异种表层角膜镜术的实验研究

在猴-猴表层板层角膜移植术成功的基础上,进行了三例人-猴异种表层角膜镜片术。术眼均获透明愈合。观察期分別为1个月、3个月和2年。对3例表层角膜镜片术的术眼角膜进行了组织学检查,发现上皮厚度及基质角膜细胞正常,进行异种表层角膜镜片术必须根据以下三个因素:供受体组织结构和屈光参数相近;供体角膜要有前弹力膜和一定的厚度。另外,对手术方法也进行了某些改进。     

圆锥角膜患者表面角膜镜片术后镜片细胞和神经重建的活体动态观察

目的：应用共焦显微镜观察圆锥角膜患者表面角膜镜片术（epikeratophakia,EP)后不同时期的镜片组织,了解低温冷冻脱水角膜材料移植后组织细胞和神经重建的动态过程。方法：对24例（24只眼）圆锥角膜患者行EP,应用共焦显微镜分别对术后不同时期的角膜镜片组织细胞、神经重建过程进行活体动态观察,并摄像记录。结果：镜片上皮层：术后3或4d,组织镜片表面已被角膜表层上皮细胞覆盖,镜片边缘可见翼状细胞及形态不规则、低密度的基底细胞;术后1个月,上皮基底细胞完全覆盖角膜镜片;术后6个月,基底细胞密度及形态趋于正常,可见上皮下散在神经丛;术后2年,神经丛形态接近正常。镜片基质层：术后1年内,镜片基质细胞无变化,细胞少、形态不规则;术后2年,镜片边缘偶见正常形态的基质细胞;术后5年,镜片中央仍未见正常形态的基质细胞,镜片前后基质细胞相同。镜片基质神经：术后6个月,有神经干伸入镜片基质内,术后2年,基质神经数量增多;术后5年,基质神经尚未达到正常密度。结果：冷冻的非活性角膜组织行EP后组织镜片各层细胞、神经重建的时间各不相同,但并不影响植片透明度。     

Long-term reversibility of epikeratophakia

PURPOSE: To assess the long-term reversibility of epikeratophakia. METHODS: Three human epikeratophakia lenticules (from 3 patients) were removed 7-14 years after refractive keratoplasty for aphakia (n = 1) and myopia (n = 2). Reasons for removal were irregular astigmatism (n = 1), opacities in the graft and host cornea, and progressive myopia (n = 2). After removal, 2 patients underwent cataract extraction and 1 underwent secondary implantation of AC-IOL. Visual acuity, refraction, keratometry, and corneal topography were assessed before and after removal of the lenticule, as well as after the cataract and IOL implantation, and were compared with the initial visual acuity and corneal curvature before epikeratoplasty. RESULTS: After removal of the lenticule, the 3 patients regained the initial curvature of the cornea (pre-epikeratoplasty), and remained stable during 6 months of follow-up. Initial best-corrected visual acuity and refraction before epikeratoplasty were restored after removal of the epikeratoplasty lenticule in the aphakic patient. Original best-corrected visual acuity was restored in the 2 myopic cataract patients after cataract extraction and IOL implantation. CONCLUSIONS: In a small care series, epikeratophakia was found to be a reversible procedure even after 7-14 years.     

Localization of collagen (I) and collagenase mRNA by in situ hybridization during corneal wound healing after epikeratophakia or alkali-burn.

The expression and localization of type I collagen and collagenase gene were studied by in situ hybridization using rabbit cornea during wound healing following epikeratophakia or alkali-burn. In corneas 24 days after epikeratophakia, alpha 1(I) collagen mRNA was detected in keratocytes which had migrated from the host cornea into the keratolens. In contrast, collagenase mRNA was detected in cells which seemed to be inflammatory cells around the suture between the host stroma and the keratolens. The increase of alpha 1(I) collagen mRNA in keratocytes was observed in corneas 94 days after epikeratophakia and in alkali-burned corneas 1-2 months after the burn. These results provide evidence that keratocytes synthesize collagen and that this synthesizing activity lasts for a long period during corneal wound healing.     

Innervation of tissue-engineered corneal implants in a porcine model: a 1-year in vivo confocal microscopy study

PURPOSE: To examine the pattern of nerve regeneration within tissue-engineered corneal substitutes grafted into host porcine corneas over a 1-year postoperative period. METHODS: Biodegradable corneal substitutes from cross-linked collagen were implanted into the left eyes of 12 pigs by deep lamellar keratoplasty. Regeneration of severed nerves into the central implant region was investigated with in vivo confocal microscopy. Both implant-recipient and control (right) eyes were examined before surgery and 2, 6, 10, and 12 months after surgery, to quantify the number, density, diameter, and branching of nerve fiber bundles at various corneal depths. Transmission electron microscopy was used to confirm the presence of nerve bundles. RESULTS: Two months after surgery, corneal nerve ingrowth was observed within the deep anterior stroma, with a number and density of regenerated nerves significantly higher than in nonsurgical control eyes (P < 0.01). Nerves within the superficial anterior stroma regenerated by 6 to 10 months after surgery, and the first subbasal epithelial nerves were seen 10 months after surgery. After 1 year, subbasal nerve density recovered to preoperative levels. Nerve fibers in the deep anterior stroma remained significantly thinner relative to control eyes after 1 year (P < 0.001), where both superficial anterior and subbasal nerve diameter did not change relative to control eyes. CONCLUSIONS: The pattern of reinnervation within tissue-engineered corneal substitutes has been quantified in vivo. Innervation proceeded rapidly in the deep anterior stroma, followed by repopulation of more superficial regions. One year after surgery, nerve density within the tissue-engineered cornea increased or remained unchanged relative to controls in all corneal regions examined.     

In vivo microstructural analysis of the cornea in Scheie's syndrome

PURPOSE: To analyze clinical and in vivo microstructural characteristics of both corneas of a 13-year-old male subject with Scheie's syndrome and compare the observations with the pathologic reports in the literature. METHODS: Standard clinical examination and real-time, slit-scanning in vivo confocal microscopy were performed and repeated after 1 year. RESULTS: In vivo confocal microscopy images at all cellular layers demonstrated brighter intercellular spaces than those of normal corneas. Cicatrization of the anterior stroma was identified, and the keratocytes of the middle and posterior stroma exhibited markedly altered morphology, often round or elliptical in shape, and with clearly demarcated, hyporeflective centers. The nerve fibers of the subbasal plexus were somewhat more irregular and difficult to distinguish, possibly due to underlying fibrosis. CONCLUSIONS: The potential of in vivo confocal microscopy to highlight microstructural alterations of the intact human cornea and evaluate such changes over time might reduce reliance on histopathologic investigations in such conditions and contribute to the ophthalmic management of the mucopolysaccharidoses in the future.     

Corneal sensitivity after epikeratophakia

Corneal sensitivity was tested in 60 eyes of 30 patients who underwent unilateral epikeratophakia for the correction of aphakia (20 patients) or keratoconus (10 patients). Postoperative recovery time ranged from 2 months to 21 months (mean: 10 months). Our results indicate a relative hypesthesia of the epikeratophakia lenticule when compared with the peripheral host cornea and contralateral control cornea. However, corneal sensitivity tested in 11 patients with more than 1 year follow-up was increased compared with the sensitivity of 19 patients whose postoperative recovery was less than 1 year. Histopathologic findings in two lenticules from a nonhuman primate demonstrated sparse epithelial axon terminals. Host corneal nerves appear to innervate the lenticules by intraepithelial extension and by penetration of the superficial keratectomy scar.     

In vivo microstructural analysis of the cornea in Maroteaux-Lamy syndrome

PURPOSE: This report describes the clinical and in vivo microstructural features of the cornea in a case of Maroteaux-Lamy syndrome. METHODS: A 17-year-old female with Maroteaux-Lamy syndrome was examined by slit-lamp biomicroscopy, Orbscan II slit-scanning elevation topography, and in vivo confocal microscopy. RESULTS: Slit-lamp biomicroscopy revealed bilateral, altered corneal transparency involving the posterior half of the stroma. Funduscopy revealed bilateral small, crowded optic discs, and radial macula retinal folds.On in vivo confocal microscopy, the middle and posterior stroma were clearly visualized and exhibited well-defined, unusually shaped keratocytes. These cells contained single or multiple hyporeflective regions with well-defined borders that ranged from 1 to 11.6 microm in diameter. These abnormal keratocytes were particularly abundant in the posterior stroma and sparse in the anterior stroma. CONCLUSIONS: This is the first case of Maroteaux-Lamy syndrome in which altered corneal transparency has been imaged by in vivo confocal microscopy and macula retinal folds have been described.     

Wavelike epitheliopathy after phacoemulsification: role of in vivo confocal microscopy

PURPOSE: The purpose of this report was to describe an uncommon wavelike epitheliopathy after phacoemulsification in a 59-year-old man with well-controlled diabetes mellitus. METHODS: Retrospective case report. RESULTS: A 59-year-old man with a history of well-controlled diabetes mellitus presented to us with irritation and blurring of vision in the right eye of 4-month duration after phacoemulsification with multifocal intraocular lens implantation. Slit-lamp examination revealed a wave-like epitheliopathy in the right cornea, extending nasally from the incision area. Corneal sensation was reduced. Confocal microscopy revealed reduced subbasal nerve plexus and unhealthy corneal epithelium. Topical application of preservative-free lubricants was advised. On follow-up examinations, it was observed that the disappearance of the epitheliopathy correlated with the regeneration of the nerve plexus, as shown by the in vivo confocal microscopy findings. CONCLUSIONS: This is an isolated case report of a wavelike epitheliopathy after phacoemulsification. The regeneration of the subbasal nerve plexus correlating with the disappearance of the epitheliopathy suggests an element of neurotrophic corneal damage. Other probable mechanisms are discussed along with the in vivo confocal microscopy findings.     

In vivo confocal microscopy in hydroxychloroquine-induced keratopathy

Background Vortex keratopathy, arising as a side effect of several medications, is characterized by golden-brown deposits in the cornea. Methods A 41-year-old woman treated for sarcoidosis with hydroxychloroquine therapy and suffering from vortex keratopathy was examined by in vivo confocal microscopy. Scans of both corneas were performed. Results By slit lamp examination, the left but not the right eye showed a golden-brown deposit throughout the cornea. In vivo confocal microscopy revealed the presence of highly reflective, dot-like intracellular inclusions concentrated in the basal epithelial layer. They were also detected within the anterior and posterior stroma, but not within the endothelium. In regions of the anterior stroma, devoid of inclusions, hyperreflective ramified keratocytes were observed, forming an extended interconnecting network. Conclusion In addition to the granular deposits, in vivo confocal microscopy revealed hyperreflective, possibly phagocytic keratocytes.     

The ultrastructure of well-healed lenticules in keratomileusis

Two well-healed hyperopic keratomileusis homoplastica lenticules, one 4 years old, the other 5 months old, were removed from the same patient following postoperative complications of triplopia and aniseikonia. The lenticules were examined by light and electron microscopy. Both lenticules were repopulated with keratocytes throughout the lamellae. Degenerated keratocytes were observed in the 5-month-old lenticule and recipient stroma, and in the 4-year-old lenticule; no degenerated keratocytes were seen in the recipient stroma of the 4-year-old lenticule. These findings may have resulted from toxic constituents of the solutions used to preserve the donor cornea. The basement membrane was thickened in both lenticules. The epithelial cell layer was irregular in the periphery of the lenticule where Bowman's membrane was disrupted. These findings suggest that careful surgical technique that minimizes damage to Bowman's layer and basement membrane may promote more rapid epithelial healing. The histologic results suggest that the cause of this patient's triplopia was irregular astigmatism.     

In vivo confocal microscopy of megalocornea with central mosaic dystrophy

In vivo confocal microscopy was performed on the central cornea of both eyes of a patient with megalocornea and central mosaic dystrophy. In the stroma, starting just below Bowman's membrane, polygonal, moderately reflective areas of opacification separated by diagonal hyporeflective striations were observed. The opaque areas appeared smaller in the anterior stromal layers and seemed to get larger towards the posterior stroma. The epithelium, Bowman's membrane and endothelium appeared normal. In vivo confocal microscopy is helpful in evaluating the morphological characteristics of corneal dystrophies, degenerations or developmental abnormalities of the cornea, and may prove to be helpful in understanding their pathophysiology.     

In vivo confocal microscopy in the patients with cornea farinata

PURPOSE: To report in vivo corneal confocal microscopic findings of patients with cornea farinata. PATIENTS AND METHODS: Two unrelated patients, a 47-year-old man and a 77-year-old woman, with cornea farinata were studied. Examination with a confocal microscope was performed in addition to routine slit-lamp biomicroscopy. RESULTS: In both cases, slit-lamp biomicroscopy showed numerous small, faint opacities in the deep stroma in both eyes. Using confocal microscopy, highly reflective small particles were observed in the cytoplasm of keratocytes in the deep stroma adjacent to the corneal endothelial layer. No abnormalities could be detected in the epithelial layer, in the mid-stromal layer, at the level of Descemet's membrane, and in the endothelial layer. CONCLUSIONS: In vivo corneal confocal microscopy is useful for observing stromal abnormalities in cornea farinata. Further investigation of posterior stromal opacities using confocal microscopy may be useful to understand and differentiate various corneal conditions involving primarily deep stromal layers.     

Corneal sensitivity 10 years after epikeratoplasty

PURPOSE: To assess corneal sensitivity in patients 10 or more years after epikeratoplasty for myopia, aphakia, hyperopia, and keratoconus. METHODS: A total of 45 eyes of 33 patients (age range at examination 33 to 55 yr) were included in the study. Corneal sensitivity thresholds using an electromagnetic aesthesiometer (Draeger) were measured at various locations on the lenticule and the recipient cornea. Measurements were evaluated regarding the corrective purpose, surgical technique, age, and gender of the patients and the area of measurements. RESULTS: The mean corneal sensitivity threshold in the center of the epikeratoplasty lenticule was significantly lower than on the peripheral recipient cornea (320.0 +/- 365.1 x 10(-5) N versus 0.1 +/- 0.5 x 10(-5) N). Corneal sensitivity at the 3 and 9 o'clock positions was significantly higher compared to the values at the 6 and 12 o'clock positions on the lenticule. No correlations of these values with age, gender, type, and primary indications for the surgery were observed. CONCLUSIONS: These results indicate a relative hypesthesia of the epikeratoplasty lenticule as compared to the peripheral host cornea, even 10 years after surgery. In our patients no clinically significant changes were observed that could be attributed to the reduced sensitivity over this follow-up period.     

In vivo confocal microscopy of human cornea covered with human amniotic membrane

Purpose  Amniotic membrane transplantation has been widely performed to reconstruct the surface of the eye and treat chemical burns or epithelial defects. However, we have difficulty observing the cornea through the opaque transplanted amniotic membrane by slit-lamp biomicroscopy. We investigated the use of confocal microscopy for observation of human corneas covered with amniotic membrane. Methods  Human amniotic membrane was placed onto the normal corneas of five volunteers aged 22–24 years. Then, all layers of the covered corneas were observed by in vivo confocal microscopy. Results  Confocal microscopy displayed the epithelium, basement membrane, and stroma of the amniotic membrane. It also displayed the corneal epithelium. Furthermore, corneal stromal keratocytes and the corneal endothelium were clearly observed through the amniotic membrane by confocal microscopy. Conclusions  We demonstrated that in vivo confocal microscopy enabled us to observe all layers of corneas covered with amniotic membrane in normal human eyes. Our findings suggest that confocal microscopy may have advantages for clinical examination of the ocular surface, including all layers of the cornea.     

In vivo corneal confocal microscopy in marfan syndrome

PURPOSE: To examine the cornea of patients with Marfan syndrome in comparison with a control group by using the in vivo confocal microscope. METHODS: Twenty-four eyes of 12 patients with Marfan syndrome had their corneas examined using the in vivo confocal microscope Heidelberg Retina Tomograph (HRT) II/Rostock Cornea Module. The control group included 24 eyes of 12 subjects who had their corneas examined by the same in vivo confocal microscope. RESULTS: Epithelium and neural plexus examination did not show any difference between the 2 groups. Examination of the stroma showed no significant differences concerning the morphology and density of keratocytes. The extracellular matrix of 16 of the 24 eyes of the Marfan group was clearly visible and showed thin highly reflective interconnected lines between keratocytes. In the healthy eye group, reflective lines were observed in only 5 of the 24 eyes. The endothelium of 14 corneas of the Marfan group showed brightly reflective particles. In no cornea of the control group were such particles observed. CONCLUSIONS: Highly reflective extracellular matrix of the stroma and brightly reflective particles among the endothelial cells were the 2 main corneal findings observed by using in vivo corneal confocal microscopy in patients with Marfan syndrome compared with a control group. Further studies need to be made to confirm these findings and eventually find new criteria for Marfan syndrome from the examination of in vivo corneal confocal microscopy.     

In vivo confocal microscopy of patients with amiodarone-induced keratopathy

PURPOSE: To describe the corneal findings in patients with amiodarone-induced keratopathy by means of in vivo confocal microscopy. METHODS: Twenty-two eyes of 11 patients (eight men and three women) receiving amiodarone therapy and 20 eyes of 10 healthy sex-and age-matched control subjects were selected for confocal microscopic examination. The patients were examined by use of a scanning slit corneal confocal microscope (Confoscan 2.0). Five complete scans of the entire cornea were performed for each eye with a total examination time of less than 5 minutes. RESULTS: All patients receiving amiodarone showed the presence of high reflective, bright intracellular inclusions in the epithelial layers. These findings were more evident within the basal cell layers. In the eyes with advanced keratopathy (stages 2 and 3), bright microdots were detectable within the anterior and posterior stroma and on the endothelial cell layer. In the anterior stroma, the keratocyte density in the treated group was reduced compared with values of the control group (p < 0.001), and a markedly irregular aspect of the stromal nerve fibers was found. The main characteristic of this nerve irregularity was represented by the clew-shaped appearance of the nerve trunks. CONCLUSION: Detailed examination of corneal structure by confocal microscopy shows that amiodarone keratopathy in long-term treated patients presents some findings that are consistent with higher toxicity than was expected and that involve the deep corneal layers.     

Confocal microscopy and corneal sensitivity in a patient with corneal manifestations of Tangier disease

PURPOSE: Tangier disease is an autosomal recessive disorder in which cholesterol-rich lipids are deposited at various tissues of the body including the cornea. In this case report, the corneal changes in a patient with Tangier disease are described. METHODS: A 72-year-old patient who was diagnosed with Tangier disease 25 years before received a complete eye examination including confocal microscopy and Cochet-Bonnet esthesiometry. RESULTS: Slit-lamp biomicroscopy and confocal microscopy showed bilateral corneal opacifications caused by lipid accumulation. Confocal microscopy showed that pathologic changes in the cornea in Tangier disease are limited to the stroma. Neither a reduced corneal sensation nor lid abnormalities as previously described in Tangier disease were found. CONCLUSION: Confocal microscopy helps to identify corneal changes in the stroma caused by Tangier disease easily missed in a slit-lamp examination. The ocular manifestations of Tangier disease do not necessarily include a reduced corneal sensitivity and lid abnormalities.     

Corneal innervation and cellular changes after corneal transplantation: an in vivo confocal microscopy study

PURPOSE: Although penetrating keratoplasty is generally considered a successful procedure, transplanted corneal tissue may exhibit abnormal epithelium, decreased sensation, and declining endothelial cell counts after surgery. This study aimed to use in vivo confocal microscopy to correlate corneal microstructure and recovery of the subbasal nerve plexus of the transplanted cornea with indications for, and time from, surgery. METHODS: This was a cross-sectional study comparing corneas from 42 patients after penetrating keratoplasty with those of 30 controls. Subjects were assessed by ophthalmic history and clinical examination, computerized corneal topography, and laser scanning in vivo confocal microscopy. RESULTS: Time from surgery ranged from 1 month to 40 years (mean, 85 +/- 105 months). Significant reductions in epithelial (P < 0.001), keratocyte (P < 0.001), and endothelial (P < 0.001) cell densities were noted in comparison with control corneas. Significant reductions in subbasal nerve fiber density (P < 0.001) and nerve branching (P < 0.001) were also noted. Endothelial cell density decreased with time after surgery (r = -0.472; P = 0.003), and nerve fiber density (r = .328; P = 0.034) increased. Keratoconus as an indication for transplantation was associated with higher subbasal nerve fiber densities (P = 0.003) than other indications for corneal transplantation. Neither nerve fiber nor cell density was correlated with best-corrected visual acuity. CONCLUSIONS: Laser scanning in vivo confocal microscopy highlights profound reductions in cell density at every level of the transplanted cornea and alterations to the subbasal plexus that are still apparent up to 40 years after penetrating keratoplasty.     

正常中国汉族儿童角膜的共焦显微镜研究

目的采用角膜激光共焦显微镜对正常中国汉族儿童活体角膜各层组织结构进行观察,分析正常中国汉族儿童角膜各层细胞的活体细胞形态学特征及密度。方法 49例6～13岁正常中国汉族儿童中央部角膜进行活体共焦显微镜检查,研究角膜各层结构的图象特点,并分析角膜各层细胞的密度,与成年人进行对比。结果中国汉族儿童正常角膜上皮表层细胞排列疏松,边界清楚,胞体较大,核反光较强,又称翼状细胞;基底层细胞排列紧密,呈规则的蜂巢状,胞质反光弱,正常情况下未见细胞核,细胞平均密度为5947.58±769.3个/mm2。前弹力膜为无细胞结构的膜状物,可见串珠状的神经纤维走行。基质层中可见在相对较暗的背景下明亮的角膜基质细胞核。角膜基质内有神经纤维分布。前基质层角膜细胞平均密度为1621.12±123.26个/mm2,后基质层角膜细胞平均密度为984.71±113.17个/mm2,前后基质层细胞密度有显著性差别（P〈0.05）。后弹力层中无细胞结构,为均一无结构组织。内皮细胞层表现为规则的六边形结构,细胞结构清晰,细胞平均密度为3682.38±251.87个/mm2。左右眼及男女之间角膜各层细胞密度的差异无统计学意义（P=0.341～0.611和P=0.194～0.855）。各层角膜细胞的面积和密度与性别和眼别无差异。中国汉族儿童角膜各层细胞的密度较正常成人高。结论角膜激光共焦显微镜能够在实时、活体和三维空间从细胞水平清晰观察中国汉族儿童活体角膜各层细胞的形态结构,可以对角膜各层结构进行定性和定量分析,在儿童角膜病的研究和临床诊断方面是十分有用的工具。