50 myopic patients (mean: -4.5 +/- 1.35 diopters) were randomized to receive LASEK in one eye and PRK in the fellow eye. No mitomycin C eye drops were used in this study. Patients were observed daily for 4 days, then at 1 month and every 3 months up to 1 year. Uncorrected and best-corrected visual acuity (UCVA and BSCVA), manifest refraction, corneal epithelium healing time, postoperative pain, and corneal haze were evaluated. Corneal wound healing was quantified with corneal confocal microscopy. RESULTS: Refractive error, UCVA, and BSCVA were not statistically different between eyes treated with LASEK and PRK. Corneal epithelium healing time was 2.52 +/- 0.99 days in the eyes treated with PRK and 2.29 +/- 0.52 days in the eyes treated with LASEK (P=.22). The postoperative pain score was 2.17 +/- 0.87 in the eyes treated with PRK and 2.62 +/- 0.60 (P=.02) in the eyes treated with LASEK. Corneal confocal microscopy showed fewer stromal activated keratocytes and less extracellular matrix deposition in the eyes treated with LASEK than in the eyes treated with PRK at 1 month postoperatively (P=.003). CONCLUSIONS: LASEK is an effective and safe procedure for low to moderate myopia, but it seems more painful until full corneal reepithelization. In the early postoperative period, the corneal wound healing process is significantly less intense in eyes treated with LASEK than in eyes treated with PRK. The role of LASEK in corneal wound healing modulation remains controversial.     

准分子激光角膜切削术与准分子激光原位角膜磨镶术角膜创口愈合的对比实验研究

比较准分子激光角膜切削术(photorefractive keratectomy,PRK)与准分子激光原位角膜磨镶术(1aser in situ keratomileusis,LASIK)后激光对角膜组织的切削效应及角膜的愈合情况,从组织学角度探讨角膜雾状混浊(Haze)及屈光度数回退的成因。方法24只新西兰白兔按预矫屈光度数随机分为-4.00 D组和-8.00 D组,每只兔右眼行PRK,左眼行LASIK。术后10d,1、3及6个月观察Haze情况并验光,每组随机处死3只兔取角膜行光镜、电镜及免疫组化检查,检测胶原Ⅲ、胶原Ⅳ、纤维连结蛋白(fibronectine,FN)及转化生长因子-β(transforming growth factor-β1,TGF-β2)的含量。结果行PRK术的右眼术后出现不同程度的Haze及屈光度数回退,其程度与预矫正屈光度数成正比。行LASIK术的左眼术后除少数角膜瓣周围半环形混浊外,手术区域角膜透明,屈光度数回退较右眼轻。-4.00 D组右眼与左眼术后屈光度数均稳定,-8.00 D组右眼较左眼屈光度数回退明显。右眼术后角膜愈合反应重,恢复慢。6个月时角膜基质仍处于修复阶段。左眼术后除形成角膜上皮栓及对应处基质轻度增生外,手术区域角膜瓣与基质床间界面清晰,无明显增生,角膜基质愈合反应轻、恢复快。术后所有兔眼角膜均有TGF-β1表达及活化,持续时间与角膜愈合时间一致。Haze及屈光度数回退组织学改变为：角膜上皮细胞增生,基底膜不成熟,前基质角膜细胞活化、增殖,新生胶原Ⅲ合成、排列紊乱,细胞外基质FN在角膜上皮下沉积。结论LASIK矫正近视尤其是高度近视优于PRK;角膜伤口愈合特别是基质愈合的反应程度,是。Haze及屈光度数回退的关键;TGF-β1是角膜愈合过程中重要调节因子,可通过介导角膜上皮一基质相互作用,调节胶原Ⅲ及FN的含量,参与瘢痕形成。     

Effects of tranilast on cultured rabbit corneal keratocytes and corneal haze after photorefractive keratectomy

PURPOSE: In vitro and in vivo studies were performed to elucidate the effects of tranilast on cellular proliferation and collagen synthesis. METHODS: Subculturing was carried out using keratocytes from rabbits that underwent photorefractive keratectomy (PRK) and developed corneal haze, and keratocytes from normal rabbit cornea. RESULTS: Tranilast suppressed proliferation in cultured keratocytes from the corneal haze region at doses of 30 and 300 micromol/L and collagen synthesis at doses of 3, 30, and 300 micromol/L. Normal corneal cultures showed suppression of keratocyte proliferation and collagen synthesis only at a high dose of tranilast (300 micromol/L). Betamethasone suppressed proliferation of keratocytes in both haze and normal cornea at a dose of 10 micromol/L, as well as collagen synthesis at respective doses of 1 and 10 micromol/L. Diclofenac sodium suppressed collagen synthesis of keratocytes in haze cornea at a high dose of 100 micromol/L, and in keratocytes in normal cornea, at doses of 10 and 100 micromol/L. In an in vivo study, either 0.5% tranilast, 0.1% betamethasone phosphate eye drops, or a tranilast base solution (control) was instilled four times daily to rabbits that had undergone PRK. Weekly evaluation of the inhibitory effect of these drugs on the development of haze was performed 2 weeks after surgery. Tranilast suppressed haze 6-13 weeks after PRK, but betamethasone phosphate showed no effect. CONCLUSION: These results indicate that tranilast is potentially effective for inhibiting the corneal haze that occurs after PRK.     

微型角膜刀法准分子激光上皮下角膜磨镶术后兔角膜愈合的实验研究

目的探讨微型角膜刀法准分子激光上皮下角膜磨镶术（Epi-LASIK）术后角膜的愈合规律,并与准分子激光屈光性角膜切削术（PRK）比较。方法建立兔近视性Epi-LASIK及近视性改良PRK动物模型,用锥虫蓝-茜素红活性染色法观察Epi-LASIK术后角膜上皮细胞活性,裂隙灯显微镜下观察角膜瓣变化、上皮修复过程和上皮下雾状混浊（haze）形成情况,通过角膜组织病理学观察角膜上皮及基质的愈合反应。结果 Epi-LASIK术后角膜上皮愈合时间为（4.67±0.41）d,而PRK术后为（2.75±0.27）d,差异有统计学意义（t=9.550,P=0.000）。PRK术后即刻和3d、5d角膜上皮瓣细胞活性率分别为（85.83±2.07）%、（48.67±3.41）%、（91.33±3.10）%,差异有统计学意义（F=215.060,P=0.000）。Epi-LASIK术后角膜瓣逐渐融解并被新生上皮取代,上皮愈合过程较改良PRK缓慢。PRK术后haze分级高于Epi-LASIK,差异有统计学意义（Z=2.27,P=0.02）。角膜组织病理学检查显示,术后1个月时2组上皮均明显增厚,3个月时上皮细胞排列仍不规则,6个月时上皮细胞形态基本恢复正常,术后3～6个月时Epi-LASIK组角膜上皮厚度已基本接近正常,与PRK组比较差异有统计学意义（P〈0.05）。术后3～6个月时Epi-LASIK组角膜前基质细胞数明显低于PRK组（P〈0.05）。结论 Epi-LASIK术后上皮瓣活性随时间的推移而降低,上皮修复较慢。与改良PRK比较,Epi-LASIK术后上皮细胞形态变化和上皮增厚程度较轻,角膜基质细胞恢复快,haze程度明显减轻。     

Comparison of wound healing after photorefractive keratectomy and laser in situ keratomileusis in rabbits.

PURPOSE: To evaluate and compare the corneal wound-healing process after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). SETTING: Kangnam St. Mary's Hospital, Seoul, Korea. METHODS: Two surgical procedures, PRK with the VISX Star excimer laser and LASIK with a MicroTech microkeratome, were performed in 24 rabbit eyes. In the PRK group (n = 12 eyes), the rabbit cornea was treated with a 20 microns ablation. In the LASIK group (n = 12 eyes), a 20 microns laser ablation was performed after a 150 microns thick hinged corneal flap had been made. During both procedures, dichlorotriazinyl aminofluorescien (DTAF) dye was applied to the ablated stromal bed; in the LASIK group, the stromal side of the corneal flap was also stained with DTAF to differentiate regenerated collagen from normal stromal tissue. Corneal wound healing was evaluated postoperatively at 1, 4, 8, and 12 weeks using light, electron, and fluorescence microscopy. The amount of regenerated stromal tissue and the number of keratocytes were analyzed by an image-analysis system. RESULTS: In the PRK group, epithelial migration and regeneration were observed in the ablated area without any stromal regeneration 1 week postoperatively. However, newly regenerated, irregularly arranged stromal collagen, with epithelial hyperplasia in the ablated area, was observed 4 to 12 weeks postoperatively by light and fluorescence microscopy. The number of keratocytes in the surgical area was also increased. In ultrastructural observation using an electron microscope, the shape of keratocytes in the ablated area was changed, and the number of rough and smooth endoplasmic reticuli, ribosomes, mitochondria, and electron-dense vesicles in the cytoplasm were increased, suggesting that the cells were activated. In the LASIK group, there was no observed regenerated collagen between the corneal flap and the ablated stromal bed except in the wound margin. Lamellated, parallel collagen fibers in the cornealstroma were not disturbed. However, in the wound margin, corneal epithelial ingrowth between the flap and the stromal bed was observed, as was some regenerated stromal tissue. The amount of regenerated stromal tissue and the number of keratocytes in the wound area were statistically smaller than those in the PRK group (P < .05). Observation by electron microscopy showed no activated keratocytes, unlike in the PRK group. The collagen fibers in the wound area were parallel. CONCLUSION: Stromal wound healing in the LASIK group was minimal compared with that in the PRK group, except in the wound margin. These results may support the clinical findings of less corneal haze in the human cornea after LASIK.     

GM6001对兔LASEK术后角膜上皮下雾状混浊形成的影响

背景近年来基质金属蛋白酶（MMPs）在准分子激光术后创伤修复过程中的作用越来越受到学者们的关注。目的探讨MMPs抑制剂GM6001对兔准分子激光角膜上皮下磨镶术（LASEK）术后角膜上皮下雾状混浊（haze）形成的影响。方法27只新西兰白兔双眼行一10．00D激光切削的LASEK,手术后动物被随机分为GM6001组、氟米龙组和阴性对照组,术后分别用150Ixmol／L的GM6001滴眼液、质量分数0．1％氟米龙和氧氟沙星滴眼液点眼。每天在裂隙灯下对兔LASEK术后角膜上皮下haze进行观察和分级。分别于术后2、4、8周各组随机选取6只眼行角膜共焦显微镜检查,制备角膜组织切片行组织病理学检查,在透射电子显微镜下行角膜组织的超微结构检查。结果LASEK术后2周和4周,阴性对照组haze分级较高的眼数多于GM6001组和氟米龙组,差异均有统计学意义（P〈0．05）,GM6001组和氟米龙组haze分级水平均明显低于阴性对照组（P〈0．01）,但GM6001组和氟米龙组相比,差异无统计学意义（P〉0．05）;术后8周组3个组间haze分级的总体比较差异无统计学意义（P〉0．05）。术后2、4、8周GM6001组术区前部基质内角膜成纤维细胞数均较对照组和氟米龙组减少,差异均有统计学意义（P〈0．05）,各时间点对照组和氟米龙组差异无统计学意义（P〉0．05）。LASEK术后GM6001组和氟米龙组兔角膜上皮细胞的形态改变、胶原纤维排列的紊乱情况均轻于阴性对照组。结论GM6001通过抑制LASEK术后MMPs对角膜基质的降解,从而抑制角膜基质成纤维细胞的增生及胶原纤维的合成,减少haze的形成,其疗效与氟米龙相似。     

Synergistic effect of ethanol and mitomycin C on corneal stroma

PURPOSE: To investigate the combined effects of ethanol and mitomycin C (MMC) application on the corneal stroma of rabbits that underwent photorefractive keratectomy (PRK). METHODS: Twenty-four rabbits (24 eyes) underwent PRK to correct -9.00 diopters of myopia. Twelve eyes had ethanol application before removing the epithelium and 12 eyes had the epithelium manually removed without ethanol. Eyes in both groups had topical MMC 0.02% application for 12 seconds immediately after excimer laser ablation. Twelve rabbits were sacrificed at two time points -4 hours and 4 weeks after surgery--and immunohistochemistry was performed with TUNEL assay, alpha-smooth muscle actin (alpha-SMA), and DAPI. RESULTS: More TUNEL-positive cells were observed in the ethanol-treated group compared to the mechanical debridement group at 4 hours after surgery (P<.01). No significant difference in alpha-SMA-positive cells was detected between the two groups at 4 weeks after sugery. However, decreased keratocyte density in the anterior stroma was more pronounced in the ethanol-treated group compared to the mechanical debridement group (P<.02). CONCLUSIONS: Ethanol application for epithelial removal during PRK seems to produce a synergistic effect with MMC, resulting in fewer keratocytes in the anterior stroma of rabbit corneas treated with MMC and ethanol than in corneas treated with MMC alone after PRK.     

Effects of acetylcysteine and dexamethasone on experimental corneal wounds in rabbits

Corneal wound healing often leads to the development of scar tissue with loss of transparency. Reconstitution of transparent corneal stroma depends on the regulation of the biosynthetic activities of post-lesional keratocytes as well as to a large extent on the limitation of matrix degradation. It has been shown that 3% concentration of N-acetylcysteine (NAC) improves the healing time of corneal wounds but some corneal haze remains. On the other hand, topical corticosteroids may retard the corneal wound healing but decrease the haze. Thus, the aim of the study was to evaluate whether adding dexamethasone to NAC could reduce the side effects of the two drugs. In this study, experimental corneal wounds were created surgically, up to the depth of one half of the stroma in the center of both eyes of all rabbits. The left eyes were treated topically with 0.9% NaCl as controls and the right eyes were treated with a combination of one drop of 3% NAC and one drop of 0.1% dexamethasone, 6 times per day. Corneal wounds were measured by fluorescein staining every day. The results indicated that the combination of acetylcysteine and dexamethasone significantly increased the mean healing time compared to the control group (p < 0.05). Clinical and histopathologic examinations revealed that the corneal haze in the treatment group was greater than in the control group. It is concluded that treatment of the eyes by a combination of 3% acetylcysteine and 0.1% dexamethasone (if used from the first day of ulceration) may retard the corneal wound healing in rabbits.     

Relation between corneal haze and transforming growth factor-beta1 after photorefractive keratectomy and laser in situ keratomileusis.

PURPOSE: To investigate the relation between corneal haze formation and transforming growth factor-beta (TGF-beta) after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). SETTING: Department of Ophthalmology, University of Tokyo Graduate School of Medicine, Tokyo, Japan. METHODS: White rabbits were divided into 4 groups, with each group receiving 1 of the following surgeries: manual epithelial abrasion, PRK, lamellar keratotomy, or LASIK. The degree of corneal haze was quantitatively analyzed by measuring the light scattering intensity of corneas before and 4 and 12 weeks after surgery. The expression of type IV collagen and TGF-beta1 in the corneas at baseline and at 4 weeks was examined immunohistochemically. RESULTS: The light scattering intensity was significantly greater 4 and 10 weeks after PRK. In contrast, epithelial abrasion, lamellar keratotomy, and LASIK did not influence the light scattering intensity of the corneas. Type IV collagen was detected in the basal lamina of the corneal epithelium and in Descement's membrane in the normal cornea. After epithelial abrasion, there was no change in the distribution of type IV collagen. Four weeks after PRK, the expression of type IV collagen was detected in the subepithelial layer of the laser-ablated area. Four weeks after lamellar keratotomy, type IV collagen was linearly and fragmentarily detected in the corneal stroma. Four weeks after LASIK, type IV collagen was linearly and continuously detected in the corneal stroma and was detected slightly in the subepithelial region of the laser-ablated area. In the normal corneas, the expression of TGF-beta1 was not detected in the keratocytes. Four weeks after PRK, the expression of TGF-beta1 increased in the keratocytes that proliferated in the subepithelial fibrous layer. In contrast, epithelial abrasion, lamellar keratotomy, and LASIK did not change the expression pattern of TGF-beta1 in the keratocytes. CONCLUSION: The multiplier effect of epithelial abrasion and excimer laser ablation in PRK may increase the expression of TGF-beta1 in keratocytes and induce corneal haze.     

Effects of various eye drops on corneal wound healing after superficial keratectomy in rabbits

PURPOSE: The effects of various eye drops on corneal wound healing, particularly in the subepithelial haze area, investigated histologically following superficial keratectomy in rabbits. MATERIAL AND METHODS: Mechanical superficial keratectomy was performed in rabbit eyes. Tranilast, betamethasone, hyaluronic acid and diclofenac eye drops were administered after the procedure. Physiological saline was used as a control. Corneas were excised 1, 2, 3, and 4 weeks after keratectomy, labeled with 3H-thymidine or 3H-proline, and subjected to autoradiography. RESULTS: In the control and diclofenac groups, corneal haze occurred three weeks after keratectomy. Histological examination revealed accumulation of proliferating keratocytes and active synthesis of collagen in the subepithelial area. In the tranilast and betamethasone groups, formation of corneal haze was reduced compared to the controls. The proliferation of keratocytes and the production of collagen in the corneal stroma were inhibited by these drugs. In the hyaluronic acid groups, corneal haze was decreased. In this group, although the proliferation of keratocytes was activated compared to the controls, abnormal accumulation of keratocytes in the subepithelial area was not detected. CONCLUSION: Tranilast and betamethasone decrease the formation of subepithelial haze by inhibiting keratocyte proliferation and synthesis of extracellular matrix in the corneal stroma. Hyaluronic acid, on the other hand, inhibits subepithelial haze by promoting physiologic wound healing.     

Plasminogen activator activity and inhibition in rabbit tears after photorefractive keratectomy

Plasminogen activator is a normal component of tear fluid that plays a role in corneal wound healing processes. This work examines whether inhibitor-induced low levels of plasminogen activator activity (PAA) during corneal re-epithelialization after excimer laser photorefractive keratectomy (PRK) correlates with the eventual occurrence of haze in rabbit eyes. Tear samples were collected with glass capillaries from 16 eyes of eight New Zealand rabbits, using i.m. injection of pilocarpine hydrochloride for stimulation. Tears were collected before and after PRK surgery, and then daily for 5 days, and every fourth day thereafter for 3 months. Both eyes underwent PRK treatment. One eye of each rabbit was treated as a control while the contralateral eye was treated with aprotinin, a serine protease inhibitor, over the first 7 days. PAA in the tear samples was measured by a spectrophotometric method using human plasminogen and chromogenic peptide substrate S-2251. For the eight control eyes after PRK, the PAA values were significantly lower (day 1) and higher (days 2 and 3) than the equilibrium PAA (p<0.001). The corneas remained clear in each of these control eyes. For the eight contralateral aprotinin-treated eyes after PRK, the PAA values on days 1-7 were significantly lower than the equilibrium PAA (p<0.001). All eight of these aprotinin-treated eyes developed corneal haze after 2 months. There was no significant difference (p=0.06) between control and aprotinin-treated eyes for the equilibrium PAA after 19 days. We conclude that a corneal wound healing abnormality (haze) develops in rabbit eyes after PRK when PAA levels are reduced using aprotinin for a week following PRK.     

羊膜移植对家兔PRK术后伤口愈合作用的研究

目的：观察羊膜对家兔PRK术后角膜伤口愈合反应的影响。方法：对30只家兔常规施行双眼PRK手术（－8．0D切削程序），即行羊膜移植，观察术后早期角膜切削区炎性细胞的浸润和角膜上皮愈合情况，晚期角膜成纤维细胞增生和上皮增生情况，观察haze并评分；早期测量角膜SOD活力和MDA含量，对结果进行统计学分析。结果：早期移植相炎性细胞的数目比对照组少，上皮愈合时间较短，晚期上皮增生较轻，晚期移植组成纤维细胞数目较少；移植组haze评分较低；早期移植组自由基反应产物MDA的含量较低，而SOD含量较高，差异均具有显著性。结论：羊膜能促进术后上皮愈合，减轻炎性反应和自由基反应，减少成纤维细胞增生，减轻haze的形成。     

Delayed epithelial healing following photorefractive keratectomy with mitomycin C treatment

Purpose: To evaluate the epithelial healing following photorefractive keratectomy (PRK) with mitomycin C (MMC) 0.02%. Methods: A total of 1520 eyes of 760 patients with myopia with spherical equivalent between ?1.5 and ?8.0 dioptres underwent PRK during 2004–2008. The epithelium was removed mechanically, and laser ablation was followed by topical application of MMC (0.02%) for 20 seconds. A therapeutic contact lens (TCL), kept in 4°C, was fitted and worn until complete epithelialization. Antibiotic, steroid and diclofenac drops were instilled during the healing phase. Steroid drops were used for 6–8 weeks and gradually reduced. The results were compared with a retrospective analysis of 500 myopic eyes which underwent PRK without MMC therapy during 2002–2004. Results: In 30 MMC treated eyes (2%), epithelial healing was delayed with a stellate defect which healed after 12–14 days. Another fifteen eyes (1%) revealed loose midperipheral epithelium and complete epithelialization took 10–14 days after scraping. Two of these eyes developed recurrent erosion treated by scraping and TCL. Seven eyes (0.5%) revealed delayed healing with paracentral epithelial plaques which were scraped and complete healing took 12–14 days. No final haze was found in the MMC‐treated eyes. In comparison, only 0.8% of the eyes which had undergone PRK without MMC revealed epithelial problems. Haze was found in 8% of these eyes. A statistically significant difference was found between the rate of epithelial problems of the two groups (p ≤ 0.002). Conclusions: MMC (0.02%) applied for 20 seconds post‐PRK was found to delay epithelial wound healing up to 14 days in 3.5% of patients with myopia compared to 0.8% of PRK eyes without MMC application. Final haze was not found despite delayed epithelialization because of MMC treatment.     

氧化剂对准分子激光兔角膜切削术后细胞凋亡的影响

目的探讨氧自由基对准分子激光屈光性角膜切削术（PRK）后凋亡机制介导的角膜创伤愈合反应的作用,以及局部应用抗氧化剂维生素C（Vit C）、维生素E（Vit E）对术后角膜的影响。方法将28只兔分成3组,其中4只为正常对照;其余24只实验兔分成2组,每组12只,行双眼-5．0 DPRK。一组术后左眼每日结膜下注射VitC 0．1g,右眼为对照;另一组术后左眼每日结膜下注射VitE 25mg,右眼为对照。于术后1、3、7、14d测定角膜组织超氧化物歧酶（SOD）、丙二醛（MDA）及谷胱甘肽过氧化物酶（GPx）的变化,同时术后定期制作病理切片,检测角膜基质细胞数量,采用脱氧核苷酸末端转移酶介导的缺口末端标记法（TUNEL）检测角膜细胞凋亡,光镜观察凋亡细胞形态,定量统计比较凋亡水平差别。结果（1）PRK术后1、3d角膜SOD、GPx活性小于对照组（P〈0．05）,MDA的水平高于对照组（P〈0．05）。局部应用抗氧化剂组其角膜SOD、GPx活性高于单纯激光组（P〈0．05）,MDA水平低于单纯激光组（P〈0．05）。（2）角膜基质细胞凋亡在1～14d均与正常对照组有差别（P〈0．01）,在应用抗氧化剂组角膜基质细胞凋亡较单纯激光组减少（P〈0．05）。（3）术后角膜基质细胞数增加,应用抗氧化剂组角膜基质细胞增生较单纯激光组减少（P〈0．05）。结论准分子激光屈光性角膜切削术后早期,角膜存在着脂质过氧化形式介导的自由基性的组织损伤破坏,促进角膜细胞的凋亡;局部应用抗氧化剂VitC、VitE能早期减轻PRK术后炎性反应,降低角膜过氧化损伤,阻止术后细胞凋亡,减轻角膜基质反应性过度增生,降低术后屈光回退和haze形成。     

Effect of collagenase inhibitors on corneal haze after PRK

The aim of the study was to determine whether collagenase inhibitors reduce corneal haze after photorefractive keratectomy (PRK). Inhibition of the initial removal phase of healing may limit the subsequent repair and replacement phases responsible for haze and regression. Thirty rabbits received -6.00D 5 mm right PRK. They were randomized to five treatment groups: G. cysteine, G. ethylene diamine tetra-acetic acid (EDTA), G. ascorbate, Oc. tetracycline or no drops. Dichlorotriazinyl aminofluorescein (DTAF) was applied to the wound immediately after surgery in two rabbits of each group, to delineate newly-synthesized from original tissue. Corneal haze was assessed by a video-linked frame grabber with computerized grey scale analysis. Corneas were taken for histology at 1 or 3 months post-operatively. Corneal haze was not significantly different between the treatment groups and controls. The severity of the histological changes varied between individuals. Within the ablation zone the epithelium was on average 10% thicker (3--4 micro m) than outside, and in some rabbits there were irregularities of the epithelial--stromal junction. The new subepithelial tissue had a mean depth of 7.8 micro m, and the superficial stroma was disorganized to a mean depth of 49 micro m. No particular treatment demonstrated significant benefits over controls; but of the treatments used, cysteine tended to produce the best results. Eyes treated with EDTA fared worst in most respects. The collagenase inhibitors used did not improve the outcome of PRK in rabbits. It remains to be determined whether firstly, the new more potent agents would have an effect, and secondly, whether collagenase inhibitors are of benefit in humans.     

Corneal haze following PRK with mitomycin C as a retreatment versus prophylactic use in the contralateral eye

PURPOSE: To report photorefractive keratectomy (PRK) treated with mitomycin C (MMC) for previous corneal haze in one eye and PRK with MMC to prevent corneal haze formation in the fellow eye. METHODS: A 40-year-old woman underwent PRK with MMC to treat previous corneal haze (secondary to previous PRK without MMC) for residual refractive error of +0.50 +0.25 x 165 in the left eye and PRK with MMC to prevent corneal haze in the right eye. RESULTS: Postoperative slit-lamp examination revealed no haze in the right eye, but continued mild haze in the left eye. CONCLUSIONS: Treatment with PRK and MMC for previous corneal haze is not as effective as primary PRK with MMC in preventing postoperative corneal haze formation.