Background Heparanase degrade heparan sulfate side chains of heparan sulfate proteoglycans in the extracellular matrix.Heparanase induces angiogenesis and likely promotes the vascularization of tumor.ObjectiveThe present study is to investigate the expression of heparanase and perlecan in retinas with oxygen-induced retinopathy.Methods Sixty-five clean neonatal C57BL/6J mice were raised in a hyperbaric oxygen box with a volume percentage of 75%±2% for 5 days and then returned to the normal air room.Another 65 matched mice were raised in the normal environment as controls.Evans blue was infused by the superior vena cava in all the mice on postnatal days 12,13,17,21 and 30,afterwards fluorescein angiography was performed and then the mice were sacrificed.The retinas of mice were isolated and prepared and the retinal vessels were examined under a fluorescent microscope and optical microscope.Heranase and perlecan mRNA was detected using reverse transcription PCR (RT-PCR).Heranase and perlecan proteins were detected by Western blot.The analysis of variance was used to compare the mRNA and the protein levels of heranase and perlecan between the experimental and control groups.Results The expression of heparanase mRNA in the retinas of different ages of mice and the different groups showed significant differences (F_(group)=16.303,P=0.000;F_(time)=18.614,P=0.000;F_(interaction)=11.299,P=0.000),and the expression of heparanase mRNA was significantly enhanced in mice from postnatal days 12,13,17 and 21 compared with normal control mice (P=0.001,0.000,0.000,0.001,respectively).The expression of heparanase protein in the retinas of different ages of mice and the different groups followed the same tendency(F_(group)=458.134,P=0.000;F_(time)=78.466,P=0.000;F_(interaction)=71.398,P=0.000).The expression of perlecan mRNA in the retinas of different ages of mice and the different groups showed significant differences (F_(group)=7.351,P=0.013;F_(time)=9.098,P=0.000;F_(interaction)=3.349,P=0.000),and increase in differences also were clearly seen in mice from postnatal days 13,17 and 21 compared with normal control mice (P=0.048,0.000,0.003,respectively).Conclusion The expression of heparanase and perlecan is associated with the development and progression of retinal neovascularization,and perlecan and heparanase together produce a synergistic effect.Heparanase and perlecan may participate in the angiogenesis of oxygen-induced retinopathy.

Background Branch retinal vein occlusion (BRVO)often leads to macular edema as the result of elevating intravitreal VEGF level,and avastin,a anti-VEGF drug,show a good effectiveness on macular edema secondary to BRVO.ObjectiveThis study attempts to evaluate the clinical efficacy of avastin on macular edema induced by BRVO.MethodsThis is a retrospective case-observation study.The clinical data of 39 eyes from 39 patients with macular edema induced by BRVO were included in this analysis.All of the patients received intravitreal injection of 1.25mg (0.05mL)avastin without other any therapy prior to the injection of avastin.This procedure followed the Declaration of Helsinki,and written informed consent was obtained from all the patients before and initial of any management.Clinical indexes included best-corrected visual acuity(BCVA),slit-lamp examination,intraocular pressure and stereoscopic biomicroscopy examination before injection and 3,6 and 12 weeks after initial injection.Optical coherence tomography (OCT),fundus photography,fluorescein fundus angiography(FFA)were performed prior to injection and 6,12 weeks after initial injection.The follow-up period was 3-20 months.ResultsThe mean BCVA was significantly improved at 3,6,12 weeks after injection in comparison with before injection (t=-6.039,-6.182,-4.189,all P=0.000).The mean CMT showed a statistically significantly decline at 6,12 weeks after injection in comparison with before injection(t=8.684,5.019,all P=0.000).No ocular or systemic adverse events were found after intravitreal injection of avastin during the follow-up duration.The numbers of visual acuity-improved eyes were significantly increased in the patients with disease course ≤1 month duration in comparison to ones with the course ≥1 month (P<0.05)in 3 weeks after injection.CMT was obviously decreased in 12 weeks after injection in comparison to before injection between with and without macular perfusion eyes (P<0.05).ConclusionIntravitreal injection of avastin is safe and effective for macular edema induced by BRVO,especially the patients with shorter course of disease.

Background The study on the classification of fungi is very important for the diagnosis and treatment of fungal keratitis.Identifying the different species of filamentous fungi is a critical factor for the application of anti-fungal drug in treating keratitis.ObjectiveThis report studies the relationship between the genotype of filamentous fungi and the clinical factors.MethodsFifty-two patients with filamentous fungal keratitis determined by clinical and laboratory examination were recruited in Tongren Hospital from January 2006-December 2006.The lesions were graded on the severity of the corneal ulcer and the presence of hypopyon.The filamentous fungal keratitis was treated with topical and systemic administration of anti-fungal drugs or corneal transplantation.The isolates were cultured in potato culture and identified by morphological characteristics based on the Nelson criterion and genotyped by the rDNA ITS method.The clinical data was retrospectively analyzed.ResultsForty-eight species (eubacteria are bacteria,not fungi)of fungus were identified by morphological characteristics,and the filamentous fungi were divided into 4 types based on the phylogenetic relationships within the rDNA ITS of the 52 filamentous fungi.The morphological characteristics and genotype were confirmed in 48 strains of eubacteria and 31 strains of 52 filamentous fungi (90.3%).The 4 groups of fungi were classified by genotype as follows:group 1 represents 22 strains including 20 strains of Fusarium solani and 2 strains of Fusarium oxysporum;group 2 represents 12 strains including 8 strains of Fusarium moniliformis,3 strains of Fusarium proliferatum and 1 strain of Fusarium incarnatum;group 3 represents 5 strains including 1 strain of Fusarium moniliformis and 4 unknown strains;group 4 represents 13 strains including 10 strains of Aspergillus spp.and 3 strains of Alternaria spp.Significant differences were found in the disease duration (P=0.00),inducing cause (P=0.03),ulcer grade (P=0.01)and outcome of the anti-fungal treatment (P=0.035)when compared between group 1 and 2 with group 3 and 4.Conclusion Filamentous fungi that cause keratitis could be correctly identified by sequencing the internal tanscribed spacer of rDNA.There are significant clinical differences among the groups classified by genotype.

Background Human retinal pigment epithelial(RPE) cells play a critical role in the pathogenesis of proliferative vitreoretinopathy(PVR) and other related ocular diseases.Research demonstrated that epidermal growth factor(EGF) stimulates activation of RPE cells and therefore causes PVR,and integrin α_5 mediates the adhesion of cells and EGF.Objective This study aims to investigate the role of mitogen-activated protein kinase(MAPK) in regulation of EGF on integrin α_5 expression in human RPE cells.MethodsHuman RPE cells strain(CRL2302) was cultured in DMEM/F12 with 10% calf serum and passaged in 0.25% trypsin.Cultured cells were divided into DMEM/F12control group,20μmol/L PD98059 +10 ng/mL EGF＋DMEM/F12(PD98059) group and 10 ng/mL EGF＋DMEM/F12(EGF) group.The expression of integrin α_5 protein in CRL2302 cells was detected by RT-PCR and calculated as α_5 mRNA/β-actin mRNA,and the expression of integrin α_5 mRNA in CRL2302 cells was detected evaluated by flow cytometry.The MAPK phosphorylation level in each group of human RPE cells was tested by Western blot.ResultsThe expression value of integrin α_5 mRNA was 0.93±0.06 in the control group,1.06±0.07 in PD98059 group and 1.97±0.09 in EGF group,showing a significant difference among the three groups(F=458.896,P<0.01).The fluorescence intensity of integrin α5 protein in CRL2302 cells after 24 hours was 1.94±0.22,4.56±0.25,2.39± 0.14 in three groups respectively with a significant difference(F=21.05,P<0.05).After 30 minutes of culture,Western blot result showed that the strongest phosphorylation levels of ERK1/2 activation in EGF group and the weakest phosphorylation levels of ERK1/2 activation in the control group;While that in PD98059 group was significantly stronger than control group and weaker than EGF group(F=143.14,P<0.01).ConclusionEGF stimulates activation of ERK1/2 pathway in human RPE cells and the expressions of integrin α_5 mRNA and protein in human RPE cells in vitro.

BackgroundOur previous research and other reports disclosed that the expression of tissue transglutaminase(tTG)in lens epithelial cells(LECs) of patients with cataract is enhanced,indicating tTG is related to formation of posterior capsule opacification(PCO).ObjectivePresent study is to observe the effect of tTG specific inhibitor monodansyl-cadaverineon(MDC) on the expression of fibronectin(FN) and collagen Ⅳ(Col-Ⅳ) induced by TGF-β_2 in human LECs.MethodsHLE-B3 was cultured in vitro in DMEM containing 10% fetal bovine serum and then were divided into 5 groups.The free-serum culture was used as normal control group.Free-serum culture containing 10μg/L TGF-β_2 was utilized as treatment group.10μg/L TGF-β_2 plused 100μmol/L,200μmol/L and 400μmol/L MDC respectively in different concentrations as MDC-treatment group.Semiquantitative RT-PCR was used to assay the expression of tTG,FN and Col-Ⅳ in HLE-B3.A(tTG/β-actin),A(FN/β-actin) and A(Col-Ⅳ/β-actin) was calculated separately as the detecting indexes.ResultstTG,FN and Col-Ⅳ were positively expressed in cultured HLE-B3.The expression levels of tTG,FN and Col-Ⅳ in HLE-B3 were remarkably increased in the group with 10μg/L TGF-β_2 compared with normal control group(t=33.95,P<0.01;t=38.24,P<0.01;t=13.48,P<0.01).The expression levels of FN and Col-Ⅳ were gradually declined in 100,200 and 400μmol/L MDC groups in comparison with TGF-β_2 treatment(P<0.01).The significant differences were also found in the expressions of FN and Col-Ⅳ in HLE-B3 among 100,200 and 400μmol/L MDC groups(P<0.01).ConclusionMDC inhibits the expression of FN and Col-Ⅳ induced by TGF-β_2 in human LECs at a concentration-dependent manner.tTG may be involved in the formation of posterior capsule opacification through up-regulating the expressions of FN and Col-Ⅳ in human LECs.

Background The anti-proliferative effect of matrine has been demonstrated and its relevance to prevention and treatment of proliferative retinovitreopathy is concerned.Howeverthe intravitreous injection of free-matrine reiteratively may raise the risk of ocular infection.ObjectiveThe goal of the present study is to investigate the sustained releasing ability and safety of matrine polyactic acid microsphere(MAT-PLA-MSintravitreal injection.MethodsMAT-PLA-MS was prepared by Hebei Medical University and examined under the transmission electron microscope.The release of MAT-PLA-MS was monitored by ultraviolet spectrophotometry.Free-matrine with the dose of 1,2,4mg was intravitreally injected respectively in 12 eyes of New Zealand albino rabbits in free-matrine group and MAT-PLA-MS with matrine(2,4,6mg respectively was administered in 16 eyes separately in matrine microsphere group.The blank microsphere was injected in 6 right eyes as blank control group and normal saline solution was injected in 6 fellow eyes as control group.The retinal function change was evaluated by electroretinogram(ERG),and the morphological and histological change of retina following drug injection were assessed under the slit lamp biomicroscope,indirect ophthalmoscope,light microscope and transmission electron microscope.The decomposed process of MAT-PLA-MS in vitreous was recorded with ocular anterior segment and fundus color camera.Results MAT-PLA-MS containing matrine showed the spherical shape with the mean diameter of 2.28±47μm under the transmission electron microscope and the drug-loading rate 6.17% and drug-release rate 87.93% in vitro for 672 hours,presenting the controllable release characteristics.After implantation into the vitreous,the MAT-PLA-MS containing matrine decomposed gradually with the prolong of time.The b amplitudes of ERG maximum response were significantly declined in 4mg free-matrine injection group in comparison with before injection in various time points(P<0.01).However,no considerably differences were found in MAT-PLA-MS with matrine groups and control groups in various time points following the intravitreal injection(P>0.05).No obvious abnormal was seen under the slim lamp and ophthalmoscope through the study period.The changes of retinal ultrastructure were found from 1 through 28 days after injection of 4mg free-matrine,and slight retinal structural damage was seen from 7 through 28 days after injection of 6mg MAT-PLA-MS containing matrine.ConclusionThese results suggest that MAT-PLA-MS possesses good sustained release feature.MAT-PLA-MS containing matrine has less toxicity to retina than free-matrine after intravitreal injection.MAT-PLA-MS is an excellent drug delivery system.

Background It has been demonstrated that αvβ3/αvβ5 and α5β1 integrins are overexpressed in neovascular tissue.Consequently,peptide containing the RGD (Arg-Gly-Asp) sequence,which exists in ligands of integrins,is effective in targeting therapeutic reagents to neovascular endothelium.ObjectivePresent study aims to investigate the antiangiogenetive effects of liposome mediated plasmid encoding endostatin with RGD sequence on alkali burn-induced corneal neovascularization (CNV) in rabbits.MethodsCNV models induced by alkali burn were established in 72 eyes of 36 New Zealand white rabbits by putting the filter paper with 1 mol/L NaOH at the central cornea for 20 seconds.The animal models were divided into four groups randomly.0.2mL of liposome and plasmid encoding RGD-ES complex (liposome mediated pCI-RGD-ES injection group),liposome and plasmid encoding ES complex (pCI-ES injection group),liposome and carrier plasmid (pCI) complex (pCI-ES injection group),and PBS were subconjunctivally injected respectively in the models from four groups twice a week for two weeks.The growth status of CNV was observed on day 1,3,7,14 after alkali burn under the slim lamp microscope.Experimental animals were sacrificed on the 3rd,7th and 14th day and the expression of VEGF in CNV was detected by immunohistochemistry.The number of corneal microvessels was counted based on the number of CNV cross-section under the light microscope.ResultsCNV area was significantly smaller in liposome mediated pCI-RGD-ES injection group and pCI-ES injection group compared with PBS group at different time points (P＜0.01),but no significant difference was seen in CNV area between pCI-ES injection group and PBS group at different time points (P＞0.01).The changes of number of corneal microvessels was followed a similar fashion as the change of CNV area.Expression of VEGF in cornea was obviously stronger in pCI-ES injection group and PBS group than liposome mediated pCI-RGD-ES injection group and pCI-ES injection group.ConclusionEndostatin with RGD sequence could effectively inhibit corneal neovascularization,and liposome is proved to be a potent carrier in gene transfer.

Background　Culture of retinal pigment epithelium(RPE) cells is very important for establishment of proliferative vitreoretinopathy (PVR) model,prevention and treatment of PVR as well as RPE cell transplantation.Isolation of animal RPE cells by trypsinization is a critical step.ObjectiveThe present study is to establish the methods of isolation and culture of retinal pigment epithelium (RPE) cells in rabbit and comparied with that of pig RPE culture.MethodsRPE cells were isolated by trypsinization in pigmented rabbit and pig and cultured in DMEM containing 20% fetal bovine serum.Cultured RPE cells were identified by immunochemistry.The fourth generations of cells were used in this experiment.Morphology and characteristics of cultured RPE cells from rabbit and pig were examined and compared under the light microscope.ResultsIsolated RPE cells from pig were obtained by once trypsin digestion,but two times of trypsinization were needed in rabbit RPE cells isolation.The differentiation in response to trypsinization was related to anatomic difference between the two types of cells .The adherence time of pig RPE cells was 24 hours ,however,the rabbit RPE needed 48-72 hours after culture.Proliferation and vitality of cultured cells were gradually attenuated and melanin decreased after several times subculture.The morphology of culture RPE cells was obviously different between rabbit and pig because species difference.Immunohistochemistry demonstrated the positive response of RPE cells for keratin.ConclusionRPE cells can be acquired from both rabbit and pig by trypsinization and culture.The culture process of RPE cells of pig is simpler than that of rabbit.Cells within the fourth generations are suitable for experimental application.

Background Neuromyelitis optica,term of Devic's disease,is characterized by the symptoms of both optic neuritis and myelitis.In clinic,misdiagnosis rate of neuromyelitis is too high to ignore because of the unsynchronous exsist of both optic neuritis and myelitis.Objective This study aims to analyze the clinical characteristics of Devic's disease in order to reduce the misdiagnosis rate.Methods Thirty six patients diagnosed as Devic's disease in General Hospital of PLA from January 2000 through October 2008 were included in this study.The clinical data including sex,age,initial events,clinical signs of optic neuritis and myelitis,misdiagnosis status were analyzed.Results The 21 patients showed the initial events of eye and were diagnosed as neurititis.In all of the 36 patient,the ratio of the patients was from 30 to 50 years.The prevalence of atrophy of optical nerve within 1-3 months was 72.7% and that of above 3 months was 91.4% in these patients.The incidence rate was obviously increased in 1-3 months course group (χ~2=7.59,P=0.009) and ＞3 months course group(χ~2= 20.29,P＜0.001) in comparison with ＜1 month course group.Two patients without clinical signs were determined the diagnosis by visual evoked potential.In 22 patients received magnetic resonance imaging of spinal cord,the lesions of 14 patients located in cervical cord and that of 9 patients was in thoracic cord and only 1 patient in lumbar cord.Conclusion Devic's disease is more common in female patients with the age between 30-50 years.Most patients visit ophtalmologist firstly due to initial events of eye.Occurrence of optic atrophy is associated with disease course.MRI suggest that the lesions of spinal cord are often in cervical cord and thoracic cord.

Background Many researches have reported the high proliferation ability of pterygium tissue.However,the origin of cells in pterygium tissue and their role in pathogenesis and recurrence of pterygium are below understand up to now.Objective This study is to explore the role of bone marrow derived stem cells and progenitor cells in the pathogenesis of pterygium.Methods Informed consent was obtained from 12 patients with the pterygium stretching the edge of the pupil prior to this trail.Pterygium specimen was obtained from 8 patients with primary pterygium and 4 patients with recurrent pterygium during the operation.The normal conjunctival tissue was obtained from the location of 10 mm from the pterygium in the operation eye.The expression levels of marker of the primitive haematopoietic progenitors(AC133),marker of the haematopoietic progenitor cells and endothelium (CD_(34)),marker of haematopoietic and stromal progenitor cells (C KIT ),and differentiation antigen of bone marrow fibroblast cells and nonhaematopoietic progenitor cells 1 (STRO 1) were detected by using immunohistochemistry.Results Various progenitor cell markers originated from bone marrow,including CD_(34),AC133,C KIT,STRO 1,were positively expressed in pterygium specimen rather than normal conjunctival specimen mainly in the basal epithelium or stroma of pterygia tissue.Majority of the positive cells were seen in the head of pterygium.C KIT positive cells were observed mainly in the basal epithelium and stroma of pterygium.The morphology of positive cells varied upon different distribution.Fibroblast like and spindle shaped positive cells were seen in the stroma layer of pterygium,and round or ovoid shaped cells were in basal epithelial layer.The distribution and shape of positive cells for AC133 and CD_(34) were similar to ones of C KIT but numbers of cells were varied between them.C KIT,AC133 and CD_(34) positive cells were coexpressed in basal epithelium and vascular endothelium.Expression of STRO 1 was found in the stroma layer not in the basal epithelium of pterygium.Similar distribution,numbers and shape of positive cells were seen between recurrent and primary pterygium.Conclusion The cells with stem cells markers and progenitor cells markers originated from bone marrow may play a role in the pathogenesis of pterygium.