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AIM: To compare fungal culture and in vivo confocal microscopy ( IVCM ) in the diagnosis of non- primary fungal keratitis.?METHODS:The clinical data of 31 cases (31 eyes) with non- primary fungaI keratitis from September 2016 to February 2017 in our HospitaI were retrospectiveIy reviewed. The positive rate of the two methods was compared by chi-square test.?RESULTS: The positive rate by fungal culture was 58％(18/31 ) and IVCM was 19％ ( 6/31 ); the positive rate comparison difference was statistically significant between fungal culture and IVCM (x2=7. 56,P<0. 01). In the 13 eyes with positive culture results, 2 eyes were positive by IVCM;in the 25 positive IVCM eyes, 14 eyes were positive in culture.?CONCLUSION: The positive rate of fungal culture in non-primary fungal keratitis is higher than that of IVCM. Fungal culture is an essential auxiliary examination in the diagnosis of non - primary fungal keratitis. With the characteristics of fast, noninvasive and repeatable, IVCM also plays an important role in the diagnosis of non-primary fungal keratitis. The combination of the two methods can improve the positive rate of diagnosis.
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AIM: To observe the efficacy of surgical excision combined with autologous limbus conjunctival flap transplantation in the treatment of pterygium accompanied with conjunctival cyst.METHODS: Totally 126 patients 188 eyes with pterygium were hospitalized in Department of Ophthalmology of Tongji Hospital of Huazhong University of Science and Technology during August 2013 and August 2015.The patients were divided into two groups: observation group (11 eyes of 11 patients) with pterygium accompanied with conjunctival cyst and control group (177 eyes of 115 patients) with primary pterygium.All patients underwent slit lamp microscope examination, anterior segment photography, and anterior segment optical coherence tomography(OCT).The size of pterygium was calculated by multiplying neck width and length of the covered corneal.All patients underwent excision combined with autologous conjunctival flap transplantation, and the resections were performed pathological section with hematoxylin and eosin staining.All patients were followed up postoperatively for 4-28mo.RESULTS: All cases in the observation group were confirmed by postoperative pathological examination.All cyst walls were complete, and containing single layer of epithelial cells.The mean size of pterygium of the observation group was 6.9±1.7mm2, and 6.3±1.8mm2 for the control group.There was no significant difference between the two groups (P>0.05).The mean postoperative healing time of observation group was 2.1±0.9d, and 1.9±0.8d for the control group.There was no significant difference between the two groups (P>0.05).Recurrence was seen in two eyes within the follow-up period in the control group, and no recurrence in the observation group.CONCLUSION: Surgical excision combined with autologous limbus conjunctival flap transplantation is a safe and effective treatment for pterygium accompanied with conjunctival cyst.
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Abstract?AIM:To investigate the effective treatment methods of corneal injury caused by chestnut thorns and the factors affecting the disease progression.?METHODS: From Jul.2014 to Oct.2015, the clinical data of 15 patients(15 eyes) with corneal injury caused by chestnut thorns in Ophthalmology Inpatient Department of Wuhan Tongji Hospital was retrospective analyzed. The patients without fungal keratitis were treated with the surgery of removing chestnut thorn from cornea and antifungal drugs. For the patients complicated with fungal keratitis, besides surgery of removing chestnut thorn and antifungal drugs, anterior chamber irrigation and corneal stroma injection with fluconazole solution were given to treat the disease.If necessary, amniotic membrane transplantation or keratoplasty was also given to the patients complicated with fungal keratitis. After that, the effectiveness of those methods and the factors affecting progression were analyzed.?RESULTS:For 11 patients without fungal keratitis, the average time between corneal injury and receiving treatment at Tongji Hospital was 1-7 (2.42±2.15) d and for 4 patients complicated with fungal keratitis, the average time was 3-30 (18.25±4.35)d.Among 15 cases, statistics suggested that the average number of chestnut thorn in patients complicated with fungal keratitis was 4.5, and all the chestnut thorn penetrated the cornea into the anterior chamber.The average number of chestnut thorn in patients without fungal keratitis was 3.5, and the proportion of chestnut thorn penetrated the cornea into the anterior chamber was 28.5%.After treatment, all patients had no new fungal keratitis or other complications.Those results indicated that the different treatments for the patients with or without fungal keratitis were all effective.?CONCLUSION:The factors affecting the progression of cornea foreign body injury caused by chestnut thorn are the number of chestnut thorn, whether chestnut thorn penetrate the cornea into the anterior chamber, time since injury, active anti -fungal therapy. If patients complicated with fungal keratitis could be treated with antifungal agents and anterior chamber irrigation or corneal stroma injection using fluconazole solution without delay, the progress of fungal keratitis could be effectively controlled, and favorable conditions for further therapy such as amniotic membrane transplantation or keratoplasty could be provided.
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To test the safety of using cardiac death donation ( DCD ) corneas for penetrating keratoplasty surgery graft.METHODS:ln chronological order, suing DCD corneas penetrating keratoplasty, corneal endothelial cell density and best corrected visual acuity ( BCVA) were tested 3~4mo after surgery.RESULTS:A total of 14 cases of DCD while 26 corneas were included in this study. Donors age ranged 0. 5 ~61 years, averagely 38. 3 ± 15. 6 years. Causes of death included that 9 cases of traumatic brain injury, 2 cases myocardial infarction, 2 cases brain stem hemorrhage, 1 case of respiratory and circulatory failure. All 26 patients underwent penetrating keratoplasty, no rejection occurred and all grafts were transparent 3 ~ 4mo after surgery. Three to four months after surgery, corneal endothelial cell density ranged 794 ~ 4 347/mm2 , averaged 2 305 ± 827/mm2 , within which was only one case was lower than 1000/mm2 (3. 8%), while 9 cases ranged from 1000 ~ 2000/mm2 (34. 6%), 16 cases were higher than 2000/mm2 (61. 5%). The age of all the 26 receipts were from 20~80 years, mean 40. 7±17. 1 years. BCVA before surgery was light perception positive to 0. 08, with an average 0. 027±0. 024. Three to four months after surgery, BCVA were 0. 2~0. 8, with an average 0. 52± 0. 182 in contrast (t=3. 96, P<0. 001).CONCLUSlON:DCD donated corneas could be used for penetrating keratoplasty graft with high security.
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Background Whether ocular anterior and posterior chamber exist a blood-aqueous barrier is in controversy.Conventional method can not offer a good evidence because it is unable to detect the aqueous component in the posterior chamber.Objective This study was to investigate the distribution of Gadolinium-diethylene triamine pentaacetic acids(Gd-DTPA)after peripheral iridectomy with magnetic resonance imaging(MRI)in rabbit.Methods Monocular peripheral iridectomy was performed on the right eyes in 8 clean New Zealand white rabbits and the fellow eyes were as controls.0.2 ml/kg(0.5 mol/L)Gd-DTPA,a tracer of MRI,was injected into ear vein in vivo to scan the eyes with MRI for the observation of the permeability and distribution.The signal enhanced ratio of interest region associated with time were analyzed.Results The signal in ciliary body of both eyes showed an immediately sharp enhancement within 10 minutes following the injection of Gd-DTPA with a peak intensity at 30-40 minutes,and then the intensity was gradually weaken over time.The signal was stronger in the operative eyes than that in the fellow eyes.The signal in the posterior chamber was gradually increased after operation,however,that in posterior chamber of the control eyes was lower.The interest regions of Gd-DTPA were ciliary,anterior chamber and posterior chamber,and the enhanced signal intensities were consisted in the posterior chamber after operation.However,the increase of the signal was not seen in the posterior chamber in the control eyes.Conclusions The pathway of plasma protein entering into the anterior chamber is very different from that of aqueous secretion.There exists a barrier between the anterior and posterior chamber which might be an integral part of the blood-ocular barrier.
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Background The in vitro culture of retinal vascular endothelial cells is the foundation of experimental study of retinal vascular disease. Shortage of human donor eyeballs is a main limiting for the laboratory work. The culture method of rat-derived vascular endothelial cells has been established. However, this method is not enough effective because of severer cellullar injury. Objective Present study was to establish a simple and high effective method for the culture of vascular endothelial cells in vitro. Methods The retinas from 5 SPF SD rats was digested by 0. 1% collagenase and cultured with explant culture method. 20% fetal bovine serum, vascular endothelial growth factor ( VEGF) , insulin-transferrin-selenium( ITS) were composed into the endothelial cell culture medium, and enough blowing was performed to get the cells and fragments from retinal tissue. The cellular suspension was prepared and cultured consequently on human fibronectin-coated culture flasks. Cultured vascular endothelial cells were identified by anti-von Willebrand staining factor. Results The cells emerged from the tissue mass,and cells and some tissue fragments attached to the wall after 24 hours of seeding. The cells grew to show the fusiform in 4 days and merged together in 5 to 6 days,and a cell monolayer was seen in the 14th day after culture. The endothelial cells showed the positive response for von Willebrand factor. After passage, the merging-growth statue of the cells was regained in 2 hours after culture. Conclusion Use of retinal pieces and collagenase-digestion can get the vascular endothelial cells with better activity in vitro. The culture method based on highly selective endothelial cell culture medium associated to FN adhesion-promoting is helpful for gaining the purified of endothelial cells.
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<p><b>BACKGROUND</b>Delta-like 4 (DLL4) is an endothelium specific Notch ligand and has been shown to function as a regulating factor during physiological and pathological angiogenesis. It has been reported that the DLL4-Notch signaling pathway is regulated by hypoxia and may prevent excessive angiogenesis through the inhibition of angiogenic branching and by triggering vessel maturation. Choroidal neovascularization (CNV) is a pathological form of angiogenesis in which hypoxia is thought to play an important role. This study was aimed to evaluate the role of DLL4 in the development of CNV.</p><p><b>METHODS</b>We utilized chemical hypoxia induced by 200 µmol/L CoCl2 to observe the expression of DLL4 in choroid-retinal endothelial cells (RF/6A cells), which are the primary cells involved in CNV. After transfection of a DLL4 small interfering RNA (siRNA), mRNA and protein expression of DLL4 and key downstream genes, including HES1 and HEY1, in hypoxic RF/6A cells were investigated by RT-PCR, real-time PCR, and Western blotting analysis. Three controls were used: one without transfection, one with transfection reagent, and one with scrambled negative control siRNA. The effects of the DLL4 siRNA on the biological function of hypoxic RF/6A cells during angiogenesis, including cell proliferation, migration and tube formation, were investigated.</p><p><b>RESULTS</b>The results showed that hypoxic conditions led to upregulation of DLL4 expression in RF/6A cells in vitro. After transfection, siRNA-duplex1 targeting DLL4 depleted the DLL4 mRNA levels by as much as 91.4% compared with the scrambled siRNA control, and DLL4 protein expression was similarly effected. There was no significant difference in DLL4 expression among the blank control, transfection reagent control, and scrambled siRNA groups. In addition, after transfection of hypoxic RF/6A cells with the DLL4 siRNA-duplex1, the mRNA levels of HES1 and HEY1, which function downstream of DLL4-Notch signaling, were lowered by 75.1% and 86.3%, respectively, compared with the scrambled siRNA control. Furthermore, knockdown of DLL4 expression significantly promoted the proliferation of hypoxic RF/6A cells and led to their arrest in the S phase of the cell cycle. Migration and tube formation of hypoxic RF/6A cells were significantly induced by the DLL4 siRNA, with the number of migrated cells increased by 1.6-fold and total tube length increased by 82.3%, compared with the scrambled siRNA (P < 0.05).</p><p><b>CONCLUSIONS</b>DLL4 functions as a negative regulator of angiogenic branching and sprouting. Based on our results, DLL4 signaling appears to play an essential role in the biological behavior of choroid vascular endothelial cells under hypoxia. Therefore, DLL4 may represent a novel target for CNV therapy in the future.</p>
Subject(s)
Humans , Basic Helix-Loop-Helix Transcription Factors , Genetics , Metabolism , Blotting, Western , Cell Cycle , Genetics , Physiology , Cell Cycle Proteins , Genetics , Metabolism , Cell Hypoxia , Genetics , Physiology , Cell Line , Cell Movement , Genetics , Physiology , Cell Proliferation , Choroidal Neovascularization , Endothelial Cells , Cell Biology , Metabolism , Homeodomain Proteins , Genetics , Metabolism , Intercellular Signaling Peptides and Proteins , Genetics , Metabolism , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factor HES-1ABSTRACT
AIM: To observe the effect of endothelin-1(ET-1) on the cytoskeleton protein F-actin of cultured human trabecular meshwork (HTM) cells. METHODS: Cultured HTM cells were randomly divided into four groups: control group(0mol/L), low-dose ET-1(10-9mol/L) treatment group, middle-dose ET-1(10-8 mol/L) treatment group, and high-dose ET-1(10-7 mol/L) treatment group. After treated with ET-1, the expression of cytoskeleton protein F-actin in trabecular meshwork was analyzed with Western-blot and the distribution of F-actin was detected with FITC-Phalloidin probe. RESULTS: ET-1 dose-dependently and significantly increased F-actin in trabecular meshwork cells. The F-actin stress fiber and periphery actin fiber highly increased and manifested mild reorganization after treated with ET-1; and there were much more cell-to-cell and cell-to-extracellular matrix attachments formation in ET-1 treated HTM cells than that in the untreated HTM cells. CONCLUSION: ET-1 promoted the expression of cytoskeleton protein F-actin and induced the trabecular meshwork actin cytoskeleton reorganization.
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AIM: To investigate the influence of He-Ne laser on connective tissue growth factor (CTGF) expression and collagen formation of fibroblast in filtration site after trabeculectomy in rabbit, and to discuss the mechanism for preventing scar formation with He-Ne laserin vivo.METHODS: The upper nasal limbus area next to the upper rectus muscle in right eyes Received 10 minutes He-Ne laser irradiation (200mW/cm2) every day for three days, the left eyes served as control. Twenty-four hours after the last irradiation, both eyes of the rabbits were took trabeculectomy surgery. The expressions of CTGF in the filtration area were tested on the 7th, 14th and 28th day after surgery and collagen density was tested on the 14th and 28th day after surgery. Each of the time point had 7 rabbits. RESULTS: The expression of CTGF was lower than that of the control group's on the 7th and 14th day after trabeculectomy surgery (P=0.01, P=0.005). When examined on the 14th and 28th day, the collagen density of irradiation group were significantly lower than that of the control group's (P=0.013, P=0.01).CONCLUSION: Pretreating the filtration area with 200mW/cm2 He-Ne laser may be helpful in preventing scar formation after trabeculectomy in rabbit, possibly due to downregulation of the expression of CTGF and collagen synthesis in fibroblasts. He-Ne laser may be developed into a new scar preventing method in filtration surgery.
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AIM:To observe the effects on human keratocytes by cationic liposome LipofectamineTM 2000(LF2000).to investigate the efficiency and safe range applied in human keratocytes,and establish basis for gene therapy of human keratocytes.METHODS: Human keratocytes cultured in vivo within 3 to 5 passages were used in experiment after being identified.The effects on proliferation of cultured human keratocytes by LF2000 with different concentrations and time were evaluated By MTT:the effects of LF2000 on the survival rate and its relation with 5,10,20,40.80mg/L concentration and time were detected by trypan blue staining.related with concentration and time.The cellular proliferation and survival rate declined when concentration of LF2000 was above certain level,and this effect increased as time became longer.LF2000 had no effect with concentration under 40mg/L for 24 hours. CONCLUSION:LF2000 did ont cause cytotoxicity during a concentration range"tested",and it is hoped to play an important role in gene therapy of human keratocytes.