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Diabetic macular edema (DME) is one of the common causes of visual impairment. Anti-vascular endothelial growth factor (VEGF) has become the preferred therapy for DME because of significant visual improvement. Early and intensive anti-VEGF therapy combined with other individualized treatments are currently the main strategy for DME treatment. Considering the complexity of DME and limitations of anti-VEGF therapy, there are still many problems and difficulties in the treatment of DME. Optimizing treatment strategies, strengthening management of the clinical course and developing new drugs, could improve the efficacy and maintain the improvement of visual acuity and visual performance.
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Retinal vein occlusion (RVO) is one of the most common diseases severely threatening visual acuity.There is not a complete consensus on the clinical treatment strategies and schemes, and no guideline or consensus has been published in China.The publication of the latest international guidelines has provided more comprehensive and clear suggestions and advices for treatment and management strategies for RVO.Reading and understanding those guidelines could provide references for clinical practice, and offer help for standard diagnosis and treatment.
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The prevention and control of novel coronavirus pneumonia is the most priority recently, and various measures during the prevention and control period will have varying degrees of impact on the implement of clinical trials.However, various examinations in ophthalmological clinical trials need close contact between operators and patients, which put us at risk of cross-infection.This paper indicated some suggestions based on the criteria of clinical trials under major public health emergencies, the management of clinical trials during epidemic period, including the follow-up of subjects, the treatment of epidemic serious adverse event (SAE) and the management requirements of co-sponsors, as well as the requirements and management principles for environment, subjects, examiners and inspection equipment in the process of ophthalmic clinical trials.It may be helpful to the ophthalmic clinical trial researchers and subjects during the period of novel coronavirus infection.
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Retinal vein occlusion (RVO) is one of the most common diseases severely threatening visual acuity.There is not a complete consensus on the clinical treatment strategies and schemes,and no guideline or consensus has been published in China.The publication of the latest international guidelines has provided more comprehensive and clear suggestions and advices for treatment and management strategies for RVO.Reading and understanding those guidelines could provide references for clinical practice,and offer help for standard diagnosis and treatment.
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The prevention and control of novel coronavirus pneumonia is the most priority recently, and various measures during the prevention and control period will have varying degrees of impact on the implement of clinical trials. However, various examinations in ophthalmological clinical trials need close contact between operators and patients, which put us at risk of cross-infection. This paper indicated some suggestions based on the criteria of clinical trials under major public health emergencies, the management of clinical trials during epidemic period including the follow-up of subjects, the treatment of epidemic serious adverse event (SAE) and the management requirements of co-sponsors, as well as the requirements and management principles for environment, subjects, examiners and inspection equipment in the process of ophthalmic clinical trials. It may be helpful to the ophthalmic clinical trial researchers and subjects during the period of novel coronavirus infection.
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Objective To investigate changes in subretinal fluid(SRF),intraretinal fluid(IRF) and visual acuity after treatment with anti-vascular endothelial growth factor(VEGF)drugs in patients with wet age-related macular degeneration(WAMD)combined with disseminated epithelial detachment (PED).Methods Thirty-one WAMD patients with PED were retrospectively analyzed.Imaging characteristics of retinal morphological changes and changes in visual acuity were analyzed by optical coherence tomography(OCT)before and after treatment with Ranibizumab(1 mg).Results Thirtyone patients were treated with regular doses 3 to 17 times.WAMD patients with persistent/recurrent SRF,IRF and PED were treated with Ranibizumab(1 mg)1 to 3 times.There was a significant difference in the mean central retinal thickness(CRT)before and after 1 month of treatment(498.5 ±242.9 μm vs.406.0±201.9 μm,t=6.025,P＜0.01).85.7％ of patients(24 cases)had a decrease in SRF,60％(9 cases)had a decrease in IRF and 67.7％ (10 patients)had a decrease in PED,and 19 patients(32.7％)had increased visual acuity assessed by the Early Treatment of Diabetic Retinopathy Study(ETDRS) chart (≥ 5 letters),and 36 patients (61.2 ％) had stable vision.Conclusions Intravitreal injection of 1 mg Ranibizumab for the treatment of WAMD patients with PED can improve or stabilize visual acuity and reduce SRF,IRF and PED.
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Objective To compare the efficacy of intravitreal dexamethasone implant (OZURDEX（R）) in the treatment of macular edema (ME) caused by central retinal vein occlusion (CRVO) in different ages.Methods A retrospective case observation study was performed.Forty eyes of 40 patients diagnosed with ME secondary to CRVO were enrolled in the study.According to patient's age,all of the 40 eyes were divided into ＜40 years old group (20 eyes) and ≥40 years old group (20 eyes).All eyes went through best corrected visual acuity (BCVA),intraocular pressure (IOP) and optic coherence tomography (OCT) examinations.Early Treatment Diabetic Retinopathy Study chart was used to test visual acuity,and central macular thickness (CMT) was measured by OCT.Fundus fluorescein angiography (FFA) was proceeded selectively.All eyes went through intravitreal injections off intravitreal dexamethasone implant and BCVA,IOP,OCT were tested 1 month,2,3,6,9 and 12 months after injection.After 3 months follow-up,intravitreal dexamethasone implant or ranibizumab would be injected again for patients with ME recurrence or poor treatment effects.Changes of BCVA,IOP and CMT before and after injection were observed.Results BCVA and CMT in the ＜40 years old group and ≥40 years old group were compared at different time points,and the differences were significantly different (BCVA:Fgroup =2.071,P =0.044;Ftimc.=9.551,P ＜ 0.001.CMT:Fgroup =2.402,P=0.034;Ftime =13.175,P＜0.001),compared with before injection,the BCVA at each time point of post-injection was improved,and CMT was thinner than that before injection;at 2 months after injection,the BCVA was improved and CMT was thinner than those 1 month after injection,the differences were statistically significant (all at P＜0.05);at 12 months after injection,the BCVA in the ＜40 years old group was obviously better than that in the ≥40 years old group,the CMT was much thinner than that in the ≥40 years old group,the differences were statistically significant (both at P＜0.05).Compared with before injection,the IOP at 1 month,2 and 3 months after injection was increased,the IOP at 2 months after injection was higher than that at 1 month after injection,the differences were statistically significant (both at P＜0.05).The mean number of injections was (1.8±0.9) times in the ＜40 years old group and (2.7±1.4)times in the ≥40 years old group,with a statistical difference between them (t =2.569,P =0.014).Conclusions In different age groups,patients with ME caused by CRVO can be effectively treated by intravitreal dexamethasone implant,the therapeutic effect is significant at 1 month post-injection and most improvement is shown at 2 months post-injection.Compared with patients over 40 years of age,patients under 40 years of age have better long-term results,better visual improvement,thinner CMT and less intravitreal injections.
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Diabetic macular edema (DME) is a common reason for vision decrease.The concept of DME treatment regime has been changed to increase the vision-sight of patients since the onset of new treatment.Recent DME guidelines introduced the latest researches of DME,which raised new clinical suggestion and clinical pathway based on evidence-based medicine.Anti-vascular endothelial growth factor (VEGF) treatment is the first-line therapy for all types of DME,laser photocoagulation and intravitreal steroid injection are often the supplementary treatment except for some special cases in which they can be the substitution for anti-VEGF treatment.The regime for DME treatment now is combination.To understand and follow the guidelines can help us instruct and standardize our management on DME.
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Objective To investigate the factors associated with short-term elevation ofintraocular pressure after ranibizumab intravitreal injection.Methods 292 eyes of 292 patients who were diagnosed retinopathy and suitable to receive ranibizumab intravitreal injection were enrolled in this prospective clinical study.There were 157 males and 135 females.193 patients diagnosed with age-related macular degeneration and 99 other retinopathy patients.Mean age of patients was 62.75 ± 13.74 years.All subjects underwent systemic and comprehensive ophthalmology examinations.The mean BCVA was 0.68± 0.47 logMAR.Mean basal intraocular pressure was 18.1 mmHg (1 mmHg=0.133 kPa).All patients received intravitreal injection with 0.05 ml of ranibizumab (0.5 mg).The intraocular pressure were measured by non-contact tonometer at 10,30,120 minutes and 1 day after injection in a sitting position.The patients were grouped by the changes ofintraocular pressure 10 minutes after injection.The elevation was more than 10 mmHg as elevation group and less than 10 mmHg as stable group.Analyze the possible related factors with elevation of intraocular pressure after ranibizumab intravitreal injection by comparing the different datum of two groups.Results The mean intraocular pressure were 23.8,20.5,19.9 and 17.4 mmHg at 10,30,120 minutes and 1 day after injection.The significant elevation level were 5.8,2.4,1.8,-0.7 mmHg compared with basal intraocular pressure.Among 292 eyes,intraocular pressure elevation in 68 eyes and stabled in 224 eyes.The age (Z=-0.732),gender (x2=1.929),right or left eye (x2=2.910),BCVA (Z=-0.039),diseases ((2=2.088) were no significant difference between two groups (P＞0.05).The injection number (Z=-2.413,P=0.001),basal intraocular pressure (Z=-3.405,P=0.016) and elevations after injection (Z=-11.501,-8.366,-5.135,-3.568;P＜0.01) were significantly different comparing two groups (P＜0.05).By logistic regression analysis,basal intraocular pressure was positively correlated with the elevation of intraocular pressure 10 minutes after injection (B=-0.844,OR=0.43,95％CI 0.24-0.76,P=0.004).Patients with higher basal intraocular pressure may occur intraocular pressure elevation after ranibizumab intravitreal injection much probably.Conclusions The factors associated with short-term elevation of intraocular pressure after ranibizumab intravitreal injection were basal intraocular pressure.The higher basal intraocular pressure,the higher risk to gain elevation of intraocular pressure after injection.
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Objective To investigate the factors associated with vision and hole closure for idiopathic macular hole (IMH) after vitrectomy surgery.Methods Eighty-nine eyes of 89 patients with IMH were enrolled in this retrospective study.There were 15 males and 74 females.The patients aged from 42 to 82 years,with the mean age of (64.13 ± 7.20) years.All subjects underwent best corrected visual acuity (BCVA) and optical coherence tomography (OCT) examinations.The BCVA ranged from 0.01 to 0.4,with the mean BCVA of 0.12 ± 0.09.The MH stages was ranged from 2 to 4,with the mean stages of 3.56 ± 0.77.The basal diameter ranged from 182 μm to 1569 μm,with the mean basal diameter of (782.52± 339.17) μm.The treatment was conventional 25G pars plana vitrectomy combined with phacoemulsification and intraocular implantation.Fortyone eyes received internal limiting membrane peeling and 48 eyes received internal limiting membrane grafting.The follow-up ranged from 28 to 720 days,with the mean follow-up of (153.73 ± 160.95) days.The visual acuity and hole closure were evaluated on the last visit and the possible related factors were analyzed.Results On the last visit,the BCVA ranged from 0.02 to 0.8,with the mean BCVA of 0.26±0.18.Among 89 eyes,vision improved in 45 eyes (50.56％) and stabled in 44 eyes (49.44％).Eighty-six eyes (96.63％) gained MH closure but 3 eyes (3.37％) failed.By analysis,patients of early stages of MH and smaller basal diameter of MH will gain better vision outcome (t=2.092,2.569;P＜ 0.05) and patients of early stage MH will gain high hole closure rate after surgery for IMH (t=-5.413,P＜0.05).However,gender,age,duration,preoperative BCVA,surgery technique,gas types and follow-up time had no relationship with the effect after surgery for IMH (P＞0.05).Conclusions Stages of MH and basal diameter of MH may be the factors associated with the visual outcome for idiopathic macular hole after surgery.However,age,gender,duration,surgery patterns,gas types and followup time showed no effects on operational outcomes.
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Diabetic macular edema (DME) is the most common reason for visual impairment in diabetic eye diseases.DME is easy to relapse and the treatment outcome is beyond satisfied.Anti-vascular endothelial growth factor (anti-VEGF) therapy has been proposed to be a first-line or key treatment regimen in the management guideline for DME.However,in clinical practice,there still are many ambiguous and controversial views in the choice and implement of different managing paradigms,including the therapeutical goal,the assessment of treatment effectiveness,the explanation of the guidelines,the regimen of anti-VEGF therapy,the value evaluation of laser photocoagulation,the use of steroids and the management of recurrent DME.Recently,the researches of treating drugs for DME are developing and making a great progress,so eye doctors should track the research results,improve the therapeutical regimen,fully understand the advantages and disadvantages of different treating methods and standardize the treatment process in order to release the best treatment effects on DME.
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Neovascular age-related macular degeneration is also known as wet age-related macular degeneration (wAMD) , which is now one of potentially blinding diseases in elder population worldwide.The application of anti-vascular endothelial growth factor (VEGF) drugs is becoming the first-line therapy for wAMD at present.However, during the long-term follow up, we find that the vision of some patients dose not improve even falls after treatment, or the vision is unable to keep for a long term after its improvement.Hence,to find the key factors that affect the therapeutical effect is the focus issue nowadays.There are many factors that affect wAMD curative effect,including the limitation of drug itself, the personal conditions of the patients, the features of the choriodal neovascularization (CNV) , the formulation and implementation of the treatment regimen, etc.Imaging features of CNV and treatment timing can serve as the available indexs to analyze the prognosis.In addition,reasonable and optimized managing regimens for wAMD probably is an approach to improve the treating effect.Understanding of the factors that affect curative effect of wAMD is benefit for us to setup the individualized therapy and achieve the best vision.Ophthalmologists should fully recognize the importance of wAMD individualized treatment and management.
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The therapeutic effect of anti-vascular endothelial growth factor (VEGF) for neovascular age-related macular degeneration (nAMD) was determined by a number of factors.Comprehensive thorough analysis of clinical features,imaging results and treatment response can predict the potential efficacy and possible vision recovery for the patient,and also can optimize the treatment regime to make a personalized therapy plan.Precise medicine with data from genomics,proteomics and metabolomics study will provide more objective and accurate biology basis for individual precise treatment.The future research should focus on comprehensive assessment of factors affecting the efficacy of anti-VEGF therapy,to achieve individualized precise diagnosis and treatment,to improve the therapeutic outcome of nAMD.
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<p><b>BACKGROUND</b>Intravitreal ranibizumab injection is effecitve on treating myopic CNVs, but it could be a risk factor for developing more severe retinoschisis in eyes with preexisted retinoschisis and epiretinal membrane. This study aimed to explore the incidence and features of retinoschisis after intravitreal ranibizumab injection for myopic choroidal neovascularization.</p><p><b>METHODS</b>Eighty-three eyes of 81 patients with choroidal neovascularization secondary to pathologic myopia were treated with intravitreal ranibizumab injection. The best corrected visual acuity and optical coherence tomography (OCT) images were recorded at baseline and every month thereafter. Central retina thickness and maximal retina thickness were measured. The subjects were divided into three groups. Eleven eyes that had retinoschisis and epiretinal membrane were in group 1, six eyes that had simple epiretinal membrane were in group 2, and 66 eyes that had neither retinoschisis nor epiretinal membrane were in group 3. Six contralateral eyes in group 1 which had retinoschisis and epiretinal membrane but were not treated with intravitreal ranibizumab injection were set as the control group.</p><p><b>RESULTS</b>Seven of the 11 eyes in group 1 developed more severe retinoschisis, the mean maximal retinal thickness increased from (380.28 ± 90.13) to (467.00 ± 70.20) µm (P < 0.05). The retinoschisis of all 6 eyes of the control group did not aggravate. Compared with the control group, the aggravation ratio of retinoschisis increased significantly (P < 0.05). No new onset of retinoschisis took place in group 2 and group 3.</p><p><b>CONCLUSION</b>Intravitreal ranibizumab injection may be a risk factor for aggravation of retinoschisis in eyes with preexisted retinoschisis and epiretinal membrane.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Angiogenesis Inhibitors , Therapeutic Uses , Antibodies, Monoclonal, Humanized , Therapeutic Uses , Choroidal Neovascularization , Drug Therapy , Intravitreal Injections , Myopia, Degenerative , Drug Therapy , Ranibizumab , Retinoschisis , Drug Therapy , Retrospective Studies , Treatment OutcomeABSTRACT
Objective To compare the safety and effect of the phacoemulsification (PHACO) versus extracapsular cataract extraction (ECCE) in patients with intumescent senile cataract.Methods 200 eyes from patients with intumescent cataract were included and randomly divided into 2 groups:PE group (108 eyes,received PHACO) and ECCE group (92 eyes,received small incision ECCE) respectively.Superior quadrant sclera tunnel incisions were made with stabs of 2.8 mm diameters.Trypan-blue was used to show the anterior lens capsular membrane.In the PHACO group,a 4 mm diameters continuous curvilinear capsulorhexis (CCC) was made,which was enlarged to 6-7 mm after the intraocular lenses (IOL) implantation.For the ECCE group,a 8 mm-diameter CCC was made.Then the lens nucleus was either phacoemulsificated or delivered and IOL was implanted.The complications during and after surgery and the visual outcomes were recorded and statistically analyzed by SPSS 13.0 software.Results Age,sex and the hardness of the nucleus were comparable between the two groups.The best corrected vision acuity (BCVA) was 0.05 and worse in all patients before surgery.While 3 days after operation,in PE group,105 eyes (97.2％,105/108) gained postoperative vision 0.05 and better,and 82 of them were better than 0.3; in group ECCE,97.2％ (89/92) of the operated eyes gained vision 0.05 and better,72 (78.3％,72/92) eyes were better than 0.3.No statistical differences were found between the two groups in postoperative vision recovery.While,there were more failure rates of the CCC,tear of the anterior and posterior capsular,loss of the vitreous and iris injury rate in the ECCE group than in the PE group (20.7％ vs.8.3％,17.4％ vs.12.0％,7.6％ vs.0.9％,15.2％ vs.0％,P=0.01,P=0.00,P=0.02,P＜ 0.001).Prolapse of iris and discoria were found in ECCE group.Conclusions With small CCC,phacoemulsification can lead to better surgical outcomes than small incision ECCE procedures,and the operative and post-operative complications are less in PE group than in ECCE group.
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Objective To investigate the effect of the modified surgery for spontaneous rupture of esophagus (SRE) so as to improve treatmeut level.Methods Clinical data and surgical methods of 16 SRE patients including four patients with mid-esophagus ruptures and 12 with lower esophagus ruptures treated between February 1999 and June 2011 were analyzed retrospectively.All patients had only one laceration with the gap length of 1.5-5 cm (median 2.5 cm).Eleven patients had rupture into the left breast,two had rupture into the right chest,with no rupture into the chest in three patients.Ten patients suffered from hydropneumothorax and five from subcutaneous emphysema.Thc esophageal mucosas rathcr than muscular layers of all patients were sutured disconnectedly with absorbable thread.Omentum majus were embedded and fixed to muscular layer on the edge of esophagus rupture site.Fundus ventriculi were suspended and fixed to the dome of diaphragm.In the meantime,diaphragmatic hiatus were reconstructed above the esophagus rupture site for lower esophagus ruptures.Results The time from SRE attack to operation ranged from one hour to three days.Eleven patients were repaired within 24 hours of SRE onset and five patients were repaired after 24 hours of SRE onset.All patients got through the perioperative period smoothly and survived the operation with cure rate of 100％.The median hospital stay was 18.5 days.No esophageal narrow or canceration were found during follow-up (range,1-10 years),but two patients suffered from reflux which were relieved significantly after conservative treatment.Conclusion For treatment of SRE,interrupted suture for esophageal mucosal layers,omentum majus embedding instead of esophageal muscular layer suture and simultaneous anti-reflux operations can significantly reduce incidence of complications like esophageal fistula,stenosis and reflux and improve the cure rate.
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ObjectiveTo investigate the experience of resection of superior sulcus tumors using the improved antero-cervical parastemal approach.MethodsThe clinical data of 3 patients with superior sulcus tumor from July 2005 to May 2010 were analyzed retrospectively.Three patients with superior sulcus tumor underwent en bloc resection using the improved antero-cervical parastemal approach.The 1st to 3rd rib were excised,1 case with 1/5 centrum vertebra excision,1 case with the 1st transverse process of thoracic vertebra excision,1 case with the 1 st and 2nd transverse process of thoracic vertebra excision,1 case with T1 nerve root and sellate ganglion excision.ResultsAll the patients recovered well after operation.There was no serious complication except for 1 case with secondary Horner syndrome.The mild paradoxicalbreathing was found in 3 cases postoperative dressing change,but they had no dyspnea and tolerance well.The paradoxical breathing was disappeared when the mediastinum was fixed after 14 d thoracic wall pressure dressing.The pathological diagnosis after operation:2 cases with squamous-celled carcinoma,1 case with adenosquamous carcinoma,there was no mediastinal lymph node metastasis,cutting edge was negative.Stage:2 cases with T3N0M0,1 case with T4N0M0.Three cases were treated with chemotherapy (NP plan) for 4 cycles after operation without radiotherapy.Three cases were followed up for 53,37, 13 months after operation,they were all survival without recurrence and good quality life.ConclusionsFor the treatment of especially anterior and middle seated superior sulcus tumors,improved antero-cervical parasternal approach provides a safe and effective exposure.The improved antero-cervical parasternal approach is worthy of promotion.
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Objective To investigate the effect and significance of early coronary artery bypass grafting (CABG) on the expression level of nerve-related factor at infracted border zone (IBZ) in dogs with acute myocardial infarction (AMI).Methods The anterior descending coronary artery of all thirty dogs randomly assigned into experimental group(n =22) and control group(n =8)were ligated into MI model.The experimental group included those undergoing CABG 1(n =6),2 week(n =4),4 week(n =6)and 6 week (n =6) after MI,and control group(n =2) was established for every experimental group.We detected the local expression level of nerve-related factor such as GAP43 mRNA,NGF mRNA and SYN mRNA at normal myocardium and infracted border zone myocavdium by means of RT-PCR through thoracotomy at eight weeks after surgery.Results Four dogs of every experimental group and all dogs of control group survived to the end of the study.The local expression level of GAP43 mRNA,NGF mRNA and SYN mRNA in control groups was significantly higher than that in normal and experimental groups (P ＜0.01 ).The local expression level of GAP43 mRNA,NGF mRNA and SYN mRNA in 4 and 6 weeks bypass groups was significantly higher than that in normal and 1,2 weeks bypass groups (P ＜0.05).There was no statistically significant the expression level of SYN mRNA among all experimental groups (P ＞ 0.05 ).Conclusions Early CABG surgery for AMI in dogs could lessen the expression level of nerve-related factor and the sympathetic remodelling at IBZ.Especially CABG surgery two weeks after MI could lessen the sympathetic maximacily.
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Objective To establish an experimental choroidal neovascularization(CNV)model in brown norway(BN)rats by laser infusion in order to study the evolution of CNV during 3 weeks with intravitreaI injection of 1 μl(25 μg)bevacizumab,and make a preliminary investigation of the treatment effect of bevacizumab on experimental CNV. Methods Four groups of 48 BN rats were photocoagulated by krypton laser in 1 eye to induce CNV model in 1 eye,in which 4 groups received 1 μl(25 μg)bevacizumab intravitreal injection 1 week after photocoagulation and the other groups received the same volume intravitreall injection of balanced salt solution(BSS)as control.Fundus fluorescein angiography(FFA)examinations were taken on 1,2,3 week and the eyes were enucleated and processed for histopathologic examination.The areas of CNV were measured in the early phase of FFA.FⅧ-Rag protein was investigated with immunohistoehemistry and was semiquantkatively analyzed. Results The area and the leakage of CNV in bevacizumab treated BN rat eyes were obviously reduced as compared with the control group at each interval.In the treatment group,the expression of FⅧ-Rag was depressed.The examination of FⅧ-Rag and FFA verified the depression of CNV as compared with the control group at each interval.The density of treatment was lower than of control group(P＜0.01).After bevacizumab treatment for 2 weeks,the area of CNV and the expression of FⅧ-Rag were decreased[(0.920±0.634)mm2 and 35.57±10.52,respectively),as compared with the control group[(2.489±0.590)mm2 and 175.37±25.20]. Conclusions Intravitreal bevacizumab injection can inhibit the development of CNV.
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Objective This study was designed to establish an experimental Choroidal neovascularization (CNV) model in BN rats, which could exhibit high reproducibility and to evaluate the expression of pigment epithelium-derived factor (PEDF). Methods 30 rats were received krypton laser. 1 days, 3 days, 7 days, 14days, 21 days and 28 days after photocoagulation, fundus fluorescein angiography (FFA) wAS performed. Total RNA were abstracted from retinal and choroidal tissue and the mRNA of PEDF was detected by RT-PCR.Results FFA showed flurescein leakage from 1 week to 4 weeks after photocoagulation. The mRNA of PEDF were in significantly difference during 4 weeks by RT-PCR.Conclusions Kryton laser photocoagulation can be successfully used to produced CNV experimental model in the BN rat. This study gave us a effective way to evaluate the mechanism of CNV by RT-PCR.