角膜胶原交联方法联合那他霉素抗真菌效果研究

目的:研究角膜胶原交联方法联合那他霉素应用时的体内外抗真菌效果,为临床真菌性角膜炎的治疗提供治疗方法及实验基础。方法:采用黄曲霉菌、茄病镰刀菌、白色念珠菌三种常见致病真菌,实验组分为交联联合那他霉素组、那他霉素联合核黄素组、那他霉素联合紫外照射组、交联组,并用那他霉素组作为对照。将药物涂到有1. 5麦氏浊度的真菌孢子悬液的沙堡弱琼脂培养皿的中心,10min后使用胶原交联仪照射10min,之后28℃培养36h,测量抑菌圈并进行统计学分析。制备兔眼茄病镰刀菌感染模型,按随机数字表法将模型兔随机分为模型对照组、交联治疗组、那他霉素治疗组、交联联合那他霉素治疗组,每组5只;另取5只正常兔按照角膜胶原交联疗法进行照射,5只正常对照。通过前节照相、角膜刮片、共焦显微镜等观察各组治疗结果,并在治疗结束后采用电镜观察角膜超微结构改变情况。结果:体外单独应用角膜胶原交联术,对三种真菌均无效;当交联与那他霉素联合应用时,抗真菌效果均优于对照组,且差异具有统计学意义(P<0. 05);那他霉素与核黄素、那他霉素与紫外线分别联合应用时,抗菌效果与对照组比较,差异均无统计学意义(P>0. 05)。针对兔真菌感染模型,那他霉素治疗组及交联联合那他霉素治疗组病程约14d,交联治疗组病程约21d;经治疗后,各治疗组均已愈合,各组角膜上皮均无缺损,新生血管较多,角膜内无菌丝;前节照相结果显示,交联联合那他霉素治疗组治疗结果优于其它各治疗组,瘢痕组织较少,角膜愈合较好,病程相对短。结论:角膜胶原交联方法联合那他霉素,可促进角膜愈合,缩短病程,增强抗真菌效果,有望为眼科临床真菌性角膜炎的治疗提供一种新的治疗技术。

结膜印迹细胞学检查在过敏性结膜炎诊断中的价值

目的:探讨结膜印迹细胞学(CIC)检查在过敏性结膜炎(AC)诊断中的价值。方法:采用病例对照研究。将2021-01/02于我科就诊临床诊断为AC的患者40例80眼纳入研究,正常对照组40例80眼,均行双眼上睑CIC检查,观察各组结膜嗜酸性粒细胞的形态和数量,并进行Tseng分级。比较两组结果的差异,分析AC组患者中性别、年龄、病程对CIC检查嗜酸性粒细胞结果的影响。结果:AC患者上睑CIC检查嗜酸性粒细胞阳性率明显高于正常对照组(χ^(2)=57.744,P<0.001);其Tseng分级等级亦高于正常对照组(Z=-2.453,P<0.05)。性别、年龄、病程对AC组患者CIC检查嗜酸性粒细胞结果没有显著影响(均P>0.05)。结论:CIC检查简单易行,结果客观精确,在AC的诊断中具有良好的应用价值。

复方倍他米松对异种角膜板层移植术后免疫排斥反应的影响

目的:观察结膜下注射复方倍他米松对鸵鸟-兔眼板层角膜移植术后免疫排斥反应的缓释治疗作用。方法:16只6周龄健康新西兰白兔角膜(单眼)行板层角膜移植术,植片应用鸵鸟脱细胞的角膜基质,术后分成两组,实验组术毕及术后结膜下注射复方倍他米松注射液(每隔7d一次),对照组术毕及术后结膜下注射地塞米松磷酸钠注射液(每隔7d一次)。分别于术后1、2wk,1、2mo取兔角膜组织做石蜡包埋切片,进行HE染色观察组织特点,同时进行间接免疫荧光法检测CD4^+、CD8^+T淋巴细胞的变化。结果:术后2mo,实验组角膜植片在位,并保持透明,新生血管极少,组织切片HE染色和间接免疫荧光染色结果显示,仅见少许中性粒细胞浸润,未见CD4^+、CD8^+T淋巴细胞;对照组炎症反应明显,角膜植片混浊,组织切片HE染色和间接免疫荧光染色结果显示,炎性反应以中性粒细胞浸润为主,可见CD4^+、CD8^+T淋巴细胞。结论:复方倍他米松作为长效缓释剂可有效抑制鸵鸟-兔板层角膜移植术后免疫排斥反应,延长植片的存活时间。

Acellular ostrich corneal stroma used as scaffold for construction of tissue-engineered cornea

AIM： To assess acellular ostrich corneal matrix used as a scaffold to reconstruct a damaged cornea. METHODS： A hypertonic saline solution combined with a digestion method was used to decellularize the ostrich cornea. The microstructure of the acellular corneal matrix was observed by transmission electron microscopy （TEM） and hematoxylin and eosin （H＆E） staining. The mechanical properties were detected by a rheometer and a tension machine. The acellular corneal matrix was also transplanted into a rabbit cornea and cytokeratin 3 was used to check the immune phenotype, RESULTS： The microstructure and mechanical properties of the ostrich cornea were well preserved after the decellularization process, in vitro, the methyl thiazolyl tetrazoUum results revealed that extracts of the acellular ostrich corneas （AOCs） had no inhibitory effects on the proliferation of the corneal epithelial or endothelial cells or on the keratocytes, The rabbit lamellar keratoplasty showed that the transplanted AOCs were transparent and completely incorporated into the host cornea while corneal turbidity and graft dissolution occurred in the acellular porcine cornea （APC） transplantation, The phenotype of the reconstructed cornea was similar to a normal rabbit cornea with a high expression of cytokeratin 3 in the superficial epithelial cell layer, CONCLUSION： We first used AOCs as scaffolds to reconstruct damaged corneas. Compared with porcine corneas, the anatomical structures of ostrich corneas are closer to those of human corneas. In accordance with the principle that structure determines function, a xenograft lamellar keratoplasty also confirmed that the AOC transplantation generated a superior outcome compared to that of the APC graft.

Corneal collagen cross-linking and liposomal amphotericin B combination therapy for fungal keratitis in rabbits

AIM： To observe the therapeutic effect of corneal collagen cross-linking（CXL） in combination with liposomal amphotericin B in fungal corneal ulcers.METHODS： New Zealand rabbits were induced fungal corneal ulcers by scratching and randomly divided into 3groups, i.e. control, treated with CXL, and combined therapy of CXL with 0.25% liposomal amphotericin B（n =5 each）. The corneal lesions were documented with slit-lamp and confocal microscopy on 3, 7, 14, 21 and 28 d after treatment. The corneas were examined with transmission electron microscopy（TEM） at 4wk.RESULTS： A rabbit corneal ulcer model of Fusarium was successfully established. The corneal epithelium defect areas in the two treatment groups were smaller than that in the control group on 3, 7, 14 and 21d（P 〈0.05）. The corneal epithelium defect areas of the combined group was smaller than that of the CXL group（P 〈0.05） on 7 and 14 d, but there were no statistical differences on 3, 21 and 28 d. The corneal epithelium defects of the two treatment groups have been healed by day 21. The corneal epithelium defects of the control group were healed on 28 d. The diameters of the corneal collagen fiber bundles（42.960 ±7.383 nm in the CXL group and 37.040±4.160 nm in the combined group） were thicker than that of the control group（24.900±1.868 nm）,but there was no difference between the two treatment groups. Some corneal collagen fiber bundles were distorted and with irregular arrangement, a large number of fibroblasts could be seen among them but no inflammatory cells in both treatment groups. CONCLUSION： CXL combined with liposomal amphotericin B have beneficial effects on fungal corneal ulcers. The combined therapy could alleviate corneal inflammattions, accelerate corneal repair, and shorten the course of disease.

Expression of visual system homeobox 1 in human keratoconus

AIM: To investigate the expression of visual system homeobox 1(VSX1) and myofibroblast marker alpha smooth muscle actin(α-SMA) in keratoconus(KC). METHODS: Thirty corneal tissue were collected from KC patients after corneal transplantation and 15 normal donor corneas were obtained. All corneal tissues divided into 4 parts for different detections. Scanning electron microscopy was used to observe the ultrastructure of the specimens. VSX1 and α-SMA localization in cornea tissues was detected using immunofluorescence histochemistry. Reverse transcription-quantitative polymerase chain reaction(RT-qPCR) and Western blot were performed to analyze the expression level of VSX1 and α-SMA. RESULTS: Compared to normal cornea tissue, the collagen fibers in KC stroma were distortional and attenuated and keratocytes were abnormally changed. VSX1 and α-SMA located in the corneal stroma. The mRNA and protein expression level of VSX1 in KC were about 3 times as high as that of normal tissue(P<0.001). α-SMA was hardly expressed in the normal corneas, however, its expression in the KC was about 1.5 times higher than that of the normal corneas(P<0.0001).

Corneal stromal mesenchymal stem cells: reconstructing a bioactive cornea and repairing the corneal limbus and stromal microenvironment

Corneal stroma-derived mesenchymal stem cells(CS-MSCs) are mainly distributed in the anterior part of the corneal stroma near the corneal limbal stem cells(LSCs). CS-MSCs are stem cells with self-renewal and multidirectional differentiation potential. A large amount of data confirmed that CS-MSCs can be induced to differentiate into functional keratocytes in vitro, which is the motive force for maintaining corneal transparency and producing a normal corneal stroma. CS-MSCs are also an important component of the limbal microenvironment. Furthermore, they are of great significance in the reconstruction of ocular surface tissue and tissue engineering for active biocornea construction. In this paper, the localization and biological characteristics of CS-MSCs, the use of CS-MSCs to reconstruct a tissue-engineered active biocornea, and the repair of the limbal and matrix microenvironment by CS-MSCs are reviewed, and their application prospects are discussed.

The pathogenic spectrum of fungal keratitis in northwestern China

I am Na An, from the Shaanxi Key Lab of Ophthalmology, Shaanxi Institute of Ophthalmology,Xi＇an City First Hospital, Xi＇an, Shaanxi Province, China. Fungal keratitis is a severe problem in most developing countries.

Corneal ulcer caused by nocardia brasiliensis in a patient with diabetes

Dear Sir,Iam Dr.Sheng-Li Mi,from the Biomanufacturing engineering laboratory,Graduate School at Shenzhen,Tsinghua University,Shenzhen,China.I write to present a case report of nocardia brasiliensis in a patient with diabetes.Nocardia is part of a group of aerobic actinomycetes,widely distributed in soil.Nocardia asteroids and nocardia brasiliensis are the most common human pathogens.Humans infected by nocardia asteroids through the respiratory tract mainly suffer from primary suppurative pulmonary conditions.Infection of nocardia brasiliensis often occurs in the advanced stages of a progressive disease or immune disorder,especially Cushing syndrome,diabetes,or in patients using corticosteroids,immunosuppressive agents and broad-spectrum antibiotics for long time[1].To our knowledge,the reports of nocardia keratitis was very rare

Corneal ulcer possibly caused by the opportunistic pathogen Schizophyllum commune

Dear Editor,I am Dr.Xian-Ning Liu,from the Institute of Ophthalmology of Xi’an First Hospital in Xi’an,Shaanxi Province,China.I write to present a case report of corneal ulcer caused by Schizophyllum commune(S.commune).Mycotic keratitis is one of the most severe causes of corneal blindness in developing countries,such as China and India[1-2].