Corneal myofibroblast generation from bone marrow-derived cells

The purpose of this study was to determine whether bone marrow-derived cells can differentiate into myofibroblasts, as defined by alpha-smooth muscle actin (SMA) expression, that arise in the corneal stroma after irregular phototherapeutic keratectomy and whose presence within the cornea is associated with corneal stromal haze. C57BL/6J-GFP chimeric mice were generated through bone marrow transplantation from donor mice that expressed enhanced green fluorescent protein (GFP) in a high proportion of their bone marrow-derived cells. Twenty-four GFP chimeric mice underwent haze-generating corneal epithelial scrape followed by irregular phototherapeutic keratectomy (PTK) with an excimer laser in one eye. Mice were euthanized at 2 weeks or 4 weeks after PTK and the treated and control contralateral eyes were removed and cryo-preserved for sectioning for immunocytochemistry. Double immunocytochemistry for GFP and myofibroblast marker alpha-smooth muscle actin (SMA) were performed and the number of SMA+GFP+, SMA+GFP−, SMA−GFP+ and SMA−GFP− cells, as well as the number of DAPI+ cell nuclei, per 400× field of stroma was determined in the central, mid-peripheral and peri-limbal cornea. In this mouse model, there were no SMA+ cells and only a few GFP+ cells detected in unwounded control corneas. No SMA+ cells were detected in the stroma at two weeks after irregular PTK, even though there were numerous GFP+ cells present. At 4 weeks after irregular PTK, all corneas developed mild to moderately severe corneal haze. In each of the three regions of the corneas examined, there were on average more than 9× more SMA+GFP+ than SMA+GFP− myofibroblasts. This difference was significant (p < 0.01). There were significantly more (p < 0.01) SMA−GFP+ cells, which likely include inflammatory cells, than SMA+GFP+ or SMA+GFP− cells, although SMA−GFP− cells represent the largest population of cells in the corneas. In this mouse model, the majority of myofibroblasts developed from bone marrow-derived cells. It is possible that all myofibroblasts in these animals developed from bone marrow-derived cells since mouse chimeras produced using this method had only 60-95% of bone marrow-derived cells that were GFP+ and it is not possible to achieve 100% chimerization. This model, therefore, cannot exclude the possibility of myofibroblasts also developed from keratocytes and/or corneal fibroblasts.     

Stromal haze, myofibroblasts, and surface irregularity after PRK

The aim of this study was to investigate the relationship between the level of stromal surface irregularity after photorefractive keratectomy (PRK) and myofibroblast generation along with the development of corneal haze. Variable levels of stromal surface irregularity were generated in rabbit corneas by positioning a fine mesh screen in the path of excimer laser during ablation for a variable percentage of the terminal pulses of the treatment for myopia that does not otherwise generate significant opacity. Ninety-six rabbits were divided into eight groups: [see table in text]. Slit lamp analysis and haze grading were performed in all groups. Rabbits were sacrificed at 4 hr or 4 weeks after surgery and histochemical analysis was performed on corneas for apoptosis (TUNEL assay), myofibroblast marker alpha-smooth muscle actin (SMA), and integrin alpha4 to delineate the epithelial basement membrane. Slit-lamp grading revealed severe haze formation in corneas in groups IV and VI, with significantly less haze in groups II, III, and VII and insignificant haze compared with the unwounded control in groups I and V. Analysis of SMA staining at 4 weeks after surgery, the approximate peak of haze formation in rabbits, revealed low myofibroblast formation in group I (1.2+/-0.2 cells/400x field) and group V (1.8+/-0.4), with significantly more in groups II (3.5+/-1.8), III (6.8+/-1.6), VII (7.9+/-3.8), IV (12.4+/-4.2) and VI (14.6+/-5.1). The screened groups were significantly different from each other (p < 0.05), with myofibroblast generation increasing with higher surface irregularity in the -4.5 diopter PRK groups. The -9.0 diopter PRK group VI had significantly more myofibroblast generation than the -9.0 diopter PRK with PTK-smoothing group VII (p < 0.01). Areas of basement membrane disruption were demonstrated by staining corneas for integrin alpha4 and were prominent in corneas with grade I or higher haze. SMA-positive myofibroblasts tended to be present sub-adjacent to basement membrane defects. Late apoptosis was detected at 1 month after surgery within clusters of myofibroblasts in the sub-epithelial stroma. In conclusion, these results demonstrated a relationship between the level of corneal haze formation after PRK and the level of stromal surface irregularity. PTK-smoothing with methylcellulose was an effective method to reduce stromal surface irregularity and decreased both haze and associated myofibroblast density. We hypothesize that stromal surface irregularity after PRK for high myopia results in defective basement membrane regeneration and increased epithelium-derived TGFbeta signalling to the stroma that increases myofibroblast generation. Late apoptosis appears to have a role in the disappearance of myofibroblasts and haze over time.     

Reduction in corneal haze and apoptosis by amniotic membrane matrix in excimer laser photoablation in rabbits

PURPOSE: To determine whether preserved human amniotic membrane can reduce corneal haze and keratocyte apoptosis induced by excimer laser photoablation in rabbit corneas. METHODS: Excimer photoablation was performed bilaterally in 30 rabbits with a 6.0 mm ablation zone and 120 microm depth using the VISX Star laser with the phototherapeutic keratectomy (PTK) mode. One eye was randomly covered by preserved human amniotic membrane secured with 4 interrupted 10-0 nylon sutures, and the other eye served as the control. The amniotic membranes were removed at 1 week, and corneal haze was graded with slitlamp biomicroscopy by 3 masked corneal specialists biweekly for the ensuing 12 weeks until the rabbits were killed. Another 18 rabbits were divided into 4 subgroups and received PTK alone, PTK with membrane, PTK with sham sutures, or PTK with tarsorrhaphy. All eyes were studied histologically, and 3 eyes in each group were studied by in situ terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling assay at 1, 3, and 7 days and 12 weeks, respectively. RESULTS: A consistent grading of differences in corneal haze scoring between the control corneas and the amniotic-membrane-covered corneas was noted among the 3 masked observers. Organized reticular post-PTK corneal haze peaked at 7 weeks in both groups, and the corneal haze score in the amniotic-membrane-covered group was significantly less than in the control group from 7 to 12 weeks (all P < .001). Compared to the control corneas, the amniotic-membrane-covered corneas had less inflammatory response at 1 and 3 days, less keratocyte apoptosis in the ablated anterior corneal stroma at 1, 3, and 7 days (P < .001), and less stromal fibroblast cellularity and epithelial hyperplasia at 12 weeks. CONCLUSIONS: Amniotic membrane matrix introduced at an early stage of the corneal wound healing process effectively reduced corneal haze induced by excimer laser photoablation in rabbits. Studies linking suppression of apoptosis in the acute wound-healing process with reduction of subsequent corneal scarring may have useful clinical applications.     

Growth factor expression in corneal wound healing after excimer laser keratectomy.

PURPOSE: To develop a rabbit model of reproducible corneal haze after excimer laser keratectomy and to characterize expression of transforming growth factor beta (TGFbeta) and basic fibroblast growth factor (bFGF) in rabbit corneas during haze formation. METHODS: Seven rabbits underwent a 100 microm deep phototherapeutic keratectomy (PTK) in one eye and a 15-microm shallow PTK in the contralateral eye. Corneal haze was compared at 1-20 weeks after surgery. Subsequently, 16 rabbits underwent 100-microm PTK in one eye and 15-microm PTK in the contralateral eye. Four rabbits were killed at 1, 2, 3, and 4 weeks, respectively, after surgery. Immunohistochemistry was performed on the corneas to localize the expression of TGFbeta and bFGF. Control subjects were rabbits that underwent either epithelial debridement alone or no surgery. RESULTS: A 100-microm PTK resulted in significantly more corneal haze than a 15-microm PTK at every postoperative examination (p < 0.05). Both TGFbeta and bFGF were expressed in the scars at 1-4 weeks after deep and shallow excimer ablations. bFGF was expressed in the keratocytes of both treated and control corneas. Minimal TGFbeta was detected in the keratocytes of the control corneas, whereas prominent TGFbeta expression was noted in the keratocyte-like cells adjacent to the postkeratectomy scars. CONCLUSIONS: The 100-microm PTK ablation resulted in significantly more corneal scarring than the 15-microm PTK ablation. Even though there was no immunohistochemical difference in the pattern of TGFbeta and bFGF expression after deep and shallow ablations, there was an association between the expression of the growth factors and corneal scarring after excimer laser keratectomy.     

Interleukin-1 receptor role in the viability of corneal myofibroblasts

The purpose of this study was to investigate the role of interleukin-1 (IL-1) in modulating myofibroblast viability in mouse corneas with stromal opacity. Twenty-four female B6; 129S1-Il1r1tm1Roml/J homozygous IL-1RI knockout mice and 24 control B6129SF2/J mice were included in this study. Each mouse had opacity-generating irregular phototherapeutic keratectomy (PTK) performed with an excimer laser in one eye. Groups of 8 mice from each group were euthanized at one month, three months and six months after surgery and the eyes cryo-preserved. The contralateral eye served as unwounded control. Immunohistochemistry was performed for α-smooth muscle actin (SMA) in central sections of all corneas. The TUNEL assay for apoptosis was performed on 8 sections of four eyes from each group. No SMA+ cells were detected in the stroma of unwounded control or knockout corneas. SMA+ myofibroblast density was significantly higher (p?相似文献   

Effects of amniotic membrane on epithelial wound healing and stromal remodelling after excimer laser keratectomy in rabbit cornea

AIMS: To investigate if the amniotic membrane (AM) promotes epithelial migration while inhibiting stromal remodelling associated with corneal haze after excimer laser keratectomy. METHODS: A wound 150 microm in depth and 6.0 mm in diameter was produced in 40 rabbits using an excimer laser. One eye was randomly chosen to be covered by the AM while the other eye served as a control. Epithelial wound healing was evaluated, together with any morphological changes of the anterior stroma connected with corneal haze. These morphological changes were histopathologically analysed using dichlortriazinyl aminofluorescein (DTAF), Masson trichrome staining, and an image analyser. RESULTS: The AM group had a short latent phase followed by fast epithelial healing (p<0.001) during the early wound healing period and a significant decrease in the inflammatory response, together with a smaller change in the number of keratocytes than the control group. The mean thickness of the regenerated stroma was significantly thinner in the AM group than in the control group at 8 weeks (p<0.0001). The AM group had a more regular architecture of regenerated stromal lamella at 8 weeks and significantly less haze after 4 weeks than the control group (p<0.05). CONCLUSION: Use of the AM as a dressing on a corneal wound created by excimer laser surgery, in which severe haze is expected, may induce rapid epithelial healing with less inflammatory response. The AM may inhibit the irregular synthesis of stromal collagen that is associated with corneal haze.     

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.     

高三尖杉酯碱对PRK后角膜浑浊的影响

目的　了解兔眼准分子激光角膜屈光切削术 (PRK)后 ,局部应用高三尖杉酯碱 (HHT)对角膜上皮下雾状浑浊(haze)抑制作用的效果。方法　将 3 0只健康家兔随机分为A、B、C 3组 ,每组 10只 ( 2 0眼 )。每组兔眼均用准分子激光屈光切削 -10 0DS。A组术后即以浸有 1mg/mLHHT的滤纸贴附于角膜切削区 5min ,用量约 0 1mL ,并用平衡盐液冲洗 ;B组自术后第 3天起双眼滴用 0 1mg/mLHHT ;C组为对照组 ,滴用生理盐水。分别观察 3组术后不同时期的眼部反应及角膜haze形成差别 ,并行组织病理学观察。结果　术后观察 3月 ,A组haze明显轻于B、C两组 (P <0 0 5 )。病理结果显示 ,A组角膜成纤维细胞增生程度明显轻于B、C两组 ,且未见明显毒副作用。结论　PRK术后应用浸有HHT的滤纸贴附切削区可抑制haze形成。     

Mitomycin C in Corneal Refractive Surgery

Mitomycin C has played a deciding role in the current revival of excimer laser surface ablation techniques. We review the literature regarding mechanism of action of mitomycin C, histological effects on the cornea, and indications, dose, exposure time, and toxicity of mitomycin C in corneal refractive surgery. Mitomycin C is an alkylating agent with cytotoxic and antiproliferative effects that reduces the myofibroblast repopulation after laser surface ablation and, therefore, reduces the risk of postoperative corneal haze. It is used prophylactically to avoid haze after primary surface ablation and therapeutically to treat pre-existing haze. There is no definite evidence that establishes an exact diopter limit or ablation depth at which to apply prophylactic mitomycin C. It is usually applied at a concentration of 0.2 mg/ml (0.02%) for 12 to 120 seconds over the ablated stroma, although some studies suggest that lower concentrations (0.01%, 0.002%) could also be effective in preventing haze when treating low to moderate myopia. This dose of mitomycin C has not been associated with any clinically relevant epithelial corneal toxicity. Its effect on the endothelium is more controversial: two studies report a decrease in endothelial cell density, but the majority of reports suggest that the endothelium is not altered. Regarding mitomycin C's effect on keratocyte population, although animal studies report keratocyte depletion after its use, longer follow-up suggested that the initial keratocyte depletion does not persist over time.     

Corneal optical coherence tomography before and after phototherapeutic keratectomy for recurrent epithelial erosions(2)

PURPOSE: To study the representation of corneal structures with optical coherence tomography (OCT) before and after excimer laser phototherapeutic keratectomy (PTK) for recurrent epithelial erosions. SETTING: Departments of Ophthalmology, Vivantes Klinikum Neuk?lln, Berlin, and Medizinische Universit?t, Lübeck, Germany. METHODS: This prospective study comprised 15 eyes of 14 patients with recurrent epithelial erosions. The central corneal and epithelial thickness as well as the wound-healing response in the anterior corneal stroma were assessed with slitlamp-adapted OCT before and after PTK. RESULTS: After PTK, the symptoms improved in all patients without loss of best corrected, glare, or low-contrast visual acuity. The mean central corneal OCT thickness was 540 microm +/-28 (SD) preoperatively, 492 +/- 36 microm immediately after epithelial debridement and PTK, and 519 +/- 25 microm after 7 weeks (P <.01). The mean central epithelial OCT thickness changed from 70 +/- 13 microm preoperatively to 60 +/- 7 microm after 7 weeks (P >.01). Changes in the light-scattering properties in the anterior subepithelial stroma revealed a hyperreflective area with a mean thickness of 46 +/- 13 microm after 7 weeks. CONCLUSIONS: Using noncontact corneal OCT, corneal and epithelial thickness changes and the wound-healing response in the anterior corneal stroma could be evaluated after PTK in patients with recurrent epithelial erosions.     

丝裂霉素C对准分子激光屈光性角膜切削术后角膜混浊的实验研究

目的探讨丝裂霉素Ｃ对准分子激光屈光性角膜切削术后角膜上皮下雾状混浊（ｈａｚｅ）的影响。方法对４５只新西兰白兔的双眼行准分子激光屈光性角膜切削术,术中、术后分别给予０．００８％丝裂霉素Ｃ、０．１％地塞米松治疗及空白对照。术后分别进行裂隙灯、角膜内皮镜、光镜和透射电镜检查。结果术后４及８周,丝裂霉素Ｃ组角膜ｈａｚｅ轻于对照组和地塞米松组;地塞米松组轻于对照组。术后１、４及８周,丝裂霉素Ｃ组术区前基质内角膜细胞数较对照组和地塞米松组少,后两组间差异不明显。三组间角膜上皮愈合时间、角膜上皮厚度及内皮细胞密度差异无显著性（Ｐ＞０．０５）。结论丝裂霉素Ｃ能通过抑制角膜细胞的生长而减轻ｈａｚｅ的形成,其效果优于地塞米松,且无明显副作用,是一种较理想的抑制ｈａｚｅ形成的药物     

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。     

Proinflammatory chemokine induction in keratocytes and inflammatory cell infiltration into the cornea.

PURPOSE: To determine the effect of interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha on cytokine, chemokine, and receptor expression in corneal stromal cells; the effect of corneal scrape injury on monocyte chemotactic and activating factor (MCAF) expression and monocyte-macrophage influx into the stroma; and the effect of MCAF and granulocyte colony-stimulating factor (G-CSF) microinjection on inflammatory cell infiltration into the stroma. METHODS: Gene array technology was used to evaluate changes in cytokine, chemokine, and receptor gene expression in stromal fibroblasts in response to IL-1alpha and TNFalpha. Expression of MCAF mRNA and protein was monitored with an RNase protection assay and Western blot analysis, respectively. Keratocyte MCAF protein expression in the rabbit cornea was detected with immunocytochemistry. After epithelial scrape injury, monocytes-macrophages were detected in rabbit corneas, by immunocytochemistry for monocyte-macrophage antigen. Inflammatory cell infiltration after MCAF and G-CSF microinjection into the stroma of mouse corneas was monitored with hematoxylin and eosin staining. RESULTS: IL-1alpha or TNFalpha upregulated the expression of several proinflammatory chemokines in stromal fibroblasts in culture. These included G-CSF, MCAF, neutrophil-activating peptide (ENA-78), and monocyte-derived neutrophil chemotactic factor (MDNCF). MCAF mRNA upregulation was confirmed by RNase protection assay, and MCAF protein was detected by Western blot analysis. MCAF protein was detected in keratocytes at 4 hours and 24 hours after epithelial injury, but not in keratocytes in the unwounded cornea. Corneal epithelial injury triggered the influx of monocytes-macrophages into the corneal stroma in the rabbit. Microinjection of MCAF and G-CSF into mouse cornea resulted in the influx of monocytes-macrophages and granulocytes, respectively, into the stroma. CONCLUSIONS: Proinflammatory chemokine induction in keratocytes is mediated by IL-1alpha and TNFalpha. The proinflammatory chemokines produced by the keratocytes probably trigger the influx of inflammatory cells into the stroma after epithelial injury associated with corneal surgery, contact lenses, or trauma.     

Retroviral gene therapy vectors for prevention of excimer laser-induced corneal haze

PURPOSE: To determine the in vivo efficacy and safety of a retroviral vector bearing an antiproliferative dominant negative mutant cyclin G1 (dnG1) construct, when used for the prevention of corneal haze after phototherapeutic keratectomy (PTK). METHODS: For in vivo efficacy studies, a 6-mm-diameter, 150-microm-deep transepithelial PTK, performed with a clinical 193-nm ArF excimer laser (VISX Star2, Santa Clara, CA) was performed on the left eyes of 20 adult New Zealand White rabbits. The surgically altered eyes were subsequently treated with eye drops containing: a retroviral vector bearing a dnG1 construct (dnG1; n = 7), a control retroviral vector (null vector) bearing only the neomycin resistance, neo(r), gene (n = 7), or a retroviral vector bearing an antisense cyclin G1 (aG1) construct (n = 6). The time of closure of the corneal epithelial defect was monitored daily with fluorescein staining. Corneal haze was evaluated before surgery and at 2, 3, and 4 weeks after surgery, with a digital imaging system. Biodistribution studies for detection of potential vector dissemination to nontarget organs were conducted by PCR-based assay. RESULTS: The re-epithelialization rate was similar among treatment groups, with complete closure of the corneal epithelial defect at 72 hours (P > 0.05). Significant corneal haze developed in the null and aG1 vector-treated groups (P 相似文献   

Transplantation of tissue-engineered epithelial cell sheets after excimer laser photoablation reduces postoperative corneal haze

PURPOSE: To apply tissue-engineered cell sheet transplantation after excimer laser keratectomy as a novel approach for the reduction of postoperative corneal haze. METHODS: Limbal biopsy specimens were obtained, and epithelial cells were cultured on temperature-responsive culture inserts without the use of feeder cells. Laser keratectomy (7.0-mm ablation zone and 160-microm depth) was performed in the contralateral eye, and autologous epithelial cell sheets were transplanted to the ablated corneal stroma. Transplant and control group eyes were assessed by slit lamp biomicroscopy, and corneal haze was scored in a masked fashion, according to the Fantes grading scale. For further examination histologic and immunohistochemical analyses were performed. RESULTS: Tissue-engineered cell sheets produced stable attachment to the laser-ablated sites, resulting in epithelialization, 5 minutes after transplantation. Conversely, control corneas required 3 to 5 days for complete re-epithelialization. At both 1 and 2 months after surgery, corneal haze was significantly inhibited in the transplant group. Histologic analyses showed that the number of keratocytes undergoing apoptosis was decreased in the transplant group at 3 days after surgery. Similarly, the expression of both collagen III and alpha-smooth muscle actin, which may enhance corneal haze, were diminished in the transplant group at 2 months. CONCLUSIONS: The transplantation of tissue-engineered epithelial cell sheets can successfully prevent the development of corneal haze after excimer laser keratectomy.     

准分子激光原位角膜磨镶术后角膜瓣下角膜上皮内生的治疗

目的　探讨准分子激光治疗性角膜切削术 (phototherapeutickeratectomy ,PTK)治疗准分子激光原位角膜磨镶术 (laserinsitukeratomileusis,LASIK)术后角膜瓣下角膜上皮内生的疗效。方法对LASIK术后发生角膜瓣下角膜上皮内生患者 6例 (6只眼 )行PTK ,术中重新掀开角膜瓣 ,刮除植入的角膜上皮组织 ,采用直径 7mm光斑 ,以 5～ 10个脉冲切削角膜瓣基质面和基质层表面 ,层间冲洗 ,复位角膜瓣 ,采用直径 3mm光斑 ,以 5 0～ 75个脉冲沿角膜瓣边缘进行切削。术后随访时间 5～ 12个月。结果　 5只眼表现为颞侧角膜瓣边缘下出现匍行性灰白色物质 ,局部角膜瓣边缘水肿和融解 ;1只眼表现为角膜瓣下局限性灰白色团块状物质 ,角膜瓣下基质层局部融解。全部术眼PTK术后无角膜瓣下角膜上皮内生复发 ;除 1只眼残留轻度角膜瘢痕外 ,余 5只眼角膜恢复透明 ;术后视力均达到LASIK术前最佳矫正视力。结论　采用PTK治疗LASIK术后角膜瓣下角膜上皮内生 ,不仅效果良好 ,而且可有效防止复发     

Mitomycin-C assisted photorefractive keratectomy in the treatment of buttonholed laser in situ keratomileusis flaps associated with epithelial ingrowth

The prophylactic intraoperative use of mitomycin-C (MMC) to prevent haze and scarring after excimer laser surface ablation (phototherapeutic/photorefractive keratectomy [PTK/PRK]) in an eye with a previous laser in situ keratomileusis (LASIK) flap buttonhole with epithelial ingrowth is described. A well-centered buttonhole measuring 2.0 mm in diameter was cut within a thin LASIK flap in an amblyopic eye. Over the next 8 weeks, corneal haze and progressive epithelial ingrowth formed centrally. An early transepithelial PTK/PRK approach was chosen to manage the buttonhole together with the epithelial ingrowth and to treat ametropia before the onset of scarring. The approach included epithelial removal with PTK, application of MMC 0.02% for 1 minute, irrigation, a short waiting period to allow for diffusion, PRK correction of -4.0 diopters without nomogram adjustment, and bandage contact lens. A regimen of prednisolone acetate 1% and ofloxacin 0.03% 5 times a day for 1 week (steroid tapered) was prescribed. Epithelial ingrowth was removed successfully. Minimal haze formation was visible 2 weeks after the retreatment but did not reduce best spectacle-corrected visual acuity (BSCVA) and resolved within the next few weeks. After 6 weeks, uncorrected visual acuity was equal to BSCVA preoperatively (20/50). There was no evidence of recurrent epithelial ingrowth or central scarring after 24 months. Transepithelial PTK/PRK was effective in managing central epithelial ingrowth in a buttonholed LASIK flap. Prophylactic intraoperative use of MMC may reduce haze formation and corneal scarring in early treatments and may also prevent recurrent epithelial ingrowth. This approach may offer faster visual recovery and no risk for a repeated buttonhole creation compared with the widespread recutting a new flap after a couple of months. The optimal application time and concentration of MMC need to be established.     

Intraepithelial phototherapeutic keratectomy and alcohol delamination for recurrent corneal erosions--two minimally invasive surgical alternatives

BACKGROUND: Phototherapeutic keratectomy (PTK) has become established as a successful therapy for recurrent corneal erosions. After epithelial debridement, Bowman's lamella and anterior stroma are ablated by the Excimer laser. We have evaluated two alternative stroma-sparing treatment options, intraepithelial PTK, and alcohol delamination. PATIENTS AND METHODS: All treatments were performed in the relapse-free period. 17 eyes with recurrent corneal erosions were treated with intraepithelial PTK: from the intact epithelium, 12 - 25 microm of tissue were ablated by the Excimer laser (group I). Alcohol delamination was performed in 13 eyes (group II). Follow-up time was between 6 months and 7 years (mean 4.2 years). RESULTS: Both methods turned out to be safe, no refractive changes were detectable. After intraepithelial PTK, we saw a cumulative recurrence rate of 12 % after 1 year, 18 % after 2 years, and 24 % after 3 years, and a temporary subepithelial scaring was seen. Alcohol delamination resulted in a recurrence rate of 15 % during the whole follow-up time (no statistically significant difference compared to intraepithelial PTK), showing no haze or scarring. CONCLUSION: Both minimally invasive, stroma-sparing methods were effective for the treatment of trauma-associated recurrent erosion. The ablation of Bowman's lamella or anterior stroma does not seem to be necessary. However, for basal membrane dystrophy, we recommend PTK after epithelial debridement for the partial ablation of Bowman's lamella.     

Long-term results of phototherapeutic keratectomy for corneal map-dot-fingerprint dystrophy (Cogan-Guerry)

PURPOSE: Patients with corneal map-dot-fingerprint dystrophy suffer typically from recurrent corneal erosion, disturbed vision, or both. The purpose of this study was to assess the morphologic and functional long-term results of minimal invasive subepithelial phototherapeutic keratectomy (PTK) for corneal map-dot-fingerprint dystrophy. METHODS: Of a total of 390 PTKs performed between October 1994 and January 2004, 15 PTKs on 15 eyes of 11 patients were included in this single-center study. All patients had symptoms of recurrent corneal erosion; in 12 eyes, reduced visual acuity was observed. The median duration of complaints was 18 months. Using 193-nm excimer laser (MEL 60/70; Carl Zeiss-Meditec), a manually guided spot profile was applied in 7 cases (pulse energy, 12 mJ; repetition rate, 2/s or 3/s; 189-425 pulses). In 8 cases, a scanning slit mode was chosen (intended ablation, 1 microm/scan; repetition rate, 20/s; 150-483 pulses). In each case, a broad deepithelialization of the Bowman layer was followed by application of defocused overlapping laser pulses. RESULTS: Complete epithelial closure was achieved after an average of 3.5 +/- 0.6 days (median, 3 days). The mean follow-up was 4.8 +/- 3.0 years, with a maximum of 9.3 years. Best corrected visual acuity increased from 0.7 +/- 0.26 preoperatively to 0.9 +/- 0.16 postoperatively. The keratometric central power remained constant (preoperatively, 43.0 +/- 1.6 D; postoperatively, 42.6 +/- 1.0 D). The average keratometric astigmatism remained constant (1.3 +/- 0.9 D, preoperatively; 1.0 +/- 0.5 D, postoperatively). In the early postoperative stage, subtle superficial corneal opacities ("haze") were observed in 6 eyes (40%), being completely reversible during the follow-up in 5 cases. No recurrence of corneal erosion was observed during the follow-up. Asymptomatic dystrophic signs in the midperiphery became visible in 2 eyes 3 and 5 years after PTK. CONCLUSION: For corneal map-dot-fingerprint dystrophy, PTK using an excimer laser with low pulse energy and low number of pulses can be considered an effective and minimal invasive treatment modality to achieve a fast and durable epithelial closure, to prevent recurrent corneal erosions, and to increase visual acuity in most patients.     

Effect of TGFβ and PDGF-B blockade on corneal myofibroblast development in mice

The purpose of this study was to investigate the role of transforming growth factor beta (TGFβ) and/or platelet-derived growth factor-B (PDGF-B) blockade on the differentiation of vimentin and alpha-smooth muscle actin (αSMA)-expressing myofibroblasts associated with haze in mice. Mouse corneas had haze-generating irregular PTK (phototherapeutic keratectomy) and topical treatment with the vectors. Six study groups of PTK treated corneas, with four corneas per group in each experiment, were Group 1) treated with TGFβ-KDEL vector interfering with TGFβ signaling through anomalous sorting of cytokine bound to the expressed altered receptor; Group 2) treated with PDGF-B-KDEL vector interfering with PDGF signaling through anomalous sorting of cytokine bound to the expressed altered receptor; Group 3) treated with both TGFβ-KDEL vector and PDGF-B-KDEL vector to interfere with signaling of both cytokines; Group 4) empty pGFPC1 vector; Group 5) empty pCMV vector; and Group 6) no vector treatment control. At one month after surgery, the corneas were analyzed by immunocytochemistry (IHC) for central stromal cells expressing myofibroblast markers vimentin and αSMA. The stroma of corneas treated with the TGFβ-KDEL vector alone (p < 0.05) or both the TGFβ-KDEL and PDGF-B-KDEL vectors (P < 0.05) had significantly lower density of vimentin-positive cells compared to the corresponding control group. The central stroma of corneas treated with the TGFβ-KDEL vector (p < 0.05) or the PDGF-B-KDEL vector (p < 0.05) had lower density of αSMA-positive cells compared to the corresponding control group. The density of αSMA-positive stromal cells was also significantly lower (p < 0.05) when both the TGFβ-KDEL and PDGF-B-KDEL and vectors were applied together compared to the corresponding control groups. This study provides in situ evidence that TGFβ and PDGF-B have important roles in modulating myofibroblast generation in the mouse cornea after haze-associated injury.