两种脉络膜血管铺片法对实验性脉络膜新生血管定量研究的比较
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摘要
目的比较两种脉络膜血管铺片法对实验性脉络膜新生血管(CNV)进行定量研究的效果。设计实验研究。研究对象BN大鼠。方法正常BN大鼠24只随机分为两组,采用相同直径激光斑诱导CNV模型,于光凝后1、2、4周分别采用荧光素异硫氰酸酯-葡聚糖(FITC-D)灌注法及凝集素法进行脉络膜血管铺片,激光共聚焦显微镜照相,Image J图像分析软件测量CNV面积。以BN大鼠麻醉开始,到脉络膜血管铺片结束计时,计算两种方法所需实验时间。以BN大鼠进行脉络膜血管铺片所消耗的FITC-D或者凝集素的剂量,计算每只标本实验费用。主要指标CNV面积、实验时间及实验费用。结果 BN大鼠光凝后1、2、4周,FITC-D灌注法及凝集素法的CNV面积(μm~2)分别为5524±1847、50653±7265、121623±7128及14560±4508、144209±8411、204066±8546;实验时间(单位:分钟)分别为119±21及390±34;实验费用(单位:元)分别289±26.3及32±4.2。结论两种方法因实验原理不同,造成CNV面积、实验时间及实验费用等方面均存在差异,实验者可以根据各自实验目的选择相应方法进行CNV相关研究。(眼科,2019, 28:120-124)
Objective To compare two methods of choroid flat mounts in quantitative study for experimental choroidal neovascularization(CNV). Design Experimental study. Participants Brown-Norway(BN) rats. Methods Normal 24 BN rats were randomly divided into two groups equally and CNV models were induced by laser photocoagulation with the same diameter spot. Choroid flat mounts were performed with fluorescein isothiocyanate-dextran(FITC-D) perfusion method or isolectin method at 1 week(W), 2 W, and 4 W after photocoagulation respectively. The photo of choroid flat mounts was taken under laser confocal microscope and the area of CNV was measured with Image J software. The experimental time was recorded form the start of anaesthesia to the end of choroid flat mounts. The expense of one choroid flat mount was calculated by the dose of FITC-D or lectin consumed in the experiment. Main Outcome Measures Area of CNV, experimental time and expense. Results At 1 W, 2 W, and 4 W after photocoagulation, the areas(μm~2) of CNV by FITC-D perfusion method or isolectin method were 5524±1847, 50653±7265, 121623±7128 or 14560±4508, 144209±8411, 204066±8546 respectively. The experimental time by two different methods was 119±21 min or 390±34 min respectively, while the experimental expense was 289±26.3$ or 32±4.2$ respectively. Conclusion There were obvious differences in the area of CNV, experimental time and expense between the 2 methods of choroid flat mounts. The appropriate method of choroid flat mounts for experimental CNV should be carefully selected according to the research purposes.(Ophthalmol CHN, 2019, 28: 120-124)
Objective To compare two methods of choroid flat mounts in quantitative study for experimental choroidal neovascularization(CNV). Design Experimental study. Participants Brown-Norway(BN) rats. Methods Normal 24 BN rats were randomly divided into two groups equally and CNV models were induced by laser photocoagulation with the same diameter spot. Choroid flat mounts were performed with fluorescein isothiocyanate-dextran(FITC-D) perfusion method or isolectin method at 1 week(W), 2 W, and 4 W after photocoagulation respectively. The photo of choroid flat mounts was taken under laser confocal microscope and the area of CNV was measured with Image J software. The experimental time was recorded form the start of anaesthesia to the end of choroid flat mounts. The expense of one choroid flat mount was calculated by the dose of FITC-D or lectin consumed in the experiment. Main Outcome Measures Area of CNV, experimental time and expense. Results At 1 W, 2 W, and 4 W after photocoagulation, the areas(μm~2) of CNV by FITC-D perfusion method or isolectin method were 5524±1847, 50653±7265, 121623±7128 or 14560±4508, 144209±8411, 204066±8546 respectively. The experimental time by two different methods was 119±21 min or 390±34 min respectively, while the experimental expense was 289±26.3$ or 32±4.2$ respectively. Conclusion There were obvious differences in the area of CNV, experimental time and expense between the 2 methods of choroid flat mounts. The appropriate method of choroid flat mounts for experimental CNV should be carefully selected according to the research purposes.(Ophthalmol CHN, 2019, 28: 120-124)
引文
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[8]刘璐,莫宾,焦剑,等.组织病理学切片与频域OCT对大鼠视网膜组织形态的对比研究.眼科,2013, 22(5):344-348.
[9] Gholipour MA, Kanavi MR, Ahmadieh H, et al. Intravitreal topotecan inhibits laser-induced choroidal neovascularization in a rat model. J Ophthalmic Vis Res, 2015, 10(3):295-302.
[10]李娜,窦国睿,张萍,等.Notch信号对激光诱导的小鼠CNV生成过程中巨噬细胞极化表型及功能的调控.中华实验眼科杂志,2015, 33(3):207-215.
[11] Lambert V, Lecomte J, Hansen S, et al. Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice. Nat Protoc, 2013, 8(11):2197-2211.
[12] Lecomte J, Louis K, Detry B, et al. Bone marrow-derived mesenchymal cells and MMP13 contribute to experimental choroidal neovascularization. Cell Mol Life Sci, 2011, 68(4):677-686.
[13] Biber K, Neumann H, Inoue K, et al. Neuronal'On'and'Off'signals control microglia. Trends Neurosci, 2007, 30(11):596-602.
[14] Santiago AR, Baptista FI, Santos PF, et al. Role of microglia adenosine A(2A)receptors in retinal and brain neurodegenerative diseases. Mediators Inflamm, 2014, 2014:465694.
[15] Eter N, Engel DR, Meyer L, et al. In vivo visualization of dendritic cells, macrophages, and microglial cells responding to laser-induced damage in the fundus of the eye. Invest Ophthalmol Vis Sci, 2008, 49(8):3649-3658.
[16] Liu J, Copland DA, Horie S, et al. Myeloid cells expressing VEGF and arginase-1 following uptake of damaged retinal pigment epithelium suggests potential mechanism that drives the onset of choroidal angiogenesis in mice. PLoS One, 2013, 8(8):e72935.
[17] Ma W, Zhao L, Fontainhas AM, et al. Microglia in the mouse retina alter the structure and function of retinal pigmented epithelial cells:a potential cellular interaction relevant to AMD. PLoS One,2009, 4(11):e7945.
[18] Checchin D, Sennlaub F, Levavasseur E, et al. Potential role of microglia in retinal blood vessel formation. Invest Ophthalmol Vis Sci, 2006, 47(8):3595-3602.
[19] Fischer F, Martin G, Agostini HT. Activation of retinal microglia rather than microglial cell density correlates with retinal neovascularization in the mouse model of oxygen-induced retinopathy. J Neuroinflammation, 2011, 8:120.
[20] Rymo SF, Gerhardt H, Wolfhagen SF, et al. A two-way communication between microglial cells and angiogenic sprouts regulates angiogenesis in aortic ring cultures. PLoS One, 2011, 6(1):e15846.
[2] Grossniklaus HE, Kang SJ, Berglin L. Animal models of choroidal and retinal neovascularization. Prog Retin Eye Res, 2010, 29(6):500-519.
[3] Edelman JL, Castro MR. Quantitative image analysis of laser-induced choroidal neovascularization in rat. Exp Eye Res, 2000, 71(5):523-533.
[4] Campos M, Amaral J, Becerra SP, et al. A novel imaging technique for experimental choroidal neovascularization. Invest Ophthalmol Vis Sci, 2006, 47(12):5163-5170.
[5] Park JR, Choi W, Hong HK, et al. Imaging laser-induced choroidal neovascularization in the rodent retina using optical coherence tomography angiography. Invest Ophthalmol Vis Sci, 2016, 57(9):T331-T340.
[6] Qiu F, Matlock G, Chen Q, et al. Therapeutic effects of PPARalpha agonist on ocular neovascularization in models recapitulating neovascular age-related macular degeneration. Invest Ophthalmol Vis Sci, 2017, 58(12):5065-5075.
[7] Jiao J, Mo B, Wei H, et al. Comparative study of laser-induced choroidal neovascularization in rats by paraffin sections, frozen sections and high-resolution optical coherence tomography. Graefes Arch Clin Exp Ophthalmol, 2013, 251(1):301-307.
[8]刘璐,莫宾,焦剑,等.组织病理学切片与频域OCT对大鼠视网膜组织形态的对比研究.眼科,2013, 22(5):344-348.
[9] Gholipour MA, Kanavi MR, Ahmadieh H, et al. Intravitreal topotecan inhibits laser-induced choroidal neovascularization in a rat model. J Ophthalmic Vis Res, 2015, 10(3):295-302.
[10]李娜,窦国睿,张萍,等.Notch信号对激光诱导的小鼠CNV生成过程中巨噬细胞极化表型及功能的调控.中华实验眼科杂志,2015, 33(3):207-215.
[11] Lambert V, Lecomte J, Hansen S, et al. Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice. Nat Protoc, 2013, 8(11):2197-2211.
[12] Lecomte J, Louis K, Detry B, et al. Bone marrow-derived mesenchymal cells and MMP13 contribute to experimental choroidal neovascularization. Cell Mol Life Sci, 2011, 68(4):677-686.
[13] Biber K, Neumann H, Inoue K, et al. Neuronal'On'and'Off'signals control microglia. Trends Neurosci, 2007, 30(11):596-602.
[14] Santiago AR, Baptista FI, Santos PF, et al. Role of microglia adenosine A(2A)receptors in retinal and brain neurodegenerative diseases. Mediators Inflamm, 2014, 2014:465694.
[15] Eter N, Engel DR, Meyer L, et al. In vivo visualization of dendritic cells, macrophages, and microglial cells responding to laser-induced damage in the fundus of the eye. Invest Ophthalmol Vis Sci, 2008, 49(8):3649-3658.
[16] Liu J, Copland DA, Horie S, et al. Myeloid cells expressing VEGF and arginase-1 following uptake of damaged retinal pigment epithelium suggests potential mechanism that drives the onset of choroidal angiogenesis in mice. PLoS One, 2013, 8(8):e72935.
[17] Ma W, Zhao L, Fontainhas AM, et al. Microglia in the mouse retina alter the structure and function of retinal pigmented epithelial cells:a potential cellular interaction relevant to AMD. PLoS One,2009, 4(11):e7945.
[18] Checchin D, Sennlaub F, Levavasseur E, et al. Potential role of microglia in retinal blood vessel formation. Invest Ophthalmol Vis Sci, 2006, 47(8):3595-3602.
[19] Fischer F, Martin G, Agostini HT. Activation of retinal microglia rather than microglial cell density correlates with retinal neovascularization in the mouse model of oxygen-induced retinopathy. J Neuroinflammation, 2011, 8:120.
[20] Rymo SF, Gerhardt H, Wolfhagen SF, et al. A two-way communication between microglial cells and angiogenic sprouts regulates angiogenesis in aortic ring cultures. PLoS One, 2011, 6(1):e15846.