Experimental Study on the Effect of Perfluorodecalin on Rabbit Corneas

Purpose: To study the effects of perfluorodecalin on the cornea of the rabbit eyes. Methods: Perfluorodecalin (0.05 ml/each) was injected into the anterior chambers of eighteen rabbit eyes. Corneal morphology and endothelial cells were monitored clinically by slit-lamp biomicroscope and specular microscope for 26 weeks. Animals were sacrificed in 1 st, 2nd, 4th, 10th, 16th, 22nd, and 26th week after injection, respectively, and the corneas were examined under the light microscope. Results: Perfluorodecalin droplets looking like “ fish eggs ” were found at about 1/4-1/2 of the corneal height in the inferior anterior chamber. Corneal opacification on the area contacted with perfluorodecalin was observed in five eyes five weeks after injection, and all in the 22nd week. Mutton fat KPs in one eye were seen in the 6th week firstly, and in all eyes in the 7th week. Corneal pannus formation in one eye was present in the 4th week, two eyes in the 5th week and three eyes in the 6th week. Retrocorneal fibrous membrane in one eye was detected at the 6th week and 3 eyes at the 7th week respectively. After injection of perfluorodecalin, endothelial cell density was sighificantly decreased (2789 ± 192 vs. 2341 ± 658, P < O. O1 ) and corneal thickness was increased. Conclusions: Perfluorodecalin injected into anterior chamber can lead to corneal damage and inflammatory reaction. Eye Sciemce 2001; 17:16 - 20.

Riboflavin/ultraviolet A-induced collagen cross-linking in rabbit corneal scar

AIM: To evaluate the biomechanical stability of the corneal scar treating with riboflavin and ultraviolet A(UVA). METHODS: Totally 86 New Zeal rabbits were divided into control group(group A, n=8) and trauma groups [group B(n=27), group C(n=24) and group D(n=27)]. Then groups B, C and D were divided into three sub-groups according to the time points of sacrifice, i.e. groups Ba, Ca and Da(4 wk, n=8); Bb, Cb and Db(6 wk, n=8); Bc(n=11), Cc(n=8) and Dc(8 wk, n=11). The right corneas of these 78 rabbits in the trauma groups were penetrated. Group B were only sutured. Group C were treated with corneal cross-linking(CXL) immediately after suturing. Group D were treated with CXL seven days after suturing. The corneal scar strips of 4.0×10.0 mm2 were cut and the stress and Young's modulus at 10% strain were evaluated. Samples from the three rabbits of group Bc and three of group Dc were used to measure the expression of alpha smooth muscle action(α-SMA). RESULTS: The mechanical strength of the corneal scar increased with time, and was strongest at 8 wk after the injury. The ultimate stress of corneal scar(group D) were 2.17±0.52 MPa, 2.92±0.63 MPa, and 4.21±0.68 Mpa at 4 wk, 6 wk and 8 wk, respectively; Young's modulus were 10.94±1.57 MPa, 11.16±2.50 MPa, and 13.36±2.10 Mpa, which were higher than that of other groups except for normal control. The expression of α-SMA in group B and group D were 0.28±0.11 and 0.65±0.20, respectively, and the difference was statistically significant(P=0.048). CONCLUSION: CXL with riboflavin/UVA at seven days after suturing improved the biomechanical properties of corneal scars most effectively in the present study.

A HISTOPATHOLOGIC IMMUNOHISTOCHEMICAL AND ULTRASTRUCTURAL STUDY OF RABBIT CORNEA AFTER EXCIMER LASER PHOTOREFRACTIVE KERATECTOMY

Objective To evaluate the corneal healing of non-epuality diopter response histopathologically, immunohistochemically and ultrastructurally after excimer laser photorefractive keratectomy(PRK) with SVS APEX PLUS(Summit Technology Inc. USA) excimer laser, and the effects of corticosteroid on the healing. Methods PRK on 6 white rabbits(12 eyes) was performed on right eye or the rabbit for an attampted correction or -4.00 diopter and on left eye for an attempted correction of -8.00 diopter. The rabbits were divided into 2 groups randomly and each group included 6 eyes: Group FLM (3 rabbits, 6 eyes) and group CM (3 rabbits, 6 eyes). Fluoromethalone was given to group FLM,and chloromycetin to group CM. On 10d, 30d and 100d,the eyes of one rabbit in each group were enucleated randomly. Half of each cornea was prepared for electron microscope observation (SEM and TEM) and the rest embedded in OCT compound for immunohistochemical study to examine Ⅲ-C and FN. Results All eyes were reepithelialized within 3d after PRK. Subepithelial corneal haze was observed on 15d,which was dominant on 30 or 60d. On 100d postoperatively,corneal hazes of 11 eyes were grades 0 or 0.5,only 1 eye(the left eye or group CM) was denser haze (grade 1). On 3d postoperatively, one or two layers of corneal epithelial cell covered the ablation zone. On 30d after PRK,the epithelial cells showed hyperplastic changes. The cells were larger and increased from normal 5 or 6 layers to 7 or 8 layers of cells on l00d after PRK,epithelium was clear with more bright epithelium. Mi- croplicae and microvilli were less than before. The expression of Ⅲ-C and FN in group CM was significantly more evi- dent than that in group FLM. Conclusion The study show that despite recovery of a continuous and smooth epithelial layer and nearly normal corneal tissues on 100d after PRK,abnormalities of both epithelium and superficial stroma can be detected in the area of ablation. The ablation depth of stroma can influence the appearance of corneal haze af- ter PRK. The microplicae and microvilli of rabbit cornea epithelium become less after PRK,which can be one of cause leading to ocular dry sensation in some patients.

Observation on ultrastructure and histopathology of cornea following femtosecond laser-assisted deep lamellar keratoplasty for acute corneal alkaline burns

AIM： To demonstrate the changes in ultrastructure and histopathology of the cornea in acute corneal alkaline burns after femtosecond laser-assisted deep lamellar keratoplasty.·METHODS： The New Zealand white rabbits treated with alkaline corneal burn were randomized into two groups,Group A（16 eyes） with femtosecond laser-assisted deep lamellar keratoplasty 24 h after burn and Group B（16 eyes）without keratoplasty as controls. All eyes were evaluated with transmission electron microscopy（TEM） at 1, 2, 3,and 4wk follow-up, then all corneas were tested by hematoxylin and eosin staining histology.· RESULTS： The corneal grafts in Group A were transparent, while those in Group B showed corneal stromal edema and loosely arranged collagen fibers. One week after treatment, TEM revealed the intercellular desmosomes in the epithelial layers and intact non-dissolving nuclei in Group A. At week 4, the center of the corneas in Group A was transparent with regularly arranged collagen fibers and fibroblasts in the stroma. In Group B, squamous cells were observed on the corneal surface and some epithelial cells were detached.· CONCLUSION： Femtosecond laser-assisted deep lamellar keratoplasty can suppress inflammatory responses, prevent toxic substance-induced injury to the corneal endothelium and inner tissues with quicker recovery and better visual outcomes.

Evaluation of artificial tears on cornea epithelium healing

AIM： To observe the efficacy of different artificial eye drops on corneal epithelium healing in rabbit. METHODS： Thirty-five rabbits with 6 mm diameter central corneal epithelium removed were randomly assigned to six groups： 0.9% normal saline（NS） group, 0.1% hyaluronate（HA） group, 0.3% HA group, Tears Naturale Free？（TNF） group, 0.4% polyethylene glycol（PEG） group, 0.5% carboxymethyl cellulose（CMC） group and blank control group. Treatments were administered topically four times daily. Corneal epithelium healing was evaluated by the percentage reduction in wound area at 24, 36, 48, 60, and 72 h after removal of the corneal epithelium. Cornea re-epithelialization was also assessed by histological analysis and electron microscopy. RESULTS： All corneal wounds completely re-epithelialized in less than 72 h. The average re-epithelialization time was 47.61±4.25 h in the 0.3% HA group and 49.72±1.05 h in the 0.9% NS group, followed by 0.1% HA, TNF, 0.4% PEG, 0.5% CMC, and lastly by the control group. Compared to the control group, there were significant differences among 0.3% HA, 0.9% NS, PEG, and TNF（P〈0.05） groups. At the first 24 h, re-epithelialization at the 0.3% HA, TNF, and 0.9% NS treatment groups were significantly faster than the other groups. At 48 h post-wounding, corneal epithelium is nearly completing re-epithelialization at 0.3% HA and 0.9% NS treatment groups. Electron microscopy revealed that there were a large number of vacuoles in the cells of the 0.9% NS group at 72 h. CONCLUSION： Artificial tears promote corneal reepithelium varied in the efficacy. Obviously, all artificial eye drops better than blank group. In the process of corneal healing, corneal epithelium cells suffered from hypoxia caused by NS.

A Study on HSV-1 Corneal Potential Infection by in Situ Nucleic Acid Hybridization

Purpose:To evaluate the possibility of HSV-1 corneal latency by in situ nucleic acid hybridization in animal models.Methods:20 normal New Zealand White(NEW)rabbits were used,14of them were inoculated bilaterally with 3×10PFU/ml of McKrae strain HSV-1by in-trastromal injection,22/28eyes developed typical herpes simplex keratitis(HSK) diseases.At 60day postoperation(PI),4latent corneas were transplanted to one eye of 4noninfected NZW rabbits and removed2weeks PI,Corneas at all time intervals of infection and two weeks after PKPwere detected for presence of HSV-1antigen and nucleic acid sequences by using clonal IgGHSV-1antibody and biotinylated HSV-1DNAprobe individually.Results:The results showed that the HSV-1DNA sequences were retained with-in the corneal epithelium and anterior stromal keratocytes during acute diseases,while the corneas during latent infection and postoperation,the HSV-1DNAse-quences were retained only within the stromal layer with negative HSV-1antigne staining.Conclusions:These results strongly suggest that the cornea may be capable of harburing latent HSV-1.Eye Science 1995;11:117-119.

Ocular penetration of topically applied 1% tigecycline in a rabbit model

AIM：To evaluate ocular penetration of topically applied 1% tigecycline.METHODS：Forty-two New Zealand White rabbits were divided into 3 groups. A 50 μL drop of 1% tigecycline was administered in group 1. In groups 2 and 3, the drop was administered every 15 min for 60min（keratitis protocol）. Aqueous humor samples in groups 1 and 2 were collected under general anesthesia at 15, 30, 45, 60, 120, and 180 min after the last drop. All animals in group 3 were euthanatized. Cornea, vitreous and blood samples were collected 60 and 120 min after the last drop. Tigecycline concentrations were measured using high performance liquid chromatography-mass spectrometry（LC-MS/MS）.RESULTS：The peak aqueous humor tigecycline concentration [mean 0.73±0.14 mg/L（SD） and 2.41±0.14 mg/L, respectively] occurred 45 min after topical drug application in groups 1 and 2. Group 3 mean values in the cornea, and vitreous, were 3.27±0.50 μg/g, and 0.17±0.10 mg/L at 60 min and 3.17±0.77 μg/g and 0.20±0.07 mg/L at 120 min, respectively. Tigecycline serum concentrations were negligible.CONCLUSION：Tigecycline levels in the aqueous humor in groups 1 and 2, and in the cornea in group 3 exceeded the minimum inhibitory concentrations of most grampositive organisms that cause bacterial keratitis and endophthalmitis.

兔眼角膜着染对眼刺激性试验结果评价的影响

目的观察正常实验兔眼经荧光素钠染色后的着染现象对眼刺激性试验结果评价的影响。方法在进行针对氯化钠滴眼液、四味珍层冰硼滴眼液、玻璃酸钠滴眼液、色甘酸钠滴眼液和复方门冬维甘滴眼液的实验兔眼刺激性试验(每组4只,雌雄各半)中,将给予生理盐水的兔左眼(自身阴性对照)和给予药物制剂的右眼(给药实验)进行1%荧光素钠染色,然后用裂隙灯显微镜连续31 d观察兔眼刺激反应并评分,记录兔角膜上皮着染的形态变化并计算着染发生率。观察结束后对眼球及哈氏腺进行组织病理学检查,并将左眼着染发生率检查结果与给予相应药物的右眼进行统计学对比分析。结果给予生理盐水的左眼和给予药物制剂的右眼均无眼刺激反应,评分均为0,角膜着染总发生率分别为3%、1%,左右眼每天和总着染发生率无明显差异(P>0.05)。角膜上皮着染形态有单点状着染、散在点状着染、局限性着染和大片融合着染的无规律变化,眼球及哈氏腺未见组织病理学改变,结果评价为无刺激性。结论实验兔眼无规律间断出现的角膜上皮着染对眼刺激性试验的结果评价无影响。

羊膜移植抑制兔角膜碱烧伤后新生血管增殖的研究

目的 比较采用保存羊膜和新鲜羊膜移植抑制角膜碱烧伤后新生血管增殖的效果 ;探讨应用保存人羊膜移植防治角膜碱烧伤后新生血管的手术时机。方法 制备角膜碱烧伤后新生血管增殖的动物模型 ;2 2只家兔 ( 2 2眼 )随机分为4组 :A组 ( 4眼 )作为对照组 ;B组 ( 6眼 )在碱烧伤的急性期行新鲜羊膜移植 ;C组 ( 6眼 )在急性期行保存人羊膜移植 ;D组 ( 6眼 )在瘢痕期行保存人羊膜移植。应用计算机彩色图像处理系统测定角膜新生血管面积。结果 3个移植组和对照组比较 ,角膜新生血管面积的差异均有显著性意义 (P <0 0 1) ;B组与C组比较无显著性差异 (P >0 0 5 ) ;C组的新生血管面积明显少于D组 (P <0 0 1)。结论 保存羊膜和新鲜羊膜移植均能有效地抑制角膜碱烧伤后新生血管的增殖 ,治疗效果无显著性差异 ;在角膜碱烧伤的急性期施行羊膜移植防治新生血管增殖的效果要优于在瘢痕期手术。

实验动物兔角膜Orbscan-Ⅱ检测

目的了解兔眼角膜的解剖学特征,为眼的动物实验提供参考数据。方法对46只新西兰白兔的92只眼角膜行Orbscan-Ⅱ检测,并分析各项检查参数。结果兔眼的平均角膜直径为(11.72±0.65)mm;平均角膜中央厚度为(391.91±21.78)μm,角膜最薄点厚度平均为(371.57±22.27)μm;兔眼的平均前房深度为(2.41±0.29)mm;兔眼角膜3mm光学区的平均屈光力为(46.53±2.02)D,5mm光学区的平均屈光力为(46.16±1.88)D;兔眼角膜前后表面高度图按形状可分为中央岛、不完全桥、规则桥、不规则桥及未分类型;兔眼Kappa角为(6.59±3.26)°。结论兔眼的上述测量参数多与人眼接近,但角膜厚度比人眼薄,角膜屈光力大于人眼,可作为眼表疾病和部分角膜屈光手术的实验动物。

兔眼角膜生物力学特性的实验研究

目的;了解兔眼角膜的力学特性,得出以面应变γ与角膜内外压强差p＇表达的本构方程。方法:模拟眼内压,测量了20只兔眼角膜。结果:获得了本构关系γ=γ_0exp(c/p＇)和面积模量E=A+Bp＇。结论:角膜是不易发生较大变形的粘弹性材料。

紫外线光损伤角膜细胞凋亡的实验研究

目的研究不同波长紫外线照射所致兔角膜光损伤的发生机制。方法分别用265nm和300nm紫外线照射8只白色家兔角膜,分别在24h和76h后取角膜进行光镜、透射电镜检查和应用TUNEL技术进行DNA断端标记。结果265nm紫外线照射24h和76h后,TUNEL阳性染色仅见于上皮细胞层,300nm照射24h后,TUNEL阳性染色见于上皮细胞和基质层角膜细胞,透射电镜也证实了典型的凋亡现象,表现为细胞皱缩,染色质浓缩成团块,聚集于核膜,凋亡小体形成。未发现明显的炎性反应。结论细胞凋亡是紫外线照射后角膜细胞死亡的发生机制。300nm比265nm紫外线照射引起更广泛角膜基质损伤。

内皮素-1对培养的兔角膜内皮细胞的影响

目的 研究内皮素-1(ET-1)对培养的兔角膜内皮细胞的增殖能力和移行能力的影响。 方法 采用MTT方法并通过损伤模型观察不同浓度ET-1加药48 h后,对体外培养的兔角膜内皮细胞增殖和细胞移行的能力。光镜下和透射电镜下观察ET-1对兔角膜内皮细胞形态的影响。采用免疫组化染色法和计算机图像分析系统检测不同浓度ET-1对细胞PCNA表达的影响。结果 一定浓度ET-1促进培养的兔角膜内皮细胞的增殖和移行,且呈剂量相关性。10 pmol／L时起作用,200 pmol／L时发挥最大作用。光镜下和透射电镜下发现ET-1对兔角膜内皮细胞的形态学特征和超微结构无影响。兔角膜内皮细胞PCNA表达阳性,ET-1促进其表达,且呈剂量相关性。 结论 ET-1可作为一种生长因子,一定浓度下可促进培养的兔角膜内皮细胞增殖和移行能力。

β-榄香烯抑制胶质瘤兔角膜移植模型血管生成的实验研究

目的复制兔角膜移植瘤血管生成模型,检测β-榄香烯体内抗血管生成效应。方法裸鼠皮下种植人脑胶质瘤细胞系SHG44细胞,形成实体胶质瘤。将所得胶质瘤实体小块移植于兔角膜,复制角膜移植瘤血管生成模型。经兔耳缘静脉注射β-榄香烯,干预两周后应用Ⅷ因子相关抗原免疫组织化学染色及图像分析技术,检测移植胶质瘤组织内微血管密度变化。结果兔角膜移植瘤血管生成模型成功复制;β-榄香烯处理组移植胶质瘤微血管密度明显低于对照组。结论兔角膜血管生成模型是定量研究肿瘤血管生成的良好工具;β-榄香烯具有体内抗肿瘤血管生成作用。

胶原罩的实验性研究

介绍一种胶原罩(collagen shield)制作的新方法。其含水率达62.1%,配戴于兔眼后观察,溶解时间为12小时至24小时,连续配戴30天,取下兔眼角膜进行扫描电镜观察,上皮及内皮的形态结构无异常改变。

兔角膜成纤维细胞培养及其生长抑制的实验研究

目的了解丝裂霉素Ｃ（ＭＭＣ）及透明质酸（ＨＡ）对准分子激光屈光性角膜切削术（ＰＲＫ）术后及正常兔角膜成纤维细胞增殖的影响。方法对兔角膜成纤维细胞体外培养，观察ＭＭＣ及ＨＡ对细胞生长的影响。结果ＭＭＣ及ＨＡ对兔角膜成纤维细胞生长具有明显的抑制作用。结论从细胞生物学角度揭示ＭＭＣ及ＨＡ作为能有效抑制体外培养角膜成纤维细胞增殖的药物，作为ＰＲＫ及其它眼科手术后用药的可能性，以期有助于防止或减少手术并发症。

玻璃酸钠眼前房注入的实验研究

对２４只兔眼前房注入１％玻璃酸钠，并用生理盐水作自身对照，观察了该药对兔眼角膜的保护作用及眼前房反应，并进行了病理切片检查，证明了玻璃酸钠注射液无任何毒性反应，是一种理想的粘弹性物质。

VEGF siRNA对兔眼碱烧伤后角膜新生血管的抑制作用

目的:研究血管内皮生长因子小干扰RNA(VEGF small in-terfering RNA,VEGF siRNA)对兔眼碱烧伤后角膜新生血管的抑制作用。方法:普通家兔25只以碱烧伤法(1mol/LNaOH溶液)诱导角膜新生血管(CNV)生成。碱烧伤后立即以脂质体(LF2000)为载体,右眼球结膜下注射VEGF siRNA重组质粒,左眼球结膜下注射pSilencer 2.1-U6 hygro空白质粒作为阴性对照。碱烧伤后1,3,5,7,14d,形态学分析评价角膜新生血管的生长情况。结果:与对照眼相比,碱烧伤后球结膜下注射VEGF siRNA重组质粒,实验眼在各时间段(3,5,7,14d),CNV长度明显变短,面积明显变小,差异有统计学意义(P<0.05)。结论:VEGF siRNA能有效地抑制碱烧伤后CNV的形成。

层粘连蛋白对兔角膜内皮细胞bcl-2表达的影响

目的:观察层粘连蛋白(LN)对兔角膜内皮细胞bcl-2表达的影响。方法:将体外培养的兔角膜内皮细胞分成3组:正常对照组、层粘连蛋白(LN)处理组、阴性对照组,采用免疫组化法和酶联免疫法分别检测3组的染色深浅及吸光度大小,以显示各组间bcl-2表达的水平。结果:免疫组织化学染色及评分结果显示LN组bcl-2为强阳性表达,正常对照组bcl-2为弱阳性表达,阴性对照组bcl-2为阴性表达。酶联免疫吸附试验检测3组吸光度分别为1.21±0.18(LN组)、1.05±0.14(正常对照组)和0.04±0.01(阴性对照组)。结论:层粘连蛋白能促进兔角膜内皮细胞bcl-2基因表达,从而可以有效地抗细胞凋亡。

兔死后角膜厚度与死亡时间的关系

目的研究兔死后角膜厚度变化规律与死亡时间（postmortem interval,PMI）的关系。方法用空气栓塞法建立兔死亡模型。在兔死后0－72h,每隔6h进行角膜取材,切片后常规HE染色,用光学显微镜摄取全角膜图像,结合Motic Images Plus 2.0图像分析软件观察角膜上皮层厚度（x1）、角膜基质层厚度（x2）、全角膜厚度（x3）3项指标的变化值,并作统计学分析,建立与PMI（y）的回归方程。结果在兔死后72 h内,角膜基质层厚度和全角膜厚度均在死后12h开始增加,并在死后54h达到高峰,两者与PMI正相关,回归方程分别为y=-0.070 2 x22＋11.398 x2＋1 634（R2=0.712 2,P〈0.05）,y=-0.074 9 x32＋12.036 x3＋1 819.4（R2=0.675 0,P〈0.05）。结论角膜基质层厚度和全角膜厚度与PMI之间呈较好的线性关系,且角膜基质层厚度优于全角膜厚度,可作为推断死亡时间的指标。