BACKGROUND:Acellular porcine corneal stroma (APCS) is a promising alternative to allografts; however, its potential requires verification. To evaluate the efficacy and safety of novel cross-linked APCS transplantation for the treatment of infectious corneal disease. METHODS:This prospective, multicenter, single-arm clinical trial was conducted from 2015-2019. Ninety patients with infectious corneal disease who had undergone anterior lamellar transplantation with an APCS across four centers in China were enrolled. All patients had previously received ineffective drug treatment and had a preoperative best-corrected visual acuity (BCVA) < 20/400. The primary outcome was the deblinding rate at 180 days compared to the target rate of 65%. The secondary outcomes included BCVA, corneal transparency, and epithelial healing. Safety endpoints included corneal graft rejection, blood count, liver and kidney function, immunoglobulin levels, concomitant medications, and additional treatments. Follow-up was performed for at least 6 months postoperatively. RESULTS:A total of 100% (90/90) and 88.9% (80/90) of the cases were included in the safety analysis and efficacy per protocol sets, respectively. Eighty percent (72/90) of patients completed 180 days of follow-up after APCS transplantation. The deblinding rate at 180 days was 90.3% (95% confidence interval 81-96%), exceeding the target ( P < 0.001). At 180 days, 55% of the grafts were completely transparent and 82.5% of the epithelium had no defects. The log of the minimum angle of resolution vision improved from 2.19 ± 0.25 preoperatively to 1.10 ± 0.3. No serious adverse events occurred. CONCLUSION:APCS demonstrated excellent corneal transparency and epithelialization following keratoplasty, confirming its safety and efficacy. Six-month data suggest its potential as an alternative treatment for infectious corneal disease. Further long-term trials are necessary to establish its comparative effectiveness and evaluate chronic complications, offering valuable insights for future clinical practice.

Fungal keratitis (FK) is a persistent and vision-threatening disease. The potential of vitamin C (VC) in tissue protection is well-recognized; however, its specific role in FK and its interaction with antifungal drugs are not well defined. In this study, we investigated the role of VC in FK in vitro and in vivo. It revealed that VC substantially decreases clinical scores and corneal perforation rates in a murine model of FK. VC promoted a concentration-dependent increase in neutrophil infiltration via mast cells in vitro and confirmed this effect in in vivo experiments. Moreover, VC enhanced fungicidal activity by boosting infiltrated neutrophils, without influencing reactive oxygen species (ROS) production or neutrophil extracellular traps (NETs) formation. VC also mitigated the cytokine storm within bone marrow-derived macrophages and increased neutrophil apoptosis, thereby facilitating the efficient clearance of senescent neutrophils. The combination of VC with amphotericin B (AmB) demonstrated additive antifungal effects, reducing fungal load and corneal perforation. These results indicate that VC is pivotal in defending against fungal corneal infections by promoting neutrophil chemotaxis through mast cells (MCs) and modulating the inflammatory response, without suppressing neutrophils' antifungal capability. The combination of VC with AmB may present a new therapeutic avenue for FK.

Objective:To investigate the mechanism of tissue damage caused by neutrophil matrix metalloproteinase-8 (MMP-8) in Fusarium keratitis. Methods:A total of 108 male C57BL/6J SPF grade mice, 6-8 weeks old, were selected to establish a model of Fusarium keratitis (FK) in the right eyes.Corneal inflammation in mice was observed and scored under a slit lamp microscope.Based on the corneal inflammation scores, the modeling eyes were divided into 0, 12, 24, 48, and 72-hour groups post-modeling.At the corresponding time points, mice were euthanized, and corneal tissues were collected.The expressions of MMP-8, adenylate-activated protein kinase (AMPKα) and its serine 172-site phosphorylated form (p-AMPKα) proteins in corneal tissues were detected by Western blot.The neutrophil count in mice corneal tissues at each time point was determined using hematoxylin and eosin staining.The co-localization of neutrophils and MMP-8 protein in the cornea was observed by immunofluorescence staining.In the in vitro corneal collagen degradation experiment, corneal tissues were divided into MMP-8 group, buffer group, and normal saline group, which were treated with 100 μl of activated recombinant MMP-8, detection buffer, and normal saline, respectively.Hydroxyproline content in corneal tissues was determined using a hydroxyproline assay kit, and the mass fractions of hydroxyproline were compared among the groups.Peripheral blood neutrophils were isolated from human blood samples, and Fusarium spores were collected for experiments.Human neutrophils were divided into four groups, negative control group (cultured neutrophils), co-culture group (neutrophils co-cultured with spores), AICAR-treated group (neutrophils co-cultured with spores and treated with p-AMPK protein kinase activator AICAR), and compound C-treated group (neutrophils co-cultured with spores and treated with the inhibitor compound C).The MMP-8 protein expression levels in each group of human neutrophils were assessed via immunofluorescence staining.The use and care of animals complied with the ARVO statement and Regulations for the Administration of Affairs Concerning Experimental Animals.The animal experiment protocol was approved by the Animal Ethics Committee of Henan Eye Hospital (No.HNEECA-2017-04-02).One healthy adult volunteer was selected and 10 ml of peripheral venous blood was collected.The clinical study protocol was approved by the Clinical Ethics Committee of Henan Eye Hospital (No.HNEECKY-2019[16]). Results:At 24 hours post-modeling, corneal opacification was observed in the modeling eyes, and corneal perforation occurred in 72-hour post-modeling group.The corneal inflammation scores in 24, 48, and 72-hour post-modeling groups were all higher than those in 12-hour post-modeling group, and the differences were statistically significant (all at P<0.001).The relative expression levels of MMP-8 protein in the cornea were higher in 12, 24, and 48-hour post-modeling groups compared to 0-hour group, with statistically significant differences (all at P<0.001).There was a moderate positive correlation between the relative expression level of MMP-8 protein in the cornea and the inflammation scores of the modeling eye ( rs=0.50, P<0.05).In the cornea, the p-AMPKα (Thr 172)/AMPKα ratio was higher in 24, 48, and 72-hour post-modeling groups than in 0-hour group, and the differences were statistically significant (all at P<0.05).The p-AMPKα(Thr 172)/AMPKα ratio in the cornea was moderately positively correlated with the relative expression level of MMP-8 protein ( r=0.54, P<0.01).The number of neutrophils in the cornea was significantly higher in 24, 48, and 72-hour post-modeling groups than in 0-hour group, with statistically significant differences (all at P<0.001).The number of neutrophils in the cornea was strongly positively correlated with the inflammation score ( rs=0.77, P<0.001), and was moderately positively correlated with the relative expression level of MMP-8 protein ( r=0.56, P<0.05).MMP-8 protein expression in the cornea of the modeling eyes showed a high degree of co-localization with neutrophils.The hydroxyproline content in the cornea was (0.52±0.02)μg/mg, (0.51±0.03)μg/mg, and (0.27±0.02)μg/mg in buffer group, normal saline group and MMP-8 group, respectively, with a significant overall difference among them ( F=156.63, P<0.01).The corneal hydroxyproline content was lower in MMP-8 group compared to buffer and normal saline groups, and the differences were statistically significant (all at P<0.05).In the experiment involving the infection of cultured Fusarium spores with human neutrophils, the fluorescence intensity of MMP-8 expression was significantly higher in AICAR-treated group than in negative control group and compound C-treated group, with statistically significant differences (all at P<0.05). Conclusions:The MMP-8 secreted by neutrophils in mice with fungal keratitis can degrade corneal stromal collagen fibers, leading to corneal opacification or perforation.The variations in MMP-8 protein expression levels in human neutrophils may be associated with AMPK activation.

Objective:To investigate the expression of adenosine 5'-monophosphate-activated protein kinase (AMPK) phosphorylation in corneal epithelial cells and the effects of fungus on AMPK phosphorylation and interleukin-6 (IL-6) production in corneal epithelial cells.Methods:The human immortalized corneal epithelial cell line was selected.The safe concentration range of AMPK agonist 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) (100, 300, 500, 1 000 μmol/L) and inhibitor Compound C (10.0, 12.5, 15.0, 17.5, 20.0 μmol/L) on corneal epithelial cells was screened by multi-function real-time unlabeled cell analyzer.Corneal epithelial cells without any treatment were used as the normal control group, and those co-cultured with spores were used as the spore control group.Corneal epithelial cells co-cultured with spores were treated with AICAR and Compound C for 4 hours in the AICAR group and Compound C group, respectively.The expression of phosphorylated AMPK (p-AMPK) and AMPK in corneal epithelial cells was detected by Western blot, and the concentration of IL-6 in the culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA).Results:After treatment with different concentrations of AICAR for different periods, there was no statistical significance in the cell index of corneal epithelial cells (all at P>0.05). The cell index of corneal epithelial cells was increased with 10.0 μmol/L and 12.5 μmol/L Compound C treatment compared with that of the normal control group.The expression levels of p-AMPK were 0.67±0.15, 2.57±0.12, 3.67±0.58 and 1.50±0.50, respectively, in the normal control group, spore control group, AICAR group and Compound C group, showing a statistically significant difference among them ( F=32.820, P<0.001). The expression level of p-AMPK was significantly higher in the spore control group compared with the normal control group ( P<0.001). The expression level of p-AMPK in the AICAR group was higher than that in the spore control group, and the expression level of p-AMPK in the Compound C group was lower than that in the spore control group, and the differences were statistically significant (both at P=0.010). There was no significant difference in the relative expression level of AMPK among the four groups ( F=0.120, P=0.950). The expression levels of IL-6 concentration in the normal control group, spore control group, AICAR group and Compound C group were (107.81±17.15), (156.32±9.94), (167.96±14.16) and (127.42±19.75)pg/ml, respectively, showing a statistically significant difference among them ( F=15.210, P<0.001). The IL-6 concentration of the spore control group was higher than that of the normal control group, and the difference was statistically significant ( P<0.001). The IL-6 concentration of the AICAR group was higher than that of the spore control group, but the difference was not statistically significant ( P=0.260). The IL-6 concentration of the Compound C group was lower than that of the spore control group, and the difference was statistically significant ( P=0.010). Conclusions:In corneal epithelial cells, AMPK phosphorylation is found, which is enhanced after fungal spores stimulation, and the secretion of IL-6 increases.

Purpose To determine whether Activated 5'AMP-activated protein kinase (AMPK) activation enhances Fusarium solani (F. solani) fungicidal capacity of neutrophils.Methods The expression of AMPK and phosphorylated-AMPK (p-AMPK) proteins was tested using Western Blot. Plate counting studied the effects of the fungicidal capacity of neutrophils enhanced by AMPK activation. Phagocytized spores by neutrophils were assessed by immunostaining and inhibited hyphal growth images were captured by JULI Stage real-time cell history recorder. Flow cytometry assay tested Reactive Oxygen Species (ROS) production and the percentage of apoptosis neutrophils. ROS Assay Kit also tested ROS production at different time points. The F. solani keratitis murine model was established, and slit-lamp microscopy captured corneal photographs.Results Our experiments were divided into the following groups. Neutrophils (N), neutrophils + spores (N + S), neutrophils + spores+ 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) (N + S + A), neutrophils + spores + Compound C (N + S + C). AMPK activator AICAR significantly increased the expression of p-AMPK in neutrophils. The plate counting experiment showed that the number of colonies in the N + S + A was significantly less than in the N + S group. Immunostaining results showed phagocytized spores were significantly increased in the N + S + A group compared with the N + S group. Captured photographs by a real-time cell history recorder camera showed F. solani hyphal growth in the N + S + A group was significantly inhibited than in the N + S group. ROS release in the N + S + A group was significantly higher in the N + S + A group than in other groups. The percentage of apoptosis neutrophils in the N + S + A group was decreased than in the N + S group. Captured photographs by slit-lamp showed that AICAR eye drop treatment alleviated the severity and decreased clinical score at 12 and 24 hours post-infection (h.p.i)Conclusion AMPK activation enhances the efficacy of neutrophils in killing F. solani in vitro and in vivo.

近视是一种常见的屈光不正.大多数人认为其只是简单的屈光不正,可以通过佩戴眼镜或者屈光手术进行矫正.事实上,近视是一种眼部疾病,其特征是眼轴异常伸长,并且无法通过光学镜片和屈光手术治愈.近视的并发症较多,严重的甚至会导致失明.近期的一项荟萃分析预估,2050年全球近一半人口将患近视,其中10％将会发展成为高度近视 [1].随着近视的患病率不断增加,已经成为公共卫生领域的一个重要问题 [2].尤其是学龄期儿童,发病越早越容易进展为高度近视 [3].近视对公众健康有很大影响,因近视致盲的患者会给家庭和社会带来沉重负担,而且失明对患者心理影响巨大.因此制定干预措施,预防近视以及控制近视进展、防止近视患者发展成为高度近视很有必要.现对近视的诊断标准、发病原因、预防及治疗方法等进行综述.

角膜瘢痕是各种角膜损伤疾病后期形成的组织纤维化,可引起视功能下降,是世界范围内致盲的主要原因之一,目前对其发生发展机制的认识仍有限.已有研究证明,白细胞介素-11(IL-11)与纤维化疾病密切相关.IL-11在多个系统的生理过程中发挥着多功能的作用.近年一些研究表明,抑制IL-11可以减轻器官的组织纤维化,这为我们治疗角膜纤维化提供新的思路.本文主要就IL-11在纤维化疾病中的作用机制进行综述,并在此基础上对IL-11在角膜瘢痕的应用前景进行展望.

PURPOSE:To evaluate the effects of deep anterior lamellar keratoplasty (DALK) with cross-linked acellular porcine corneal stroma (APCS) and post-operative topical tacrolimus treatment in patients with fungal keratitis. METHODS:This multicenter prospective study involved 25 cases of fungal keratitis that were treated by DALK with cross-linked APCSs and post-operative topical tacrolimus from December 2013 to November 2014 at the Wenzhou Eye Hospital and the Henan provincial Eye Hospital. Signs of post-operative inflammation, corneal reepithelialization, corneal neovascularization, and graft rejection were assessed, and best corrected visual acuity (BCVA), intraocular pressure (IOP), and APCS graft transparency were monitored for the 12-month follow-up period. RESULTS:All 25 patients underwent DALK without Descemet's membrane perforation. Corneal epithelium recovered completely in 17 patients in the first week, and APCS grafts maintained transparency in 18 patients at 1-year follow-up. The mean BCVA significantly improved from 2.16 ± 0.32 (LogMAR) at baseline to 1.56 ± 0.70 at 1-week (P < .001), 0.95 ± 0.57 at 1-month (P < .001), and 0.70 ± 0.51 at 3-month follow-ups (P < .001). The BCVA kept stable at 6-month and 12-month follow-ups. Post-operative topical tacrolimus alleviated the ciliary injection, except in one case which acute stromal rejection occurred. One patient developed fungal reinfection and underwent penetrating keratoplasty. Graft rejection occurred in three patients. No case was noted with graft splitting, elevated IOP or tacrolimus intolerance. CONCLUSIONS:DALK using cross-linked APCS combining topical tacrolimus treatment is safe and effective in managing fungal keratitis. It may ameliorate the shortage of corneal donation globally.

背景:角膜上皮干细胞定位于角膜缘,又称之为角膜缘干细胞,临床上由于眼表严重热烧伤、化学性烧伤、慢性炎症等原因引起的角膜缘干细胞缺乏或功能障碍治疗较为棘手.目前利用组织工程技术体外培养角膜上皮干细胞并进行临床移植成为新型有效的治疗方向.目的:探讨在无血清培养条件下采用改良组织块培养法培养人角膜上皮干细胞的可行性.方法:人角膜缘组织来自河南省眼库,植片直径小于8 mm角膜移植术后的供者剩余眼球材料,手术显微镜下剖取角膜缘上皮层外2/3区域,采用2种方法培养人角膜上皮干细胞,常规组织块培养组是将组织块上皮面向上贴壁,加入K-SFM培养液后置于37℃、体积分数为5％CO2细胞培养箱中培养;改良组织块培养组是先将组织块浸泡于K-SFM培养液中,置于细胞培养箱中孵育12 h,然后组织块上皮面向下贴壁培养.组织块周边有细胞游离出贴壁生长记作"培养第1天",每日相差显微镜下观察细胞生长变化.利用免疫荧光染色技术检测改良组织块培养第5,10,14天时原代细胞中p63及K3的表达.结果与结论:①改良组织块培养组出膜时间明显短于常规组织块培养组(P<0.05),出膜率明显高于常规组织块培养组(P<0.05);②改良组织块培养组细胞生长状态良好,培养第10天可见小体积细胞较多,聚集成灶状分布;培养第14天可见细胞克隆灶,克隆灶内细胞体积较小,形态均一;③培养第5天,K3表达量较多,p63表达量较少;培养第10天,K3和p63表达量均增多;培养第14天,K3表达量未见明显增多,p63表达量明显增多;④在无血清培养条件下,改良组织块培养法能显著促进角膜上皮干细胞的游离,提高体外培养细胞数量,为人角膜缘上皮组织片的构建提供种子细胞.

目的 探讨mTOR通路抑制剂雷帕霉素对真菌性角膜炎小鼠角膜瘢痕化的影响.方法 取96只SPF级C57BL/6J雄性小鼠,随机分为雷帕霉素组和对照组,每组各48只.两组小鼠同时建立真菌性角膜炎模型.雷帕霉素组模型制作前1 d按6.0 mg·kg-1雷帕霉素对小鼠进行腹腔注射预处理,之后按0.2 g·L-1浓度在结膜下注射5μL,持续3 d;对照组注射PBS溶液.造模后对各组小鼠进行角膜临床评分,Western blot和实时荧光定量PCR分别检测造模后各组小鼠不同时间角膜LC-3Ⅱ、α-SMA和TGF-β1表达情况.结果 造模后24 h、48 h、72 h、96 h、120 h、144 h、336 h,对照组小鼠角膜临床评分均明显高于雷帕霉素组,差异均有统计学意义(均为P<0.05).造模后144 h、216 h雷帕霉素组小鼠角膜LC-3Ⅱ表达上调,LC-3Ⅱ蛋白和mRNA相对表达量与对照组相比,差异均有统计学意义(均为P<0.05);而在造模后72 h及336 h两组LC-3Ⅱ蛋白和mRNA相对表达量差异均无统计学意义(均为P>0.05).与对照组相比,造模后144 h、216 h雷帕霉素组小鼠角膜α-SMA蛋白及mRNA相对表达量下降,差异均有统计学意义(均为P<0.05);造模后144 h、336 h雷帕霉素组TGF-β1 mRNA相对表达量亦下降,与对照组相比,差异均有统计学意义(均为P<0.05).结论 雷帕霉素通过促进自噬作用下调角膜瘢痕化相关因子的表达,减轻了真菌性角膜炎模型小鼠角膜瘢痕化程度.

AIM: To investigate the effect of intestinal fungal dysbiosis induced by antifungal drug on corneal wound healing in mice.METHODS: Male C57BL/6J mice(free of eye disease)were divided into two groups randomly: control(Ctrl)group and amphotericin B treated(Amph)group, The Ctrl group was given a normal diet, and the Amph group was supplemented with amphotericin B to induce intestinal fungal dysbiosis. After 4wk intervention, corneal epithelial trauma was implemented in both groups. Corneal fluorescein staining was used to evaluate the corneal wound area dynamically. Immunofluorescence staining was applied to observe the changes of corneal epithelial cells and inflammatory cells. HE staining was used to assess the change of corneal thickness.RESULTS: Compared with Ctrl group, Amph group had delayed re-epithelialization rate and wound repair, less inflammatory cells and thinner corneal.CONCLUSION: Intestinal fungal dysbiosis delays the corneal wound healing, leading to a weak inflammatory response.

Objective To study the response of neutrophils of fungal keratitis in mice under different light conditions.Methods 93 male SPF C57BL/6 mice aged 6-8w were selected and bred under the following light conditions,LD group mice received light from 7:00 to 19:00 (light intensity 110-210IW/cm2),LL group received 24h light (light intensity 110 -210IW/cm2),DD group received 24h dark, all mice were bred under LD environment for 7 days,the mice were divided into three groups, LD group, LL group and DD group, the mice were fed for 12-15 days under the corresponding light conditions. To make fungal keratitis model, among which 12 mice were only used for clinical observation, respectively 0h before modeling, 12h,18h,24h,36h,48h,72h,120h,168h after modeling, and slit lamp microscope was used for anterior segment photography and clinical scoring.The rest of the 81 mice was performed after the above time point executed based respectively,immunofluorescence staining was performed on the whole cornea,using a single photon laser confocal microscope,the whole corneal film was scanned under 40x oil lens,the infiltration of neutrophils in cornea of mice treated at different time points and under different experimental conditions was calculated by using image processing software. Results The total keratitis score of LD group was higher than that of DD group,at 24 h after modeling, the clinical score of corneal inflammation of LD group was significantly higher than that of DD group (P=0.008).The total keratitis score of LL group was higher than that of DD group,at 18h, 24h, 36h and 168h after modeling ,the clinical scores of corneal inflammation of LL group were significantly higher than those of DD group (P=0.013, 0.004, 0.008, 0.022 < 0.05),at 12 h and 48 h after modeling, the infiltration of neutrophils in the cornea of LD group was significantly higher than that of DD group (P=0.033, 0.009 < 0.05).The infiltration of neutrophils in the cornea of LL group was higher than that of DD group,at 12h, 24h and 48h after modeling, the infiltration of neutrophils in the cornea of LL group was significantly higher than that of DD group (P=0.002, 0.022, 0.001 < 0.05).The infiltration of neutrophils in the three groups was positively correlated with clinical scores (r = 0.192, 0.454, P = 0.019, 0.000).Conclusion In the stage of fungal infection in mice, light helps to increase the recruitment of neutrophils ,more neutrophils infiltrated, leading to more severe inflammation of fungal keratitis in mice.

Bacterial keratitis is an aggressive infectious corneal disease. With the continuing rise in antibiotic resistance and a decline in the discovery of new antibiotics, new antimicrobial drugs are now required. In the present study, we determined the antibacterial activity of diacerein, an anti-inflammatory drug, against 76 Gram-positive cocci isolated from bacterial keratitis patients in vitro and anti-Staphylococcus aureus activity in a mouse bacterial keratitis model in vivo The MICs of diacerein were tested using the broth microdilution method in vitro A BALB/c Staphylococcus aureus keratitis animal model was selected and the corneal clinical observation, viable bacteria, and hematoxylin-eosin and Gram staining of infected corneas were measured to evaluate the antibacterial efficacy of diacerein eye drops in vivo An in vivo eye irritation study was carried out by a modified Draize test in rabbits. Our in vitro results showed that diacerein possesses satisfactory antibacterial activity against the majority of Gram-positive cocci (60/76), including all 57 tested Staphylococcus spp. and 3 Enterococcus spp. The in vivo experiment showed that diacerein eye drops reduced bacterial load and improved ocular clinical scores after topical administration of diacerein drops on infected corneas. The ocular irritation test revealed that diacerein eye drop had excellent ocular tolerance. These results indicated that diacerein possesses in vivo anti-Staphylococcus aureus activity. We suggest that diacerein is a possible topically administered drug for Staphylococcus aureus-infected patients, especially those with ocular surface inflammatory disorders.

目的 探讨真菌性角膜溃疡患者采用不同角膜移植术治疗的效果及预后情况.方法 回顾性分析54例(54眼)真菌性角膜溃疡患者的临床资料,全部患者均接受角膜移植术治疗,总结治疗效果,同时开展5～20个月时间随访,对预后情况进行记录.结果 本组患者在接受治疗后眼球均得到保全,其中50例患者接受手术治疗后患眼视力得以提升,4例患者患眼视力无提升;本组患者接受一次角膜移植术治疗后病情均得以治疗,治愈率为100.00％;本组患者术后6例患者出现真菌性角膜炎复发,6例患者出现继发性青光眼,22例患者的角膜移植片出现排斥反应,11例患者出现并发性白内障;45例患者植片透明,7例患者植片半透明,2例患者植片浑浊.结论 临床上对真菌性角膜炎溃疡开展治疗时,角膜移植效果显著,但手术治疗后需加强并发症监测,使角膜移植术治疗成功率得以提升.

目的 探究高糖饮食对小鼠真菌性角膜炎的影响.方法 选取健康无眼疾的雄性C57BL/6J小鼠78只,随机分为高糖饮食组和模型对照组,每组36只,模型对照组给予正常饮用水,高糖饮食组给予含体积分数10％果糖溶液,每2d测量两组小鼠体质量及血糖,10 d后建立真菌性角膜炎模型.造模后24h、36 h、48 h、72 h、96 h、120 h、168 h裂隙灯显微镜下对角膜进行临床评分并拍照.处死小鼠后,取角膜组织进行HE染色和PAS染色;测定角膜内中性粒细胞和巨噬细胞浸润体积.利用酶联免疫吸附实验对小鼠角膜内的白细胞介素-1β含量进行测定.结果 造模后0～14 d,两组小鼠体质量与血糖差异均无统计学意义(均为P＞0.05).造模后24h、36 h、48 h、120 h、168 h,高糖饮食组小鼠角膜临床评分均明显高于模型对照组,差异均有统计学意义(均为P＜0.05).高糖饮食组小鼠角膜穿孔率79.5％,高于模型对照组的40.9％,差异有统计学意义(P =0.000).造模后各时间点,高糖饮食组中性粒细胞浸润体积均高于模型对照组,差异均有统计学意义(均为P =0.000).造模后72 h、96h、120 h、168 h,高糖饮食组巨噬细胞浸润体积均高于模型对照组(均为P=0.000).角膜组织病理学检查结果示,高糖饮食组炎症反应更重,角膜组织破坏更早且更为严重.造模后24h、48h高糖饮食组白细胞介素-1β含量均明显高于模型对照组(均为P＜0.05).结论 高糖饮食加重了真菌性角膜炎感染的严重程度,增强了中性粒细胞、巨噬细胞的趋化,促进了IL-1β的分泌.

Bacterial keratitis is an aggressive infectious corneal disease. With the continuing rise in 14 antibiotic resistance and a decline in the discovery of new antibiotics, new antimicrobial 15 drugs are now required. In the present study, we determined the antibacterial activity of 16 diacerein, an anti-inflammatory drug, against 76 Gram-positive cocci isolated from bacterial 17 keratitis patients in vitro and anti- Staphylococcus aureus activity in mouse bacterial keratitis 18 model in vivo. The minimum inhibitory concentrations (MICs) of diacerein were tested using 19 the broth microdilution method in vitro. A BALB/c Staphylococcus aureus keratitis animal 20 model was selected and the corneal clinical observation, viable bacteria and 21 Hematoxylin-eosin and Gram staining of infected corneas were measured to evaluate 22 antibacterial efficacy of diacerein eye drops in vivo. An in vivo eye irritation study was 23 carried out by a modified Draize test in rabbits. Our in vitro results showed that diacerein 24 possesses satisfactory antibacterial activity against the majority of Gram-positive cocci 25 (60/76), including all 57 tested Staphylococcus and 3 Enterococcus . The in vivo experiment 26 showed that diacerein eye drops reduced bacterial load and improved ocular clinical scores 27 after topical administration of diacerein drops on infected corneas. The ocular irritation test 28 revealed that diacerein eye drop had excellent ocular tolerance. These results indicated that 29 diacerein possesses in vivo anti- Staphylococcus aureus activity. We suggest that diacerein is a 30 possible topically administered drug for Staphylococcus aureus -infected patients, especially 31 those with ocular surface inflammatory disorders. The data show that diacerein and rhein is more susceptible to Staphylococcus antibacterial activity against Gram-positive cocci. We determined the antibacterial activity of diacerein against 76 Gram-positive cocci isolates from bacterial keratitis patients in vitro and anti- Staphylococcus aureus activity in mouse bacterial keratitis model in vivo. Our results showed that possessed effective antibacterial activity against Staphylococcus and Enterococcus in vitro and reduced bacterial load and improves ocular clinical scores in a murine Staphylococcus aureus keratitis model in vivo.The literature reports that diacerein is entirely converted into before our results that diacerein transfers into rhein quickly in the cornea In the in vitro study, we tested the antibacterial activity of diacerein and its metabolite rhein against all strains of Gram-positive cocci isolated from bacterial keratitis patients between our Our results showed that diacerein and rhein possesses the same satisfactory in vitro antibacterial activity against the majority of Gram-positive cocci (60/76), including all 57 tested Staphylococcus and 3 Enterococcus . 57 Staphylococcus 38 , 7 , 6 , 2 , 2 , 1 and . MIC results applied model to evaluate theantibacterial efficacy of three 291 dose of diacerein eye drops in vivo. The results showed that diacerein reduced bacterial load 292 and improved ocular clinical scores after topical administration of diacerein drops on infected 293

Objective To compare the removal efficiency of γδT cells between cornea and ear skin and develop an alternative method for dynamic monitoring of γδT cells in mouse cornea in vivo using 2-photon laser scanning microscopy.Methods The γδT cells in mouse ear skin were monitored before and after antibody neutralization,and the mice corneas were excised and stained for counting γδT cells at 6 h,12 h,24 h after antibody neutralization by using 2-photon laser scanning microscopy,followed by comparison of the removal efficiency of γδT cells between the cornea and ear skin.Results The γδT cells in normal mouse cornea were often distributed in the limbal epithelium and superficial stromal layer.The irregular morphology of γδT cells in the epithelial layer was often accompanied by protuberances,while the stromal γδT cells were mostly round or oval and the number of cells was approximately 27 ± 4.After antibody neutralization,the number of γδT cells in the cornea of mice gradually decreased,and the number of cells at 6 h,12 h and 24 h was significantly lower than that of before depletion (P =0.03,0.00,0.00),and the removal efficiencies were 48％,78％,and 96％,respectively.The γδT cells in ear skin of the normal mice were ellipse or stellate with cell processes and they were located in epidermal layer,and the cell number was about 60 ± 9.After antibody neutralization,the number of γδT cells were significantly reduced at 6 h,12 h and 24 h compared with before depletion (P =0.000,0.000,0.000) and the removal efficiency were 43％,72％ and 95％,respectively.Conclusion The number of γδT cells in the cornea and ear skin is gradually decreased after antibody neutralization,and their removal efficiency is consistent with time.Therefore,monitoring the γδT cells in the mouse ear skin is an ideal alternative to dynamically monitoring the changes in the number of γδT cells in the cornea in vivo.

The role of mast cells (MCs) in fungal infection is largely unknown. This study was to explore a protective role and mechanism of MCs in fungal keratitis. Experimental fungal keratitis (FK) mouse model was developed. Mice untreated (UT) or receiving corneal wound without fungal infection (Mock) were used as controls. Large number of connective tissue MCs was found in normal mice. MC activation with degranulation was largely observed, and the percentage of degranulated/total cells was high in FK. Dilated limbal vasculature with increased permeability, as well as largely infiltrated neutrophils with stimulated ICAM-1 protein levels were observed in corneas of FK mice, when compared with Mock and UT mice. Interestingly, pretreatment with cromolyn sodium (Block) significantly blocked MC degranulation, dramatically suppressed vascular dilation and permeability, and markedly reduced neutrophil infiltration with lower ICAM-1 levels in FK mice at 6–24 hours. Furthermore, the Block mice manifested prolonged disease course, increased pathological damage, and vigorous fungus growth, with much higher corneal perforation rate than FK mice at 72 h. These findings reveal a novel phenomenon that MCs play a vital role in protecting cornea against fungal infection through degranulation that promotes neutrophil infiltration via stimulating ICAM-1 production and limbal vascular dilation and permeability.

目的 评价Worth四点法测量主导眼的临床价值.方法 选择178例受试者在屈光不正全矫的基础上均使用Worth四点法和卡洞法测量主导眼,在该方法测得的主导眼前加+0.25DS球镜使该眼处于欠矫状态,再次用两种方法测量主导眼,比较主导眼欠矫前后测量结果,用SPSS对检查数据进行卡方检验分析.结果 屈光全矫状态下用worth四点法和卡洞法测量结果差异有显著性(P＜0.05);在主导眼前加+0.25DS球镜前后用worth四点法测量结果差异有显著性(P＜0.05);主导眼前+0.25DS前后用卡洞法测量结果差异无显著性(P＜0.05).结论 在屈光不正矫正状态下worth四点法检测主导眼的结果与经典卡洞法检测结果有显著差异,卡洞法检测主导眼结果不受屈光矫正状态影响,而worth四点法检测主导眼结果受屈光矫正状态的影响,不适合作为主导眼的检测方法.

Objective To study the therapeutic effect and safety of orthokeratology in the teenagers with high myopia.Methods A retrospective cohort study was carried out.The clinical data of 120 eyes from 60 high myopia patients wearing orthokeratology contact lens for 1 year in Henan Eye Hospital from June 2013 to June 2014 was analyzed,and the data of contemporaneous 118 eyes from 59 matched high myopia patients wearing conventional glasses were used as controls.The initial spherical equivalent (SE) of all the patients was-6.00 to-9.00 D.The maximum correction amplitude of the orthokeratology contact lens was set as 6.00 D based on the Regulation of China Food & Drug Administration.The contact lenses were worn for 1 month firstly and then the conventional glasses were used at daytime and contact lenses were consecutively worn at night for 1 year in the contact lenses group,and only glasses were worn in the conventional glasses group.The uncorrective visual acuity (UCVA),SE,corneal fluorescein staining were examined 1 week,1 month,3 months,6 months and 12 months after wearing lenses.The changes of corneal endothelial cells were examined 6 and 12 months after wearing lenses.Results The UCVA was improved and SE was reduced 1 month and 3,6 and 12 months after wearing lenses in comparison with 1 week after wearing lenses in the contact lenses group (all at P＜0.01);while no significant change was seen in UCVA in the conventional glasses group (all at P＞0.05).Compared with wearing contact lenses,the SE was increased in various time points after wearing glasses in the conventional glasses group (all at P＜0.01).The axial length and SE extended by (0.08±0.12)mm and (0.19±0.21)D in the contact lenses group and those in the conventional glasses group were (0.29±0.14)mm and (0.69±0.27)D,showing significant differences between the two groups (t =10.024,5.691,both at P＜0.01).No significant differences were found in corneal endothelial cell density,percentage of hexagonal cells and coefficient of variation of corneal endothelial cells in various time points after wearing contact lenses (corneal endothelial cell density:Fgroup =0.090,P =0.769;Ftime =0.133,P =0.563.percentage of hexagonal cells:Fgroup =0.071,P=0.836;Ftime =1.091,P =0.203.coefficient of variation:Fgroup =0.107,P =0.734;Ftime =1.948,P =0.156).The incidence of corneal fluorescein staining was 31.67％ in the contact lens group,which was much higher than 3.51％ in the conventional glasses group (x2 =97.910,P =0.001).Conclusions Compared with wearing only conventional glasses,orthokeratology can improve UCVA and control axial length extension in juvenile with high myopia,but orthokeratology is more likely to damage the corneal superficial layer.