LASIK induces minimal regrowth and no haze development in rabbit corneas

PURPOSE: To quantify central corneal regrowth and haze development after LASIK in rabbits. METHODS: New Zealand White rabbits received an 89 microm (-8 diopters) myopic LASIK and were evaluated during 4 months using slit-lamp and in vivo confocal microscopy to monitor changes in central corneal morphology, epithelial and stromal thickness, flap and bed thickness, and corneal light backscattering (haze). At various time-points, corneas were processed for histology. RESULTS: Using in vivo confocal microscopy, LASIK induced no detectable morphological changes besides a slightly elevated light backscattering at the interface. Correspondingly, all corneas remained clear with no haze development by slit-lamp biomicroscopy. Corneal thickness was stable by 8 weeks after an increase of 17 +/- 4 microm that consisted of a 13 +/- 3 microm stromal regrowth and a 4 +/- 2 microm epithelial hyperplasia. At the LASIK interface, less than 4 microm new extracellular matrix was deposited. Accordingly, all LASIK flaps were easily pulled off by 6 months. CONCLUSIONS: LASIK induces a minimal wound healing response in rabbit corneas with no haze development and a regrowth (regression) of only 17 microm of an 89-microm photoablation. Three main factors contributed to the observed regrowth: epithelial hyperplasia (approximately 4 microm), matrix deposition at the LASIK interface (approximately 4 microm), and stromal growth outside the interface within the flap and wound bed (approximately 9 microm).     

Corneal stromal changes induced by myopic LASIK

PURPOSE. Despite the rapidly growing popularity of laser in situ keratomileusis (LASIK) in correction of myopia, the tissue responses have not been thoroughly investigated. The aim was to characterize morphologic changes induced by myopic LASIK in human corneal stroma. METHODS: Sixty-two myopic eyes were examined once at 3 days to 2 years after LASIK using in vivo confocal microscopy for measurement of flap thickness, keratocyte response zones, and objective grading of haze. RESULTS: Confocal microscopy revealed corneal flap interface particles in 100% of eyes and microfolds at the Bowman's layer in 96.8%. The flaps were thinner (112 +/- 25 microm) than intended (160 microm). The keratocyte activation in the stromal bed was greatest on the third postoperative day. Patients with increased interface reflectivity due to abnormal extracellular matrix or activated keratocytes at > or = 1 month (n = 9) had significantly thinner flaps than patients with normal interface reflectivity (n = 18; 114 +/- 12 versus 132 +/- 22 microm, P = 0.027). After 6 months the mean density of the most anterior layer of flap keratocytes was decreased. CONCLUSIONS: Keratocyte activation induced by LASIK was of short duration compared with that reported after photorefractive keratectomy. The flaps were thinner than expected, and microfolds and interface particles were common complications. The new findings such as increased interface reflectivity associated with thin flaps and the apparent loss of keratocytes in the most anterior flap 6 months to 2 years after surgery may have important clinical relevance.     

Stromal wound healing explains refractive instability and haze development after photorefractive keratectomy: a 1-year confocal microscopic study

PURPOSE: To evaluate the mechanism(s) producing refractive instability and corneal haze development after photorefractive keratectomy (PRK). DESIGN: Prospective, nonrandomized, comparative case series, self-controlled. PARTICIPANTS: Seventeen eyes of 17 patients with low- to moderate-grade myopia (-2.88 to -9.13 diopters [D]) were included. METHODS: Surgical intervention was a standardized, 6-mm diameter PRK procedure using the Meditec MEL 60 excimer laser (Aesculap-Meditec, Heroldsberg, Germany). The photoablation center was evaluated before surgery and at 1, 3, 6, 9, and 12 months after PRK using rapid, continuous z-scans of confocal images, termed confocal microscopy through focusing (CMTF). MAIN OUTCOME MEASURES: Simultaneous epithelial and stromal thickness analysis and objective assessment of corneal light backscattering were obtained from digital image analysis of the CMTF scans. Corneal reinnervation and anterior stromal keratocyte density and wound healing morphologic features were evaluated on high resolution, in vivo confocal images. Manifest refraction was measured and corneal clarity was graded by slit-lamp biomicroscopy. RESULTS: Epithelial thickness averaged 45+/-10 microm at 1 month, 50+/-8 microm at 3 months, and 52+/-6 microm at 12 months after PRK, as compared with 51+/-4 microm before surgery, demonstrating complete restoration of the preoperative thickness without compensatory hyperplasia. Interestingly, epithelial rethickening had no significant correlation with refractive regression. By contrast, stromal regrowth (from 1-12 months) averaged 6+/-12 microm (range, 27 microm thinning-22 microm rethickening) and correlated closely (r = 0.84, P<0.001) with changes in refraction that averaged 0.84+/-1.23 D, ranging from -1.63 D (hyperopic shift) to +3.38 D (myopic regression). Stromal rethickening increased proportionally with the actual photoablation depth (r = 0.63, P<0.01); linear regression analysis suggested an average regrowth rate of 8% per year for the entire study group. Stromal rethickening was not associated with CMTF haze development over time, suggesting that haze and regression were caused by two independent wound healing mechanisms. In agreement with these findings, all "hazy" corneas showed increased numbers of anterior stromal wound healing keratocytes with increased reflectivity of both nuclei and cell bodies, suggesting that cellular-based reflections, as opposed to extracellular matrix deposition, are the major origin of increased corneal light scattering after PRK. CONCLUSIONS: Taken together, these data indicate that keratocyte-mediated regrowth of the photoablated stroma appears to be the main cause of myopic regression in humans treated with a 6-mm diameter PRK, whereas hyperopic shifts appear to be a direct consequence of stromal thinning. By contrast, the corneal epithelium appeared to restore its preoperative thickness without contributing significantly to the refractive changes after PRK. Finally, this study also provides strong evidence that the development of haze after PRK is directly associated with increased cellular reflectivity from high numbers of wound healing keratocytes.     

Corneal response to femtosecond laser photodisruption in the rabbit

In this report we evaluated the effect of femtosecond laser energy on the development of corneal haze and keratocyte activation in rabbits following intra-stromal photodisruption to create LASIK flaps using a modified commercial femtosecond surgical laser. Three groups of flap parameters were studied: 1.5 microJ/pulse with 10 microm spot separation and complete side cut (Group 1); 3.5 microJ/pulse with 14 microm spot separation and complete side cut (Group 2); 3.5 microJ/pulse with 14 microm spot separation and partial (50 microm) side cut (Group 3). All flaps were left attached without lifting to avoid epithelial contamination. Rabbits were then evaluated pre- and post-operatively by quantitative in vivo and ex vivo confocal microscopy. The achieved flap thickness 1 week after surgery averaged 88.9+/-12.8, 90.8+/-6.9 and 86.5+/-6.8 microm for Groups 1-3 respectively (p=NS). Interface thickness was significantly greater (p<0.05) in the higher energy groups averaging 40.0+/-11.2 and 37.7+/-5.7 microm for Groups 2-3 compared to 28.6+/-4.5 microm for Group 1. Corneal haze was barely detectible and not significantly different between groups, although haze was detected in the region of the side-cuts in Groups 1 and 2. No clinically significant changes in stromal or epithelial thickness were noted. Laser confocal microscopy showed the presence of small diameter cells within the flap interface that resided within disrupted regions of the corneal collagen lamellae. Keratocyte activation was only detected in regions of the 100% side cut and not over the flap interface. In conclusion, the results of this study indicate that photodisruption of the corneal stroma alone without flap elevation regardless of laser energy does not induce significant corneal haze in the rabbit. However, a thicker stromal interface was seen with the higher energy suggesting greater stromal damage.     

Immunohistology of corneal wound healing after photorefractive keratectomy and laser in situ keratomileusis. wachtlin@ukbf.fu-berlin.de.

PURPOSE: The aim of the study was to compare corneal wound healing after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) using conventional, immuno- and enzymohistologic methods. METHODS: Sixteen white Russian rabbits in each group underwent PRK or LASIK. Keratocyte density was recorded from 1 week to 6 months post-operatively on conventional histological sections. Immunohistologic cellular fibronectin and tenascin were used as markers of early epithelial and stromal wound healing in the cornea. The cell damage was demonstrated enzymohistologically using alkaline phosphatase. RESULTS: The reaction was similar in quality with both methods and occurred at sites of simultaneous epithelial and stromal injury. Mild scarring was found around the edge of the flap after LASIK; PRK-treated corneas developed a central subepithelial haze and scarring. A hypocellular region was found in the anterior part of the ablation zone shortly after PRK. Fibroblast migration later led to hypercellularity and subsequent clinical haze formation. After LASIK this reaction was limited to the peripheral entry point of the microkeratome blade around the edge of the corneal flap, where cellular fibronectin and tenascin reactions were positive. An acellular zone was found anterior to the interface after LASIK. The keratocyte damage visualized by alkaline phosphatase was more extensive after PRK than after LASIK. CONCLUSION: The stromal reaction to surgery was more extensive after PRK than after LASIK. A cytokine-mediated interaction between the epithelium and stroma was suggested as the cause of keratocyte cell migration and scar formation.     

Confocal assessment of the corneal response to intracorneal lens insertion and laser in situ keratomileusis with flap creation using IntraLase

PURPOSE: To assess the response of the cornea to hydrogel intracorneal lens (ICL) insertion or laser in situ keratomileusis (LASIK) with IntraLase (IntraLase Corp.) at the cellular level. SETTING: Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, USA. METHODS: Twenty patients (29 eyes) were evaluated by in vivo confocal microscopy 1 to 6 months postoperatively: 20 eyes had LASIK with flap creation by IntraLase, and 9 eyes had ICL insertion (8 following IntraLase). RESULTS: For LASIK with IntraLase, keratocyte activation and/or interface haze was detected in 8 of 20 eyes. The remaining eyes had interface particles but no cell activation. Keratocyte activation was generally limited to a few cell layers adjacent to the interface. However, 2 patients exhibited multiple layers of activation and increased extracellular matrix (ECM) reflectivity (haze) surrounding the interface by confocal microscopy. Both patients also had clinical haze and photophobia. For ICLs, following insertion, 5 of 9 eyes had activated keratocytes adjacent to the implant surfaces. The largest amount of cell activation and ECM haze detected by confocal microscopy was in 2 patients with significant clinical haze. Structures with an epithelioid morphology were detected on some implant surfaces. Epithelial thickness was 33.3 microm +/- 2.3 (SD) in the ICL eyes and 49.2 +/- 6.5 microm in the LASIK with IntraLase eyes. CONCLUSIONS: Both LASIK with IntraLase and ICL insertion following IntraLase induced keratocyte activation, which may underlie clinical observations of haze in some patients. Intracorneal lens implant also induced thinning of the overlying corneal epithelium.     

Assessment of keratocyte activation following LASIK with flap creation using the IntraLase FS60 laser

PURPOSE: To assess the response of corneal keratocytes to the IntraLase FS60 femtosecond laser using attenuated steroids. METHODS: Thirty patients (30 eyes) who underwent LASIK with the IntraLase FS60 were assessed by clinical examination and confocal microscopy 3 months postoperatively. Postoperative steroid regimen was Econopred Plus (Alcon Laboratories Inc) every hour for 1 day and four times daily for 7 days. RESULTS: No cornea had clinically significant flap interface haze. Two corneas had trace haze at the interface detected by slit-lamp examination; both showed significant keratocyte activation by confocal microscopy. Overall, some degree of keratocyte activation was detected at the flap interface in 10 of 30 eyes. The measured interface reflectivity was 328.8 +/- 85.0 confocal backscatter units (CBU) in eyes with activated keratocytes and 88.9 +/- 74.5 CBU for the remaining 19 eyes (P < .001). CONCLUSIONS: With attenuated steroids, keratocyte activation was found in a significant number of eyes, although interface haze was subclinical. A higher steroid dosage might therefore be indicated.     

Evaluation of corneal stromal changes in vivo after laser in situ keratomileusis with confocal microscopy

PURPOSE: To assess by in vivo confocal microscopy the modifications of the corneal stroma after laser in situ keratomileusis (LASIK) for myopia. DESIGN: Nonrandomized comparative (self-controlled) trial. PARTICIPANTS: Sixteen eyes of 13 patients were examined before surgery and at days 8, 30, and 90, and 9 eyes were examined at 6 months postoperatively using an in vivo confocal microscope. TESTING/INTERVENTION: Stromal morphologic changes, keratocyte density, flap thickness, and subclinical haze were evaluated and compared at different time points. LASIK was performed with a Flapmaker microkeratome (Solan Ophthalmic products, Jacksonville, FL) and a Lasersight LSX excimer laser (LaserSight Technologies Inc., Winter Park, FL). MAIN OUTCOME MEASURE: Confocal microscopy results. RESULTS: Microfolds at the Bowman's layer were found in most eyes, as well as variable reflectivity particles (pa) located at the interface level in all eyes examined postoperatively. The density of these particles significantly decreased with time with, respectively, 504 +/- 101 pa/mm2 at day 8 and 380 +/- 111 pa/mm2 at day 30 (P = 0.003), 332 +/- 100 pa/mm2 at month 3 and 312 +/- 40 pa/mm2 at month 6. The mean flap and the activated-cells area thicknesses were, respectively, 102 +/- 26 microm and 61 +/- 19 microm and showed significant negative correlation (P < 0.0001). The intensity of the added peak (47.3 microm 8.6%), corresponding to the subclinical haze, realized by Z-scan measure, was also negatively correlated with flap thickness (P = 0.01). Keratocyte (k) density quantified in the posterior stroma significantly increased from day 0 (480 +/- 67 k/mm2) to day 8 (701 +/- 41 k/mm2, P < 0.0001 compared with day 0) and day 30 (917 +/- 143 k/mm2, P = 0.0006, compared with day 0) but significantly decreased at 3 months postoperatively (597 +/- 56 k/mm2, P < 0.0001 compared with day 30) to reach the initial level at month 6 (502 +/- 41 k/mm2, nonsignificant compared with day 0). There was no correlation between preoperative or postoperative spherical equivalent and the density of particles, keratocytes, and the haze intensity. CONCLUSIONS: This study confirms the presence of microfolds and particles at the interface level, as well as subclinical impairment. Evaluation of keratocyte density constitutes a major contribution of confocal microscopy toward an understanding of the keratocyte response to corneal wound healing after corneal refractive surgery. Moreover, flap thickness seems to be involved in the postoperative cellular activation with a higher response when thin.     

Effect of exogenous keratinocyte growth factor on corneal epithelial migration after photorefractive keratectomy

PURPOSE: To investigate the effect of topical keratinocyte growth factor (KGF) on wound healing after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). SETTING: Department of Ophthalmology, Rayne Institute, St. Thomas' Hospital, London, United Kingdom, St. Erick's Eye Hospital, Stockholm, Sweden, and the University of Regensberg, Regensberg, Germany. METHODS: In a placebo-controlled trial, 24 New Zealand white female rabbits were divided into 3 equal groups. Group 1 (n=8) had myopic PRK (6.0 diopters [D]) using the Technolas 217z laser (Bausch & Lomb). Group 2 and Group 3 had myopic LASIK (6.0 D) with a flap depth of 140 microm and 180 microm, respectively. Topical KGF (20 microg/mL) was administered to half the treated eyes in each group intraoperatively and postoperatively; the other half received placebo eyedrops. Epithelial closure, corneal haze, and keratocyte activation in the rabbit eyes were analyzed and compared with those in placebo-controlled eyes for 5 weeks postoperatively. RESULTS: In Group 1, the mean reepithelialization after PRK was 0.10 mm2/h +/- 0.02 (SD) in the KGF group and 0.33 +/- 0.05 mm2/h in the control group (P=.001). There was no significant difference in the mean backscatter between the KGF eyes (154 +/- 45.95) and the control eyes (141 +/- 38.45) after PRK (P=.42). Histology revealed reduced epithelial cell layers in the KGF group and comparable keratocyte density as in the control group. In Groups 2 and 3, there was no significant difference in backscatter, epithelial layers, and keratocyte density between KGF and control eyes after LASIK. CONCLUSIONS: Topical KGF (20 microg/mL) delayed reepithelialization after PRK. It had no effect on stromal wound healing in LASIK eyes with an intact epithelial barrier.     

VisuMax飞秒激光准分子激光角膜原位磨镶术术后角膜反应的共焦显微镜观察

目的观察VisuMax飞秒激光制瓣的准分子激光角膜原位磨镶术（LASIK）术后角膜反应的动态变化。方法等效球镜为-6.75～-12.50D的近视患者6例12眼行VisuMax飞秒激光制瓣的LASIK。飞秒激光参数设置为脉冲频率200kHz,脉冲能量100～150nJ,瓣直径7.9～8.3mm,预计瓣厚度80～100μm。分别于术后1周、1个月、3个月用裂隙灯检查角膜中央区域的形态,并用共焦显微镜观察中央区域的角膜反应。结果术后1周、1个月、3个月裂隙灯检查所有手术眼未见haze或瓣皱褶,共焦显微镜下观察所有眼未见明显角膜上皮细胞的形态改变。术后1周,4例6眼观察到近切削界面的基质细胞轻度激活,表现为细胞核反光增强,细胞轮廓可见,切削面反光轻度增强,其余眼未观察到明显的基质细胞激活。角膜基质细胞激活在术后1个月减轻,至术后3个月基本消失。术后所有手术眼均可在切削面持续观察到高反光沉积物,1眼（预期角膜瓣厚度80μm）在共焦显微镜下可见角膜微皱褶。结论 VisuMax飞秒激光LASIK术后存在角膜细胞水平反应的变化,其角膜基质细胞激活程度较轻可能与脉冲能量较低有关。     

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.     

Prospective evaluation of PermaVision intracorneal implants using in vivo confocal microscopy

PURPOSE: To report effects of the PermaVision intracorneal lens at the cellular level using in vivo confocal microscopy. METHODS: Four eyes implanted with intracorneal lenses beneath an IntraLase flap for correction of hyperopia were evaluated preoperatively and 1 to 6 months postoperatively. RESULTS: Intracorneal lenses were tolerated in three eyes with little or no haze observed clinically and good visual results. Minimal keratocyte activation was detected by confocal microscopy, and cell density was decreased posterior to the implants. Epithelial thinning was observed 1 month after implantation. Thickness stabilized by 6 months but remained thinner than baseline (33 +/- 2 microm vs 48 +/- 8 microm, P < .01). The fourth eye had a complicated course with early flap displacement followed by diffuse lamellar keratitis. Confocal microscopy revealed activated keratocytes throughout the anterior stroma. The implant was removed, and recovery was promising. CONCLUSIONS: Implantation of intracorneal lenses can induce side effects of epithelial thinning, keratocyte loss, and keratocyte activation.     

Effects of topical tranilast on corneal haze after photorefractive keratectomy

PURPOSE: To determine whether topical tranilast might reduce corneal haze through suppression of transforming growth factor (TGF)-beta1 synthesis in keratocyte after photorefractive keratectomy. SETTING: Department of Ophthalmology, Korea University College of Medicine, Seoul, Republic of Korea. METHODS: Photorefractive keratectomy was performed on 48 eyes of 28 white rabbits and 24 eyes in a tranilast group were treated with tranilast solution, and the other 24 eyes in control group were treated with saline after laser ablation. The grades of corneal haze at 1, 2, 4, and 8 weeks after surgery were evaluated in 10 eyes of each group for comparison. Immunohistochemistry was performed on 10 eyes of each group, and Western blot analysis was done on 4 eyes of each group for studying TGF-beta1 expression at postoperative day 7. RESULTS: There was no statistically significant difference in corneal haze between 2 groups from week 1 to week 4 after surgery, but a significant difference was found at week 8 after photorefractive keratectomy (P=.02). The mean number of keratocytes that expressed TGF-beta1 in the tranilast group was 58.3 (+/-17.2), which showed significant difference, compared with that of the control group, 104.5 (+/-23.0) (P<.01). Western blot analysis also revealed that the amount of TGF-beta1 in tranilast group was slightly less than the control group. CONCLUSIONS: Topical tranilast could reduce corneal haze by suppressing TGF-beta1 expression in keratocytes after photorefractive keratectomy.     

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.     

准分子激光兔角膜切削术后细胞凋亡和增殖

目的 寻找准分子激光角膜切削术（ＰＲＫ）术后细胞凋亡和激活增殖的动态联系,评价激光去除上皮（ＰＴＫ）和机械刮除上皮（ＭＥＳ）对凋亡和增殖的影响。方法 对１８只兔按ＰＴＫ和ＭＥＳ行ＰＲＫ（－９．９０Ｄ,６．０ｍｍ直径）,术后定期用活体共聚焦显微镜观察及制作病理切片,ＴｄＴ介导ｄＵＴＰ缺口末端标记（ＴＵＮＥＬ）原位显示凋亡细胞,激光扫描共聚焦显微镜观察凋亡细胞形态,定量统计比较凋亡水平差别。结果 ＰＲ     

The effects of epithelial viability on stromal keratocyte apoptosis in porcine corneas stored in Optisol-GS

PURPOSE: To investigate the effect of corneal epithelium on the viability of corneal stromal keratocytes in Optisol-GS. METHODS: After sterilization, corneoscleral buttons were excised and stored in Optisol-GS for various time periods. Group 1 corneas (n = 40) underwent mechanical corneal epithelial debridement before storage while group 2 corneas (n = 40) were stored with intact epithelium. Changes in corneal thickness, keratocyte density, and keratocyte apoptosis were investigated immediately, at 4 hours, and on days 1, 2, 3, 5, 7, and 14 in the preservation medium. The differences between group 1 and 2 corneas were analyzed. RESULTS: Corneal thickness increased significantly in the second week of preservation in both groups, though more substantially in group 1. Significant corneal epithelial apoptosis was noticed in the first week in group 2 corneas. Corneal stromal keratocyte density decreased with prolonged preservation time. DNA laddering was detected by ligation-mediated polymerase chain reaction throughout the experiment periods in both groups, but the increase of keratocyte apoptosis was more significant after 5 days of preservation, especially in group 1. CONCLUSIONS: Stromal keratocytes underwent apoptosis in Optisol-GS. The absence of corneal epithelium during preservation further increased the stromal keratocyte apoptosis.     

In vitro human corneal model to investigate stromal epithelial interactions following refractive surgery

PURPOSE: To develop an in vitro human corneal model to evaluate stromal epithelial interactions following corneal refractive surgical procedures. SETTING: Department of Academic Ophthalmology, Rayne Institute, St. Thomas' Hospital, London, United Kingdom. METHODS: Fifty-six human donor corneas procured from the eye bank were placed in a specially designed acrylic corneal holder and were cultured using the air-interface organ culture technique for up to 4 weeks. Corneal refractive surgical procedures such as a simple epithelial defect, 4 diopter (D) and 9 D photorefractive keratectomy (PRK), 4 D and 9 D laser-assisted subepithelial keratectomy (LASEK), and 9 D laser in situ keratomileusis (LASIK) were performed on the model. Temporal events in epithelial and keratocyte cell kinetics were evaluated using digital imaging, confocal microscopy, and light microscopy. Two-way analysis of variance and Student t tests were used to assess statistical significance. RESULTS: Epithelial healing following PRK was completed by 92 hours +/- 10 (SD) at a rate of 0.58 +/- 0.45 mm2/hour. In LASEK, the epithelial flap was replaced by regenerating peripheral epithelium that showed significant delay in epithelial closure (120 +/- 5 hours) with prolonged latency (24 +/- 4 hours, P<.0001) in comparison with PRK. The magnitude of keratocyte loss corresponded to ablation depth, and keratocyte regeneration was dependent on epithelial closure. In comparison, LASIK corneas showed a lesser percentage of keratocyte loss with poor recovery of keratocyte density in the stromal flap. Epithelial viability and keratocyte density were well preserved in the in vitro human model as observed in control corneas for up to 4 weeks. CONCLUSIONS: The temporal events in stromal epithelial interactions in the in vitro human model closely mimicked in vivo observations. The human model further avoided species-specific variations and provided a suitable test bed for evaluating newer algorithms and therapeutic regimens following refractive surgery.     

Corneal stromal changes after LASIK

PURPOSE: To assess stromal modifications after laser in situ keratomileusis (LASIK) for myopia using in vivo confocal microscopy. METHODS: Thirteen eyes from 10 patients were examined before surgery and at days 8 and 30 after surgery using an in vivo confocal microscope coupled with a Z-Scan system. Stromal morphological changes, keratocyte density, flap thickness, and subclinical haze were evaluated and compared at different time points. RESULTS: Microfolds at the Bowman's layer were found in 55%, eyes as well as variable reflectivity particles located at the interface level in all postoperative examined eyes. The mean flap and activated-cell area thicknesses were respectively, 101+/-28 micrometer and 50.5+/-14 micrometer with a significant negative correlation (r=-0.89, p=0.01). The intensity of the added peak (47.3+/-8.6% scattered light), corresponding to the subclinical haze, as measured by Z-Scan, was also negatively correlated with the flap thickness (r=-0.89, p=0.01). CONCLUSION: This study confirms the presence of microfolds and particles at the interface level as subclinical complications. Evaluating the keratocytic activation by confocal microscopy can lead to a better understanding of corneal wound healing after LASIK and can help to improve the techniques. The flap thickness seems to be involved in the cellular activation induced by LASIK.     

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.     

Normal human keratocyte density and corneal thickness measurement by using confocal microscopy in vivo

PURPOSE: To quantify keratocyte density according to stromal region and subject age and to measure the thickness of the normal human cornea and its layers in vivo. METHODS: Seventy normal corneas of 70 subjects were examined by confocal microscopy (contact lens wearers were excluded). Ages of subjects ranged from 12 to 80 years, with 10 subjects per decade. Images were recorded by continuously focusing the optical section through the full-thickness central cornea. Two independent human observers manually identified bright objects (keratocyte nuclei) against a dark background to quantify keratocyte density. This method was validated histologically in three human corneas. Thickness measurements were obtained by plotting mean reflected light intensity in images against corneal depth, and calculating distances between intensity peaks that corresponded to corneal layers. RESULTS: Full-thickness central keratocyte density was 20,522 +/- 2,981 cells/mm(3) (mean +/- SD, n = 69). The number of keratocytes in a full-thickness column of central stroma, which had a cross-sectional area of 1 mm(2), was 9624 +/- 1385 cells. Keratocyte density was highest in the anterior 10% of the stroma. Full-thickness keratocyte density was correlated with age (r = -0.62, P < 0.001), decreasing 0.45% per year. Central corneal thickness was 563.0 +/- 31.1 microm (mean +/- SD) and central epithelial thickness was 48.6 +/- 5.1 microm. CONCLUSIONS: This is the first study to quantify regional keratocyte density comprehensively in vivo across a broad age range of normal human subjects. The method was acceptable to both subject and observer, and may prove useful for quantifying keratocyte density in patients with corneal disorders or after corneal surgery.