On the influence of neutrophils in corneas with necrotizing HSV-1 keratitis following amniotic membrane transplantation

Necrotizing herpetic stromal keratitis (HSK) in mice rapidly improved after amniotic membrane transplantation (AMT). In this study we determined the fate of polymorphonuclear neutrophils (PMN) after AMT. AMT or tarsorrhaphy (T) was performed in BALB/c mice with ulcerative HSK. After 2 days, corneas were studied histologically and by transmission electron microscopy (TEM). CD11b, Gr-1, and TUNEL-positive cells were identified. Macrophages were depleted by subconjunctival injection of dichloromethylene-diphosphonate-liposomes (Cl₂MDP-LIP) before AMT. Corneas were studied for interleukin (IL)-1α, IL-2, interferon (IFN)-γ, CXCL1, CXCL2, and tumor necrosis factor (TNF)-α production by ELISA. PMN-enriched cell preparations co-cultured with amniotic membrane (AM) or with AM and such recombinant (r) cytokines as rIL-1α, rIL-2, and rTNF-α or supernatants from activated lymphocytes were investigated by flow cytometry (Annexin-V/7-AAD and TUNEL), and a dimethylthiazolyl-diphenyltetrazolium-bromide (MTT)-viability assay. Corneas in the AMT mice had less inflammation, fewer PMN-like cells and fewer CD11b+, and Gr-1+ cells (P < 0.01), but a higher ratio of apoptotic to viable PMN-resembling cells (P < 0.01) than the T mice. Phagocytic removal of apoptotic PMN-like cells by macrophages was evident in the AMT group. After Cl₂MDP-LIP treatment, the corneas had more cell debris and apoptotic cells with PMN-like morphology. The concentrations of IL-1α, IL-2, CXCL1, and TNF-α were reduced in corneas of the AMT group as compared to that of the T group, while the concentration of CXCL2 was increased. Apoptosis of PMN-resembling cells was detected following cocultivation with AM, even when proinflammatory cytokines were present. Resolution of corneal inflammation in mice with necrotizing HSK after AMT is associated with increased apoptosis of PMN-like cells, reduction of pro-inflammatory cytokines, an increase of CXCL2, and increased removal of apoptotic PMN-like cells by macrophages.     

多层羊膜移植治疗基质坏死型单纯疱疹病毒性角膜溃疡

目的 探讨多层羊膜移植联合抗病毒药物和糖皮质激素治疗基质坏死型单纯疱疹病毒性角膜炎（HSK）的临床疗效。方法 选择2003年1月至2004年4月在山东省眼科研究所青岛眼科医院就诊的13例（13只眼）基质坏死型HSK患者,病程为3～22个月,平均15个月。经过系统的局部及全身抗病毒药物治疗1周以上,角膜炎性反应迁延不愈,角膜溃疡有继续扩大或向深基质发展趋势。利用角膜刮片镜检、坏死组织培养或共焦显微镜检查排除细菌或真菌感染,行多层羊膜移植手术,术后联合抗病毒药物和糖皮质激素滴眼液进行治疗。裂隙灯显微镜观察角膜溃疡愈合和基质水肿消退情况,荧光素钠染色观察角膜上皮的愈合情况,共焦显微镜检查角膜上皮和羊膜的转归情况。随访时间3～13个月,平均10个月。结果 术后1～3周,13例患者角膜溃疡愈合,荧光素钠染色阴性;术后1个月,角膜基质水肿完全消退。术后7～10d,表层大羊膜均发生自溶或脱落;内层羊膜紧贴角膜溃疡面,并逐渐溶解吸收,最长在位时间达到术后3个月。共焦显微镜检查仍可见羊膜组织结构,其上有扁平的角膜样上皮细胞覆盖。术后3个月时,7例角膜恢复透明,4例形成角膜斑翳,2例形成角膜白斑,眼表恢复稳定。在随访期内13例患者均未见HSK复发。结论 多层羊膜移植联合抗病毒药物和糖皮质激素是治疗基质坏死型HSK的一种有效方法。     

多层新鲜羊膜移植对基质坏死型单纯疱疹病毒性角膜溃疡的疗效观察

目的:观察多层新鲜羊膜移植联合抗病毒药物和糖皮质激素治疗基质坏死型单纯疱疹病毒性角膜溃疡的临床疗效。方法:选择本院2002-10/2009-10住院确诊此病的患者18例18眼,经局部及全身系统抗病毒治疗1wk以上,角膜溃疡迁延不愈,甚至有扩大、加深趋势,予行多层新鲜羊膜移植覆盖手术,术后联合抗病毒药物和糖皮质激素治疗。结果:术后18例患者角膜溃疡愈合,其中8例患者角膜恢复透明,10例患者角膜形成局限性斑翳或白斑。其中1例溃疡穿孔的患者,行羊膜移植术后,溃疡愈合,但形成瘢痕,视力低下,1a后予行角膜移植。结论:多层新鲜羊膜移植联合抗病毒药物和糖皮质激素是治疗基质坏死型单纯疱疹病毒性角膜溃疡的一种简单、经济、有效增视性治疗方法,可让患者恢复部分视力或保持角膜完整性,为角膜移植争取了机会。     

Microbial keratitis after amniotic membrane transplantation

Purpose To determine the incidence of early-onset (<30 days) and late-onset (>30 days) microbial keratitis after treatment of persistent corneal epithelial defects with amniotic membrane transplantation (AMT) utilizing tissue acquired from a commercial laboratory or prepared by the institutional eye bank.Methods A retrospective, non-randomized, sequential, comparative study was performed for every patient with a persistent corneal epithelial defect who underwent primary AMT at KKESH between January 1, 2003 and June 30, 2004.Results A total of 142 AMT procedures were performed for persistent corneal epithelial defects during the study period. There were 72 cases using commercially prepared tissue and 70 cases using locally prepared tissue. The mean patient age was 50.3±25.6 years (range, 1–104 years). The mean follow up was 6.3±5.0 months (range, 1–21 months). There were no cases of early-onset microbial keratitis in cases in which either commercially acquired tissue or locally prepared tissue was used.Conclusion Amniotic tissue prepared in a commercial laboratory or by properly qualified eye bank personnel may be used for AMT in eyes with persistent corneal epithelial defects with minimal risk of microbial keratitis in the first postoperative month.     

Fungal keratitis following amniotic membrane transplantation

Purpose To report a case of fungal keratitis following amniotic membrane (AM) transplantation for pseudophakic bullous keratopathy. Method A 55-year-old female underwent amniotic membrane transplantation (AMT) with epithelial debridement and anterior stromal puncture in the left eye for symptomatic bullous keratopathy. She developed fungal keratitis after 4 weeks. Result Aspergillus sp. was isolated from the corneal scraping. The patient was treated with systemic and topical antifungal medication. Conclusion Although AMT is safe, there is a risk of microbial infection after the procedure especially in poor ocular surfaces.     

Influence of dimethylfumarate on experimental HSV-1 necrotizing keratitis

Background This study was performed to investigate the influence of fumaric acid esters on the course of herpes stromal keratitis (HSK).Methods The corneas of BALB/c mice were inoculated with 105 plaque-forming units of herpes simplex virus 1 (HSV-1, KOS strain). Groups of mice were treated intraperitoneally with phosphate buffered saline (PBS) (control mice), or with dimethylfumarate (DMF) at 15 mg/kg of body weight dissolved in PBS daily for 28 days pre-infection and for 14 days post-infection. The course of HSV-1 keratitis was studied clinically. Corneal sections were examined for inflammatory cell infiltration. The numbers of CD3, GR-1, CD11b and F4/80-expressing cells infiltrating the corneas were analyzed by immunohistochemistry.Results On day 14 after HSV infection, 72% of the mice in the control group had severe HSK. The development of HSK was reduced by DMF treatment in the DMF group (22%) (P=0.004). The total number of inflammatory cells and infiltration of polymorphonuclear-neutrophils (PMNs) were reduced in the corneas of DMF-treated mice. Compared to the PBS-treated mice, numbers of CD3, CD11b, GR-1 and F4/80-positive cells were reduced in the DMF group of mice.Conclusions The course of experimental herpes stromal keratitis can be improved with systemic fumaric acid ester treatment. The improvement of keratitis correlates with a reduced corneal infiltration of T cells and mononuclear cells.The authors have no financial interest in any of the reagents used in this study     

羊膜移植手术治疗真菌性角膜溃疡

目的 探讨羊膜移植手术对真菌性角膜溃疡的治疗效果。方法 对24例(24眼)真菌性角膜溃疡患者在彻底清除角膜病灶后进行羊膜移植。结果 24例真菌性角膜溃疡行羊膜移植均一次成功愈合良好，随访3～24月，平均12月，未见免疫排斥反应。结论 羊膜移植是治疗真菌性角膜溃疡的一种有效方法。     

病灶清创联合羊膜移植治疗迁延性真菌性角膜炎的疗效观察

目的 探讨病灶清创联合羊膜移植治疗迁延性真菌性角膜炎疗效的有效性和安全性.方法 回顾性研究.共收集2007年3月至2008年3月河南省眼科研究所确诊的迁延性真菌性角膜炎患者16例(16只眼).临床确诊的真菌性角膜炎患者(角膜刮片或真菌培养阳性者)经及时,规范的抗真菌药物治疗4～6周,角膜溃疡病灶仍存,荧光素染色上皮缺损至少2 mm角膜浸润病灶未累及角膜深层(裂隙灯检查浸润病灶深度＜1/3角膜厚度,无内皮斑、前房积脓)者,行病灶清创联合羊膜移植术治疗.结果 16例患者中11例(68.8％)病变处于活动期(术前共焦显微镜检查及10％氢氧化钾涂片检查:阳性),5例(31.2％)病变处于非活动期(术前共焦显微镜检查及10％氢氧化钾涂片检查:阴性)术后平均愈合时间(13.21±6.35)d(7～26)d;其中活动期病变组平均上皮愈合时间(14.22±7.05)d;非活动期病变组平均上皮愈合时间(11.40±5.03)d.两组上皮愈合时间差异无统计学意义(P＞0.05).所有病例随访期间未见严重的并发症.结论 病灶清创联合羊膜移植对治疗迁延性真菌性角膜炎有着良好的疗效,是治疗迁延性真菌性角膜炎的又一方法.     

羊膜移植治疗暴露性角膜炎的临床疗效观察

目的:观察羊膜移植治疗暴露性角膜炎的临床疗效.方法:回顾性分析2015-09/2016-09在我院就诊的经外院药物治疗无效、反复发作的暴露性角膜炎患者14例19眼,采用羊膜移植术治疗,术后观察羊膜溶解时间、角膜透明度、角膜荧光染色、视力、并发症及复发情况.结果:所有患者在术后3~5 d出现羊膜溶解,7~14 d出现羊膜脱落.裂隙灯检查示11例16眼患者角膜透明度基本恢复透明,治疗前和治疗28 d后角膜透明度及角膜荧光染色评分差异均具有统计学意义(P<0.05).所有患者视力均不同程度提高,且未出现并发症.结论:羊膜移植治疗暴露性角膜炎具有良好的临床效果.     

羊膜移植联合羊膜覆盖治疗真菌性角膜炎分析

目的 探讨角膜清创联合羊膜移植羊膜覆盖治疗真菌性角膜炎的手术适应证的选择和临床效果.方法 2008年3月至2009年11月就诊的17例真菌性角膜炎患者,视力为FC/50cm～0.2,经角膜刮片法10%KOH溶液处理后查见菌丝,常规全身、局部抗真菌治疗3周至2个月后,患者病情迁延不愈,角膜涂片仍见菌丝.手术适应证为感染深度小于1/3角膜厚度,感染范围小于1/3角膜面积.手术方式:在局麻下行角膜清创,至角膜植床无明显感染迹象.溃疡面行羊膜小植片移植,角膜表面行羊膜大植片覆盖.术后结膜下注射0.2%氟康唑注射液0.5ml.结果 16例患者术后视力为0.5～1.0,感染得到控制.羊膜小植片与角膜融合,可见角膜组织内云翳形成.1例患者术后感染灶融解,改行板层角膜移植术后治愈.结论 角膜清创联合羊膜移植羊膜覆盖可以有效治疗药物难以治愈的表浅真菌性角膜炎.良好的手术适应证的选择不仅可以治愈真菌性角膜炎,而且可以获得良好的术后视力.真菌复发和角膜融解是手术后的危险因素.     

羊膜移植对实验性HSK中基质金属蛋白酶表达的影响

目的研究羊膜移植（AMT）对单纯疱疹性角膜炎（HSK）中基质金属蛋白酶（MMP-2，-9）表达的影响。方法40只BALB／c小鼠角膜感染Ⅰ型单纯疱疹病毒（HSV-1），实验组角膜行AMT。术后第0、2、7、14d取出角膜。常规病理切片、免疫组化染色和计算机图像分析检测角膜中MMP-2及-9的表达及平均光度值的变化。结果对照组20只鼠眼中17只发生HSK；AMT组仅有9只眼发生，差异有显著统计学意义（P〈0．01）。AMT组角膜上皮、基质病变程度及新生血管发生率明显低于对照组（P〈0．05）。免疫组化及图像分析显示对照组角膜细胞和浸润炎性细胞中表达的MMP-2及-9在第2d增加，14d时达高峰。AMT组各时间点MMP-2及-9表达低于对照组，差异有显著统计学意义（P〈0．05）。结论羊膜移植可能通过抑制角膜细胞及浸润的炎症细胞产生MMPs，从而抑制HSK的发生和发展。     

Effect of amniotic membrane transplantation on the healing of bacterial keratitis

PURPOSE: To study, with the use of an animal model, the efficacy of amniotic membrane (AM) transplantation as adjunctive treatment in corneal healing after bacterial keratitis. METHODS: Staphylococcus aureus keratitis was induced in 47 rats by injection of bacteria into the corneal stroma. Treatment was started 48 hours later with one of three randomly assigned protocols: cefazolin drops (50 mg/mL) and AM transplantation (n = 16); nonpreserved 0.9% saline drops and AM transplantation (n = 15); or cefazolin without AM transplantation (n = 16). Cefazolin and saline drops were administered every 30 minutes for 6 hours, then hourly for 6 hours. AM was transplanted 24 hours after termination of cefazolin or saline treatment. Results were clinically assessed 7 days after AM transplantation or at the corresponding time in the nontransplanted animals. The rats were then killed, and their corneas were removed for bacterial counts or histopathologic examination. RESULTS: The best clinical results were observed in the group treated with cefazolin and AM transplantation, manifested by the least corneal haze and neovascularization (P = 0.007 and P = 0.014, respectively) and minimal bacterial counts (28 colony-forming units [CFU]/mL compared with 160 CFU/mL and 240 CFU/mL, respectively). Histopathologic examination showed that the central corneal vessels from rats treated with cefazolin and AM were smaller and less congested than those from the other two groups. CONCLUSIONS: AM transplantation is a useful adjunctive treatment after bacterial keratitis in this rat model. The transplanted AM improved the healing process, resulting in decreased corneal haze and less neovascularization.     

A controlled study of amniotic membrane transplantation for acute Pseudomonas keratitis

ObjectiveTo evaluate the efficacy of amniotic membrane transplantation (AMT) to improve the outcomes of acute Pseudomonas keratitis as compared with a control group.DesignProspective interventional case series with retrospective controls.ParticipantsWe studied 14 eyes with Pseudomonas keratitis as the AMT group and 11 eyes with Pseudomonas keratitis as the control group.MethodsEyes in the AMT group were treated with antibiotic therapy followed by single-layer AMT at 2 to 3 days. Eyes in the control group received only antibiotic therapy. Patients were followed for 11.1 ± 2.4 months.ResultsIn the AMT group, pain significantly decreased from a mean score of 2.4 ± 0.5 preoperatively to 1.1 ± 0.9 at day 2 postoperatively (p < 0.001). Corneal epithelial defects healed completely within 13.2 ± 2.6 days in the AMT group compared with 15.5 ± 3.4 days in the control group (p = 0.07). At final follow-up visits, the sizes of corneal opacity and deep neovascularization were not different between the 2 groups. However, the mean score for density of the corneal opacity was significantly less in the AMT group compared with the control group (2.1 ± 0.4 vs 2.5 ± 0.7, respectively, p = 0.04). Although the best corrected visual acuity using hard contact lenses was not different between the 2 groups, uncorrected visual acuity was better in the AMT group (0.45 ± 0.22 logMAR) than in the control group (0.71 ± 0.32 logMAR, p = 0.03). No patient in either group developed significant corneal thinning or perforation.ConclusionsAMT in acute Pseudomonas keratitis was associated with immediate pain relief, less density of the final corneal opacity, and better uncorrected visual acuity at the final follow-up visit.     

清创及生物羊膜移植联合药物治疗真菌性角膜炎

目的 探讨角膜清创联合生物羊膜移植并术后抗真菌药物治疗真菌性角膜炎的效果.方法 真菌性角膜炎39例（39眼）,采用角膜清创联合生物羊膜移植术,术后结膜下注射抗真菌药物及局部抗真菌滴眼液滴眼.随访3～6月,裂隙灯显微镜观察生物羊膜愈合情况、角膜溃疡情况、视力提高情况.结果 36例角膜溃疡愈合,3例因羊膜脱落,再次行生物羊膜移植术,术后继续局部治疗,溃疡愈合良好.结论 角膜清创联合生物羊膜移植并术后抗真菌药物结膜下注射是一种行之有效的治疗真菌性角膜炎方法.     

新鲜羊膜移植治疗单纯疱疹病毒性角膜炎的初步研究

目的：探讨新鲜羊膜移植治疗单纯疱疹病毒性角膜炎的可行性、手术方法和治疗效果。方法：对本院收治的23例单纯疱疹病毒性角膜炎患(25眼)进行羊膜移植，手术方法有：单层羊膜覆盖术18例(20眼)，多层羊膜移植术5例(5眼)。术后随访6～12mo。结果：术后均未见新鲜羊膜植片急性排斥反应。术后3～15d炎症得到迅速有效的控制，眼部刺激症状消失，睫状体充血消退。视力有不同程度的提高。结论：对单纯疱疹病毒性角膜炎进行新鲜羊膜移植可以有效的控制炎症，迅速减轻局部症状，重建眼表，缩短病程，防止严重并发症的发生。     

Management of acute ulcerative and necrotising herpes simplex and zoster keratitis with amniotic membrane transplantation

AIM: To report promoted healing of acute ulcerative and necrotising herpetic keratitis after amniotic membrane transplantation (AMT). METHODS: Retrospective, non-comparative case series of seven patients with acute ulcerative and necrotising herpetic stromal keratitis. Single or multilayer AMT with epithelial side facing up was performed. The main outcome measures were wound healing of the corneal ulcers and decrease of stromal inflammation. RESULTS: The mean follow up was 10.7 (SEM 1.4) months (range 5-15 months). AMT was performed once in five cases, and twice in further two. Improvement of stromal inflammation was noted within 16.4 (2.5) days (range 7-28 days). Epithelial defects healed within a mean of 17 (2.7) days (range 7-28 days). Vision improved in all but two patients. No serious side effects occurred during the follow up. CONCLUSIONS: Although performed in an uncontrolled and non-randomised series of patients, these findings indicate that the AMT shows promise in selected cases for the restoration of ocular surface integrity, reduction of stromal inflammation, and improvement of vision in acute ulcerative and necrotising herpetic keratitis.     

角膜清创联合羊膜移植治疗表浅真菌性角膜炎临床观察

目的 观察角膜清创联合羊膜移植治疗表浅真菌性角膜炎的临床疗效.方法 选择2007年9月至2010年9月在我院确诊的表浅真菌性角膜炎患者经眼局部抗真菌药物治疗无效或病程长、迁延不愈者36例(36只眼),采用角膜清创联合羊膜移植手术治疗.术后随访3~6个月,裂隙灯观察角膜与羊膜愈合情况、共焦显微镜观察羊膜转归等.结果 术后...     

First experience with amniotic membrane transplantation

BACKGROUND: The purpose of this retrospective study was to analyze the outcome of amniotic membrane transplantation (AMT) performed at the University Eye Clinic Bern during the last 12 months. PATIENTS AND METHODS: Nine men (62.4 +/- 16.7 yrs.) and four women (78.3 +/- 22.3 yrs.) were treated with an AMT and grouped according to the ophthalmologic diagnosis: Group A, chronic corneal surface defect without limbal stem cell deficiency (n = 8); Group B, conjunctival fornix reconstruction (n = 7); Group C, filtering bleb defect (n = 2). RESULTS: 11/17 (65 %) AMT's performed in 14 eyes of 13 patients showed a favorable postoperative result after a mean follow-up time of 8.7 (+/- 2.9) months. In Group A (chronic corneal surface defect) 4/8, in Group B (conjunctival fornix defect) 7/7 and in Group C (filtering bleb defect) 0/2 showed an improvement of the basic ocular problem. 4/8 patients from Group A and 7/7 patients of Group B showed postoperatively a strong reduction of the ocular inflammation. CONCLUSIONS: In the present small study, favorable results were achieved in patients with chronic corneal surface defects without limbal stem cell deficiency and conjunctival fornix defects following AMT. In patients with fornix defects, the AMT seemed to be a valuable alternative to the more complicated transplantation of mouth- or nose mucous membrane. The two eyes with filtering defects failed.     

新鲜羊膜移植治疗病毒性角膜炎疗效观察

目的:观察新鲜羊膜对难治性病毒性角膜炎的治疗效果。方法:选取2008-06/2010-06来我院治疗病毒性角膜炎的患者18例18眼,经过角膜刮片检查排除真菌及棘阿米巴感染,所有患者均有反复发作史。所选患者在经过1～3d的抗病毒治疗后接受新鲜羊膜移植术,根据患者溃疡深度采用单层或多层新鲜羊膜移植。术后仍然抗病毒及抗细菌治疗,但减少局部药物使用次数。随访1～6(平均4)mo。结果:术后1～4wk,18例患者角膜溃疡均愈合。荧光素染色阴性,角膜基质肿逐渐消退。术后3mo,1例患者角膜透明,10例患者角膜形成云翳,7例患者角膜形成斑翳。所有患者视力均较术前有不同程度提高。结论:新鲜羊膜移植能够填补角膜溃疡,减少药物对角膜的副作用,减轻角膜炎症,促进角膜愈合,是治疗难治性病毒性角膜炎的一种有效方法。