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A comparing study of quantitative staining techniques for retinal neovascularization in a mouse model of oxygen-induced retinopathy

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International Journal of Ophthalmology(English edition) 2012年第1期5卷 1-6页

作者： Xiao-Ling Liang, Liao-Xu Long Key Lab of Ophthal Ministry of Edu Zhongshan Ophthalmic Center Sun Yat-sen University Guangzhou 510060 Guangdong Province China Department of Ophthalmology Nanhai Hospital Southern Medical University Foshan 510515 Guangdong Province China Guangzhou Medical University 195# Dongfeng Xi Road Guangzhou Guangdong ProvinceChina

AIM: To explore an efficient, practical and objective quantitative method to evaluate the retinal neovascularization in mouse model of oxygen induced retinopathy (OIR). METHODS: Thirty C57BL/6J mice were explored in OIR model procedure. Eyes were removed for different staining methods including: (1) HE staining; (2) immunohistochemistry with Griffonia Simplicifolia Lectin (GSL); (3) Immunofluorescence with FITC labeled CD31 antibody; (4) Two-step immunofluorescence with purified-CD31 antibody; (5) FITC-Dextran perfusion combined with two-step purified-CD31immunofluorescence. Images of the retinal vasculature were analyzed by imaging software. ' RESULTS: GSL immunohistochemistry could clearly demonstrate the deep and superficial capillary beds. FITC labeled CD31 Immunofluorescence was blurring with high fluorescence background which was hard to distinguish retinal neovascularization in some area. Excellent detail of neovascularization and preexistent retinal vessels was provided in two-step Purified-CD31 immunofluorescence group. CONCLUSION: GSL immunohistochemistry can clearly demonstrate neovascularization tufts in deep and superficial capillary beds. Immunofluorescence of specific antigen CD31 on vascular endothelium can selectively label the neovascularization of mouse retina. When combined with computer analysis software, it is an effective and objective quantitative method to evaluate the retinal neovascularization in OIR mouse model.

关键词： neovascularization endothelial cell CD31 hematoxylin-eosin staining immunohistochemistry immunofluorescence retinopathy of prematurity

Tracking the in vivo spatio-temporal patterns of neovascularization via NIR-II fluorescence imaging

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Nano Research 2020年第11期13卷 3123-3129页

作者： Mo Chen Sijia Feng Yimeng Yang Yunxia Li Jian Zhang Shiyi Chen Jun Chen Institute of Sports Medicine of Fudan University Department of Orthopaedic Sports MedicineHuashan HospitalFudan UniversityShanghai200041China

The in vivo spatio-temporal patterns of neovascularization are still poorly understood because it is limited to multi-scale techniques from the cellular level to living animal level.Owing to deep tissue-penetration and zero autofluorescence background,the second near-infrared(NIR-II,1,000–1,700 nm)fluorescence imaging recently shows promise in breaking through this dilemma by dynamically tracking the pathophysiological process of neovascularization in vivo.Here,NIR-II fluorescence imaging was recruited for monitoring blood vessels in order to visualize the vascular injury and quantitively assess neovascularization in mouse models of acute skeleton muscle contusion and hindlimb ischemia.The temporal analysis of real-time NIR-II fluorescence intensity demonstrated that the blood flow perfusion of ischemia area was able to rapidly restore to 96%of pre-ischemic state within one week.Moreover,the spatial analysis revealed that the lower and outer quadrants of ischemia area in the mouse model of hindlimb ischemia always had relatively high blood flow perfusion compared with other quadrants during three weeks post-ischemia,and even exceeded pre-ischemic quantity at 21 days post-ischemia.In conclusion,this in vivo imaging technique has significant potential utility for studying the spatio-temporal patterns of neovascularization in vivo.

关键词： bioimaging second near-infrared(NIR-II)fluorescence nanoprobes spatio-temporal neovascularization

Effect of trapping vascular endothelial growth factor-A in a murine model of dry eye with inflammatory neovascularization

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International Journal of Ophthalmology(English edition) 2016年第11期9卷 1541-1548页

作者： Jin Woo Kwon Jin A Choi Eun Young Shin Tae Yoon La Dong Hyun Jee Yeon Woong Chung Yang Kyung Cho Department of Ophthalmology St.Vincent's HospitalCollege of Medicinethe Catholic University of Korea Research Institute of Medical Science St.Vincent's Hospitalthe Catholic University of Korea

AIM： To evaluate whether trapping vascular endothelial growth factor-A（VEGF-A） would suppress angiogenesis and inflammation in dry eye corneas in a murine corneal suture model.METHODS： We established two groups of animals, one with non-dry eyes and the other with induced dry eyes.In both groups, a corneal suture model was used to induce inflammation and neovascularization. Each of two groups was again divided into three subgroups according to the treatment; subgroup I（aflibercept）,subgroup II（dexamethasone） and subgroup III（phosphate buffered saline, PBS）. Corneas were harvested and immunohistochemical staining was performed to compare the extents of neovascularization and CD11b＋ cell infiltration. Real-time polymerase chain reaction was performed to quantify the expression of inflammatory cytokines and VEGF-A in the corneas.RESULTS： Trapping VEGF-A with aflibercept resulted in significantly decreased angiogenesis and inflammation compared with the dexamethasone and PBS treatments in the dry eye corneas（all P 〈0.05）, but with no such effects in non-dry eyes. The anti-inflammatory and antiangiogenic effects of VEGF-A trapping were stronger than those of dexamethasone in both dry eye and non-dry eye corneas（all P 〈0.05）. The levels of RNA expression of VEGF-A, TNF-alpha, and IL-6 in the aflibercept subgroup were significantly decreasedcompared with those in the PBS subgroup in the dry eye group.CONCLUSION： Compared with non-dry eye corneas,dry eye corneas have greater amounts of inflammation and neovascularization and also have a more robust response to anti-inflammatory and anti-angiogenic agents after ocular surface surgery. Trapping VEGF-A is effective in decreasing both angiogenesis and inflammation in dry eye corneas after ocular surface surgery.

关键词： dry eye aflibercept neovascularization inflammation dexamethasone

Topical dihydroartemisinin inhibits suture-induced neovascularization in rat corneas through ERK1/2 and p38 pathways

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International Journal of Ophthalmology(English edition) 2011年第2期4卷 150-155页

作者： Yan-Yan Zhong, Xiao-He Lu Department of Ophthalmology Zhujiang Hospital Southern Medical University Guangzhou 510282 Guangdong Province China Department of Febrile Disease the First Affiliated Hospital Guangzhou University of Chinese Medicine Guangzhou 510405 Guangdong Province China Department of Ophthalmology Nanfang Hospital Southern Medical University Guangzhou 510515 Guangdong Province China

AIM: To determine if topical instillation of dihydroartemisinin (DHA) inhibits corneal neovascularization (NV) in rats and to investigate the role of the extracellular regulated kinases (ERK) 1/2 and p38 pathways in this process. O METHODS: Suture-induced corneal NV was produced in rats and the eyes were topically treated with different concentrations of DHA (20mg/L, 10mg/L or 5mg/L) or normal saline 4 times a day for 7 days. The corneal NV was quantified as the proportion of NV area to the whole cornea. Western blot was used to determine the expressions of vascular endothelial growth factor (VEGF) and the phosphorylation status of VEGF receptor-2, ERK1/2 and p38 in the corneas. Immunofluorescent staining was used to determine the expressions of phospho-ERK1/2 and phospho-p38 in the corneal tissues from the eyes treated with 20 mg/L DHA (DHA group) or normal saline (control group). RESULTS: The proportion of corneal NV area in the eyes treated with normal saline or DHA at dosages of 20mg/L, 10mg/L or 5mg/L was (23.74 +/- 3.00)%, (15.73 +/- 2.88)%, (19.53 +/- 2.42)%, and (23.38 +/- 2.79)%, respectively. In the eyes treated with 20mg/L or 10mg/L DHA, the corneal NV area was significantly reduced when compared to that in eyes with normal saline (P < 0.05). Western blot analyses revealed that 20mg/L DHA significantly inhibited the expressions of VEGF and phospho-VEGFR-2. Both 20mg/L and 10mg/L DHA inhibited the expressions of phospho-ERK1/2 and phospho-p38. Immunofluorescent staining further demonstrated that 20mg/L DHA lowered the Expression levels of phospho-ERK1/2 and phospho-p38 in the corneas with suture-induced NV. O CONCLUSION: Suture-induced NV in rat corneas was significantly inhibited by topical treatment with 20mg/L and 10mg/L DHA. The results suggest that the effects could be partially dependent on the DHA-mediated inhibitions of the ERK1/2 and p38 pathways.

关键词： cornea neovascularization dihydroartemisinin extracellular regulated kinases 1/2 p38

Acetylcholinesterase inhibition ameliorates retinal neovascularization and glial activation in oxygen-induced retinopathy

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International Journal of Ophthalmology(English edition) 2020年第9期13卷 1361-1367页

作者： Qiu-Ping Liu Xian Zhang Ya-Zhou Qin Jing-Lin Yi Jing-Ming Li Affiliated Eye Hospital of Nanchang University Nanchang 330006Jiangxi ProvinceChina Department of Ophthalmology First Affiliated Hospital of Xi'an Jiaotong UniversityXi’an 710061Shaanxi ProvinceChina

AIM:To investigate whether inhibition of acetylcholinesterase(AChE)by donepezil ameliorate aberrant retinal neovascularization(RNV)and abnormal glial activation in oxygen-induced retinopathy(OIR).METHODS:A mouse model of RNV was induced in postnatal day 7(P7)mice by exposure to 75%oxygen.Donepezil was administrated to P12 mice by intraperitoneal injection.Expression and localization of AChE in mouse retinas were determined by immunofluorescence.RNV was evaluated by paraffin sectioning and hematoxylin and eosin(HE)staining.Activation of retinal M uller glial cells were examined by immunoblot of glial fibrillary acidic protein(GFAP).rMC-1.a retinal Muller cell line,was used for in vitro study.Expression of hypoxia-induced factor 1α(HIF-1α)and vascular endothelial growth factor(VEGF)were determined by Western-blot analysis,enzyme-linked immunosorbent assay(ELISA)or immunostaining.RESULTS:Aberrant RNV and glial activation was observed after OIR.Of note,retinal AChE was mainly expressed by retinal Muller glial cells and markedly increased in OIR mice.Systemic administration of donepezil significantly reduced RNV and abnormal glial activation in mice with OIR.Moreover,ischemia-induced HIF-1αaccumulation and VEGF upregulation in OIR mouse retinas and cultured rMC-1 were significantly inhibited by donepezil intervention.CONCLUSION:AchE is implicated in RNV with OIR.Inhibition of AChE by donepeizl is likely to be a potential therapeutic approach for retinal neovascular diseases.

关键词： acetylcholinesterase oxygen-induced retinopathy neovascularization glosis mice

Combined therapy with bevacizumab and photodynamic therapy for myopic choroidal neovascularization: A oneyear follow-up controlled study

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International Journal of Ophthalmology(English edition) 2014年第2期7卷 335-339页

作者： Sandro Saviano Rita Piermarocchi Pia E.Leon Alessandro Mangogna Andrea Zanei Fabiano Cavarzeran Sc Daniele Tognetto University Eye Clinic University of TriesteOspedale Maggiore Trieste 34129 Italy Eye Clinic of Padova University of Padova

AIMTo evaluate the efficacy and safety of a combined treatment for myopic choroidal neovascularization (CNV) using photodynamic therapy (PDT) and intravitreal bevacizumab and to compare it with intravitreal bevacizuma... 详细信息

关键词： bevacizumab combined therapy myopia neovascularization photodynamic therapy

Complement factor B knockdown by short hairpin RNA inhibits laser-induced choroidal neovascularization in rats

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International Journal of Ophthalmology(English edition) 2020年第3期13卷 382-389页

作者： Xin Wang Qing-Li Shang Jing-Xue Ma Shu-Xia Liu Cai-Xia Wang Cheng Ma Department of Ophthalmology the Second Hospital of Hebei Medical UniversityShijiazhuang 050000Hebei ProvinceChina Department of Pathology Hebei Medical UniversityShijiazhuang 050017Hebei ProvinceChina Department of Ophthalmology Tianjin First Central HospitalTianjin 300192China

AIM:To evaluate whether recombinant complement factor B(CFB)short hairpin RNA(sh RNA)reduces laserinduced choroidal neovascularization(CNV)in rats.METHODS:Laser-induced rat CNV model was established,and then the animals underwent fundus fluorescence angiography(FFA)and hematoxylin and eosin(HE)staining.On day 3 and 7 after photocoagulation,the expression of CFB and membrane attack complex(MAC)was detected by immunhischemistry.A recombinant CFBsh RNA plasmid was constructed.CFB and scrambled sh RNA plasmids were intravenous injected into rats via the tail vein on the day of laser treatment,respectively.On day 7,the incidence of CNV was determined by FFA,and the expression of CFB and vascular endothelial growth factor(VEGF)in retinal pigment epithelium(RPE)/choroidal tissues was detected by immunhischemistry,Western blot and/or semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR)in CFB and scrambled sh RNA groups.The possible adverse effects of CFB-sh RNA injection were assessed by transmission electron microscopy and electroretinography.RESULTS:FFA and HE results indicated that a laserinduced rat CNV model was successfully established on day 7 after photocoagulation.The expression of CFB and MAC was extremely weak in normal retina and choroid,and increased on day 3 after photocoagulation.However,it started to reduce on day 7.CFB sh RNA plasmid was successfully constructed and induced CFB knockdown in the retinal and choroidal tissues.FFA showed CFB knockdown significantly inhibited incidence of CNV in rats.Moreover,CFB knockdown significantly inhibited the expression of VEGF in RPE/choroidal tissues.CFB sh RNA caused no obvious side effects in eyes.CONCLUSION:CFB knockdown significantly inhibits the formation and development of CNV in vivo through reducing the expression of VEGF,which is a potential therapy target.The alternative pathway of complement activation plays an important role in CNV formation.

关键词： choroidal neovascularization complement factor B short hairpin RNA membrane attack complex vascular endothelial growth factor

Abrupt visual loss during anti-vascular endothelial growth factor treatment for type 3 neovascularization

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International Journal of Ophthalmology(English edition) 2019年第3期12卷 480-487页

作者： Jae Hui Kim Young Suk Chang Jong Woo Kim Chul Gu Kim Dong Won Lee Department of Ophthalmology Kim's Eye Hospital Konyang University College of Medicine Department of Ophthalmology Konyang University College of Medicine

AIM: To investigate the incidence of abrupt visual loss and its associated factors, during anti-vascular endothelial growth factor(VEGF) treatment for type 3 neovascularization.METHODS: This retrospective study included 137 eyes that were newly diagnosed with type 3 neovascularization. All eyes were treated with anti-VEGF therapy. Abrupt visual loss was defined as loss of 5 or more lines in best-corrected visual acuity(BCVA) in comparison to the previous visit. The incidence and timing of abrupt visual loss as well as the factors associated with it, were determined. In addition, the BCVA at the final follow-up was compared between the eyes with and those without abrupt visual loss.RESULTS: The mean follow-up period was 42.4±18.9 mo after diagnosis, and abrupt visual loss was noted in 22 eyes(16.1%) at a mean of 19.6±13.9 mo. Abrupt visual loss was found to be associated with subretinal hemorrhage in 11 eyes(50.0%), development of or increase in the height of pigment epithelial detachment with fluid in 8 eyes(36.4%), and tears in the retinal pigment epithelium in 3 eyes(13.6%). The logarithm of minimum angle of resolution(logMAR) mean BCVA at the final follow-up was 2.07±0.67(Snellen equivalents: 20/2349) and 1.00±0.55(20/200) in eyes with and without abrupt visual loss, respectively. BCVA was significantly worse in the eyes with abrupt visual loss(Pneovascularization and is associated with poor visual outcome. Additional studies are needed to determine how abrupt visual loss can be prevented.

关键词： age-related macular degeneration type 3 neovascularization retinal angiomatous proliferation hemorrhage tear in retinal pigment epithelium pigment epithelial detachment

Effect of nintedanib thermo-sensitive hydrogel on neovascularization in alkali burn rat model

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International Journal of Ophthalmology(English edition) 2020年第6期13卷 879-885页

作者： Yan Gong Guo-Hai Wu Ling-Yi Zhang Zhe Zhang Yan-Hong Liao Xiao-Tian Liu Department of Ophthalmology Ningbo Eye HospitalNingbo 315040Zhejiang ProvinceChina

AIM:To investigate the effects of nintedanib thermo-sensitive hydrogel(NTH)on neovascularization and related markers in corneal alkali burns of Wistar rats.METHODS:NTH was prepared by grinding,and its phase-transition temperature was determined.Thirty specific-pathogen-free Wistar rats served as a model of corneal alkali burn in the right eye were randomly divided into 3 groups(n=10,each):model group treated with 0.9%saline once a day,NTH group with 0.2%nintedanib b.i.d,and dexamethasone group with dexamethasone ointment once a day.The left eye of rats served as the controls.The corneal transparency was observed under a slit-lamp microscope,and the area of neovascularization was calculated.On day 7,the rats were sacrificed,and the cornea was removed and embedded with paraffin,then stained with hematoxylin一eosin,and the expression of vascular endothelial growth factor receptor 2(VEGFR-2)and CD31 in the corneal tissues of each group was detected by immunofluorescence.RESULTS:The phase-transition temperature ofnintedanib obtained by grinding was 37℃after adding artificial tears.The results of the alkali burn model indicated that the growth rate of neovascularization in the NTH group was slower than that in the model group,and the neovascularization area was significantly smaller than that in the model group(Pneovascularization of corneal induced by alkali burn in rats.

关键词： nintedanib alkali burn neovascularization cornea rat