PS持有加拿大研究主席在视网膜细胞生物学和爱尔康研究院新研究者奖。这项工作是由健康研究加拿大学院（221478）的资金支持，加拿大糖尿病协会（OG-3-11-3329-PS）的加拿大自然科学和工程研究理事会（418637）和基金会战斗失明加拿大。技术支持也由送货线路德RECHERCHE恩桑特德拉视觉魁北克提供。

伦理声明:所有动物实验坚持以研究协会在视觉与眼科在眼科用动物和视觉研究和动物保健的加拿大委员会（ARVO）语句建立的动物护理指引。 1。氧诱导视网膜病变（OIR） <ol><li>鼠标幼仔为P0出生记录日期。 </li><li>在进入O 2，记录动物的全部重量，以确保有足够的体重范围。注意:对于C57BL / 6小鼠在P17，体重应5和7.5 g的为最大NV 32系列 。为了保持环境的一致性，建议使用同窝对照（转基因小鼠和老鼠接受实验处理）。当评估一个病毒载体的作用，必须考虑病毒的嗜性，并允许有足够的时间对病毒交付的转基因充分表达。迅速表达病毒载体，如第三代慢病毒24，25，33，建议。 </li><li>将鼠标幼仔在P7（C57BL / 6或期望株）和CD1母亲培养成一个氧气室设定为75％的O 2 5天27。环境湿度和温度在整个O 2的曝光保持不变。注:配备的O 2源中央研究设施是理想，限制了繁琐的更换空氧气罐。如果使用转基因或基因敲除小鼠的工作，重要的是确保对照和实验小鼠被来自同一供应商获取到相同的菌株30，29内限制遗传漂移是重要的。 </li><li>在P12，从氧舱去除小鼠和动物返回到环境的O 2。 </li></ol> 2，玻璃体腔注射交货化合物的内层视网膜 （瓦时药理复合或重组蛋白的恩评估效果） <ol><li>在P14，麻醉小鼠中的氧气2升/分钟（或选择的动物保护委员会批准的麻醉剂）2％异氟醚。为了验证该麻醉的效果，顺序地捏住尾巴，后脚和耳钳。 </li><li>把鼠标在它的肚子。 </li><li>使用配有一个斜面拉到玻璃针的无菌10微升注射器进行注射1微升的溶液含有在眼睛的后缘被研究的化合物或赋形剂（生理盐水）的最大容积，具有45°的角度避开镜头。注意:该拉玻璃针是使用下拉环氧树脂附着到注射器。 </li><li>用棉签鼠标的眼睛滴一滴润滑剂眼药膏（理想的抗生素）的。 </li><li>返回鼠标回到笼子里培育的母亲。小鼠然后小心地监视，直到再覆盖和完全卧床。 </li></ol>容器灌注和屏障功能（完整性）3。通过荧光素血管造影评估<ol><li>麻醉小鼠中的氧气2升/分钟（或选择的动物保护委员会批准的麻醉剂）2％异氟醚。为了验证该麻醉的效果，顺序地捏住尾巴，后脚和耳钳。注意:该典型地在P17时再生进行评估。还随身携带了分析，在P19和P21，以确定是否血管的完整性被保留一段时间。 </li><li>一旦麻醉，称重鼠标。 </li><li>做中线的腹部切口解剖剪刀。注:解剖仪器应定期检查和激化。 </li><li>切肋骨横向和提高胸腔钳的帮助。注意:这是必要的，以切割为横向，以避免对心脏的损伤。 </li><li>去除perip后从大庆外围心脏组织，钳降主动脉与止血钳。 </li><li>慢慢地用25号针头向左心室注入荧光素 - 葡聚糖。注意:如果血管屏障功能进行了研究，70 kDa的荧光葡聚糖被采用，因为它会泄漏出的船只，当容器完整性受到损害。如果研究者希望投血管，2 MDA荧光葡聚糖被使用。关键的步骤:1）为了确保均匀的重新分区，离心机荧光葡聚糖和注入上清液，2）为了防止血管收缩，注入温热荧光葡聚糖溶液，3）它的循环时间不应该过量的4分钟。 </li><li>注射操作剪刀后斩首老鼠2分钟。 </li></ol> 4，眼球摘除和眼球固定注:在评估血管再生率，首先收集在视网膜P12和另外在P14和P17。增加采样时间点的数目S代表更精确地确定24血运重建率。 <ol><li>倾斜的鼠标头部，并将其放置在其一侧。 </li><li>去除皮肤和眼睑用解剖剪刀覆盖眼球。 </li><li>将弯钳眼睛下方轻轻向上拉，直到视神经被切断。 </li><li>把鼠标的头部到其另一侧，并执行相同的步骤（步骤4.2和4.3）。 </li><li>以确保固定剂更好的渗透，穿刺中，用30号针头在眼睛的前室中的孔。 </li><li>转移目光含4％多聚甲醛（PFA）管，且固定为1小时，在室温下进行。 </li><li>除去PFA，用冰冷的PBS溶液洗眼睛的4倍。 </li></ol> 5，视网膜解剖<ol><li>鼠标放置在眼里含冷PBS的培养皿中，并在立体显微镜下进行视网膜剥离。 </li><li>去除眼睛周围用显微解剖SCI多余的脂肪/组织ssors。 </li><li>切断与显微解剖剪刀角膜。 </li><li>使用两对钳子的，微小的剥离巩膜远离朝向视神经的周边并丢弃。 </li><li>用钳子捏住镜头（角膜下方的白色球）和眼罩提取它。用一对镊子作为支撑的，和对方的抓地力和小心地抬起并取下镜头。 </li><li>用小刷子（大小为0）和镊子分离从视网膜的内侧的玻璃体血管。 </li><li>删除使用产钳连接到视盘玻璃体血管束。 </li><li>解剖转移到视网膜开始之前染色过程含有PBS中，发生在ICE 2 ml离心管中。 </li></ol> 6，视网膜血管染色<ol><li>孵育解剖视网膜过夜，在4℃下轻轻摇动以溶液的荧光在PBS耦合凝集素B4（若丹明凝集素或其他），含1mM的CaCl 2 </子&gt;（1:100稀释的2毫克/毫升的植物凝集素B4的解决方案建议）。在整个染色过程中，覆盖管，用铝箔或不透明箔避光。 </li><li>在第二天，除去染色溶液和在PBS中于室温下10分钟，洗3次视网膜。 </li></ol>视网膜铺片7的制备<ol><li>转移视网膜，感光器的一面朝下，在显微镜载玻片上，并用外科手术刀来划分视网膜成四个相等大小的象限使得4深等距离径向切口。在切口，振奋视网膜用刷子，这样它不会移动。 </li><li>使用两把刷子浸泡在PBS中，仔细压平象限感光面朝下，沉浸在视网膜中的安装介质，以防止光漂白。然后小心地将盖玻片安装的视网膜表面上不施加压力，并确保气泡不会盖玻片下积聚。&lt;/ LI&gt; </ol> 8，成像和Vasoobliteration（VO）和新生血管（NV）的定量如前所述31 <ol><li>采取整体式视网膜图像的落射荧光显微镜在10倍的放大倍率。 </li><li>在照片编辑软件打开视网膜图像，缝合在一起，并测量视网膜总面积，无血管区。区域可以用像素表示。 </li><li>由像素的总的视网膜区域的数目除以在无血管区域的像素的数量确定VO的程度。 </li><li>除以NV的像素的数量由像素中的总的视网膜区域的描述31的数目确定NV的程度。 </li></ol>

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	<li>Carmeliet, P., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+mechanisms+of+nerve+and+blood+vessel+wiring.">Common mechanisms of nerve and blood vessel wiring.</a> Nature. 436, 193-200 (2005).</li><li>Eichmann, A., Thomas, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Parallels+between+Neural+and+Vascular+Development.">Molecular Parallels between Neural and Vascular Development.</a> Cold Spring Harb Perspect Med. 3, (2012).</li><li>Larrivee, B., Freitas, C., Suchting, S., Brunet, I., Eichmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+of+vascular+development:+lessons+from+the+nervous+system.">Guidance of vascular development: lessons from the nervous system.</a> Circ Res. 104, 428-441 (2009).</li><li>Stefater Iii, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+angiogenesis+by+a+non-canonical+Wnt-Flt1+pathway+in+myeloid+cells.">Regulation of angiogenesis by a non-canonical Wnt-Flt1 pathway in myeloid cells.</a> Nature. 474, 511-515 (2011).</li><li>Checchin, D., Sennlaub, F., Levavasseur, E., Leduc, M., Chemtob, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+role+of+microglia+in+retinal+blood+vessel+formation.">Potential role of microglia in retinal blood vessel formation.</a> Invest Ophthalmol Vis Sci. 47, 3595-3602 (2006).</li><li>Quaegebeur, A., Lange, C., Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurovascular+link+in+health+and+disease:+molecular+mechanisms+and+therapeutic+implications.">The neurovascular link in health and disease: molecular mechanisms and therapeutic implications.</a> Neuron. 71, 406-424 (2011).</li><li>Yamamoto, Y., Craggs, L., Baumann, M., Kalimo, H., Kalaria, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+molecular+genetics+and+pathology+of+hereditary+small+vessel+diseases+of+the+brain.">Review: molecular genetics and pathology of hereditary small vessel diseases of the brain.</a> Neuropathol Appl Neurobiol. 37, 94-113 (2011).</li><li>Brun, A., Englund, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+white+matter+disorder+in+dementia+of+the+Alzheimer+type:+a+pathoanatomical+study.">A white matter disorder in dementia of the Alzheimer type: a pathoanatomical study.</a> Ann Neurol. 19, 253-262 (1986).</li><li>Prat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+multiple+sclerosis+lymphocytes+through+brain+endothelium.">Migration of multiple sclerosis lymphocytes through brain endothelium.</a> Arch Neurol. 59, 391-397 (2002).</li><li>Rule, R. R., Schuff, N., Miller, R. G., Weiner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gray+matter+perfusion+correlates+with+disease+severity+in+ALS.">Gray matter perfusion correlates with disease severity in ALS.</a> Neurology. 74, 821-827 (2010).</li><li>Antonetti, D. A., Klein, R., Gardner, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+retinopathy.">Diabetic retinopathy.</a> N Engl J Med. 366, 1227-1239 (2012).</li><li>Hartnett, M. E., Penn, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+management+of+retinopathy+of+prematurity.">Mechanisms and management of retinopathy of prematurity.</a> N Engl J Med. 367, 2515-2526 (2012).</li><li>Sapieha, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinopathy+of+prematurity:+understanding+ischemic+retinal+vasculopathies+at+an+extreme+of+life.">Retinopathy of prematurity: understanding ischemic retinal vasculopathies at an extreme of life.</a> J Clin Invest. 120, 3022-3032 (2010).</li><li>Chen, J., Smith, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinopathy+of+prematurity.">Retinopathy of prematurity.</a> Angiogenesis. 10, 133-140 (2007).</li><li>Cheung, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+retinopathy+and+systemic+vascular+complications.">Diabetic retinopathy and systemic vascular complications.</a> Progress in Retinal and Eye Research. 27, 161-176 (2008).</li><li>Smith, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Through+the+eyes+of+a+child:+understanding+retinopathy+through+ROP+the+Friedenwald+lecture.">Through the eyes of a child: understanding retinopathy through ROP the Friedenwald lecture.</a> Invest Ophthalmol Vis Sci. 49, 5177-5182 (2008).</li><li>Caballero, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+vascular+damage+can+be+repaired+by+healthy,+but+not+diabetic,+endothelial+progenitor+cells.">Ischemic vascular damage can be repaired by healthy, but not diabetic, endothelial progenitor cells.</a> Diabetes. 56, 960-967 (2007).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-mediated+STAT3+activation+inhibits+retinal+vascularization+by+down-regulating+local+erythropoietin+expression.">VEGF-mediated STAT3 activation inhibits retinal vascularization by down-regulating local erythropoietin expression.</a> Am J Pathol. 180, 1243-1253 (2012).</li><li>Ritter, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+progenitors+differentiate+into+microglia+and+promote+vascular+repair+in+a+model+of+ischemic+retinopathy.">Myeloid progenitors differentiate into microglia and promote vascular repair in a model of ischemic retinopathy.</a> J Clin Invest. 116, 3266-3276 (2006).</li><li>Saito, Y., Geisen, P., Uppal, A., Hartnett, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+NAD(P)H+oxidase+reduces+apoptosis+and+avascular+retina+in+an+animal+model+of+retinopathy+of+prematurity.">Inhibition of NAD(P)H oxidase reduces apoptosis and avascular retina in an animal model of retinopathy of prematurity.</a> Mol Vis. 13, 840-853 (2007).</li><li>Connor, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+dietary+intake+of+omega-3-polyunsaturated+fatty+acids+reduces+pathological+retinal+angiogenesis.">Increased dietary intake of omega-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis.</a> Nat Med. 13, 868-873 (2007).</li><li>Banin, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T2-TrpRS+inhibits+preretinal+neovascularization+and+enhances+physiological+vascular+regrowth+in+OIR+as+assessed+by+a+new+method+of+quantification.">T2-TrpRS inhibits preretinal neovascularization and enhances physiological vascular regrowth in OIR as assessed by a new method of quantification.</a> Invest Ophthalmol Vis Sci. 47, 2125-2134 (2006).</li><li>Dorrell, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+retinal+astrocytes+normalizes+revascularization+and+prevents+vascular+pathology+associated+with+oxygen-induced+retinopathy.">Maintaining retinal astrocytes normalizes revascularization and prevents vascular pathology associated with oxygen-induced retinopathy.</a> Glia. 58, 43-54 (2010).</li><li>Joyal, J. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+neurons+prevent+vascular+regeneration+of+neural+tissue+by+secreting+semaphorin+3A.">Ischemic neurons prevent vascular regeneration of neural tissue by secreting semaphorin 3A.</a> Blood. 117, 6024-6035 (2011).</li><li>Binet, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+ER+Stress+Impedes+Myeloid-Cell-Induced+Vascular+Regeneration+through+IRE1alpha+Degradation+of+Netrin-1.">Neuronal ER Stress Impedes Myeloid-Cell-Induced Vascular Regeneration through IRE1alpha Degradation of Netrin-1.</a> Cell Metab. 17, 353-371 (2013).</li><li>Fukushima, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sema3E-PlexinD1+signaling+selectively+suppresses+disoriented+angiogenesis+in+ischemic+retinopathy+in+mice.">Sema3E-PlexinD1 signaling selectively suppresses disoriented angiogenesis in ischemic retinopathy in mice.</a> J Clin Invest. 121, 1974-1985 (2011).</li><li>Smith, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen-induced+retinopathy+in+the+mouse.">Oxygen-induced retinopathy in the mouse.</a> Invest Ophthalmol Vis Sci. 35, 101-111 (1994).</li><li>Lange, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+retinal+vaso-obliteration+and+neovascularisation+in+the+oxygen-induced+retinopathy+(OIR)+mouse+model.">Kinetics of retinal vaso-obliteration and neovascularisation in the oxygen-induced retinopathy (OIR) mouse model.</a> Graefes Arch Clin Exp Ophthalmol. 247, 1205-1211 (2009).</li><li>Connor, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+oxygen-induced+retinopathy+in+the+mouse:+a+model+of+vessel+loss,+vessel+regrowth+and+pathological+angiogenesis.">Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis.</a> Nature Protocols. 4, 1565-1573 (2009).</li><li>Stahl, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+retina+as+an+angiogenesis+model.">The mouse retina as an angiogenesis model.</a> Invest Ophthalmol Vis Sci. 51, 2813-2826 (2010).</li><li>Stahl, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-aided+quantification+of+retinal+neovascularization.">Computer-aided quantification of retinal neovascularization.</a> Angiogenesis. 12, 297-301 (2009).</li><li>Stahl, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+Weight+Gain+Modifies+Severity+and+Functional+Outcome+of+Oxygen-Induced+Proliferative+Retinopathy.">Postnatal Weight Gain Modifies Severity and Functional Outcome of Oxygen-Induced Proliferative Retinopathy.</a> Am J Pathol. 177, 2715-2723 (2010).</li><li>Cerani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-Derived+Semaphorin+3A+is+an+Early+Inducer+of+Vascular+Permeability+in+Diabetic+Retinopathy+via+Neuropilin-1.">Neuron-Derived Semaphorin 3A is an Early Inducer of Vascular Permeability in Diabetic Retinopathy via Neuropilin-1.</a> Cell Metabolism. 18, 505-518 (2013).</li><li>Sapieha, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eyeing+central+neurons+in+vascular+growth+and+reparative+angiogenesis.">Eyeing central neurons in vascular growth and reparative angiogenesis.</a> Blood. 120, 2182-2194 (2012).</li><li>Dorfman, A., Dembinska, O., Chemtob, S., Lachapelle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+manifestations+of+postnatal+hyperoxia+on+the+retinal+structure+and+function+of+the+neonatal+rat.">Early manifestations of postnatal hyperoxia on the retinal structure and function of the neonatal rat.</a> Invest Ophthalmol Vis Sci. 49, 458-466 (2008).</li><li>Dorfman, A. L., Joly, S., Hardy, P., Chemtob, S., Lachapelle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+oxygen+and+light+on+the+structure+and+function+of+the+neonatal+rat+retina.">The effect of oxygen and light on the structure and function of the neonatal rat retina.</a> Doc Ophthalmol. 118, 37-54 (2009).</li><li>Chopp, M., Zhang, Z. G., Jiang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis,+angiogenesis,+and+MRI+indices+of+functional+recovery+from+stroke.">Neurogenesis, angiogenesis, and MRI indices of functional recovery from stroke.</a> Stroke. 38, 827-831 (2007).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+and+improved+cerebral+blood+flow+in+the+ischemic+boundary+area+detected+by+MRI+after+administration+of+sildenafil+to+rats+with+embolic+stroke.">Angiogenesis and improved cerebral blood flow in the ischemic boundary area detected by MRI after administration of sildenafil to rats with embolic stroke.</a> Brain Res. 1132, 185-192 (2007).</li><li>Robinson, R., Barathi, V. A., Chaurasia, S. S., Wong, T. Y., Kern, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+animal+models+of+diabetic+retinopathy:+from+molecular+approaches+to+mice+and+higher+mammals.">Update on animal models of diabetic retinopathy: from molecular approaches to mice and higher mammals.</a> Dis Model Mech. 5, 444-456 (2012).</li><li>Chia, R., Achilli, F., Festing, M. F., Fisher, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+uses+of+mouse+outbred+stocks.">The origins and uses of mouse outbred stocks.</a> Nat Genet. 37, 1181-1186 (2005).</li><li>Jenuth, J. P., Peterson, A. C., Shoubridge, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+selection+for+different+mtDNA+genotypes+in+heteroplasmic+mice.">Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice.</a> Nat Genet. 16, 93-95 (1997).</li><li>Mattapallil, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rd8+mutation+of+the+Crb1+gene+is+present+in+vendor+lines+of+C57BL/6N+mice+and+embryonic+stem+cells,+and+confounds+ocular+induced+mutant+phenotypes.">The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes.</a> Investigative ophthalmolog., &amp; visual science. 53, 2921-2927 (2012).</li></ol>

作者什么都没有透露。

什么是刺激在缺血性神经组织中新血管的健康成长最有效的方法是什么？它是治疗有效的干预和加速自然发生的血管再生？在神经缺血性疾病，如缺血性视网膜病或中风，血管退化与减少神经元功能35-38相关联。因此，应对早期损伤，疾病的直接/早段期间复原区域微循环可能证明是有益的。在眼部背景下，实验范式是加速缺血性视网膜血管重建减少病理性新生血管形成21-25，34，?...

整个中枢神经系统发育，神经，免疫细胞和血管建立显着耦合网络，以确保有足够的组织灌注，并允许感官信息1-5传输。血管系统，导致组织氧合不足和代谢受到影响供给和故障日益被视为一个重要因素神经退行性疾病6的发病机制。血管漏失和神经血管单元的大脑内的劣化，例如，与血管性痴呆，脑7的白质血管病变和阿尔茨海默氏病与小动脉和小血管8的狭窄相关联。此外，受损的血管屏障功能被认为有助于多发性硬化症9和肌萎缩性侧索硬化症10。 直接的相关性，以在本协议中所述，致盲的视网膜模型的疾病，如糖 ​​尿病性视网膜病变11和早产儿12的视网膜病变，13的特征是早期血管退化的相位。对神经血管视网膜随后的缺血性应激触发过度和病理血管生成中的第二阶段中可能源于作为一种代偿性反应重新死刑犯氧和能量供应14-16。一个有吸引力的策略来克服缺血性应激是中央到疾病进展是恢复功能性血管网络特别是在神经视网膜（ 图2和3）的局部缺血区。挑起控制血管生成的响应可能会遇到如反直觉对于其中的抗血管生成治疗，如抗的VEGF被认为是适于处理的条件。然而，证据表明这种方法的有效性安装。例如，加强ischem"生理状"血管再生IC视网膜病变已经通过引进内皮前体细胞17，抑制缪勒细胞表达VEGF诱导的其他血管生成因子18，注射髓系祖细胞19，抑制NADPH氧化酶诱导的细胞凋亡20，增加饮食中ω-3多不饱和脂肪酸下调的优雅展现酸摄入21，具有色氨酸tRNA合成酶22的羧基末端片段和VEGF或FGF-2的保护胶质细胞23的直接给药治疗。此外，我们已经表明，调节神经古典的指导线索，如脑信号或导蛋白在缺血性视网膜病变加速视网膜内的良性血管的血管再生，从而减少病理性血管生成24，25。直接的临床意义，几个上述动物研究提供的证据表明，促进血管再生成期间视网膜病的早期缺血阶段可以显著减轻威胁视力的视网膜前新生血管形成19，23，24，26，有可能通过对缺血性负担的减轻。 制定刺激的功能性血管再生治疗策略仍然是血管生物学家一个显著的挑战。在这里，我们描述了一个实验系统，它采用的氧诱导视网膜病变（OIR）小鼠模型，探讨策略，视网膜内调节血管再生。 Smith 等人开发的。于1994年27，这种模式可作为人类增生性视网膜病变的代理，由露出P7鼠标幼仔至75％的O 2，直到P12和随后重新引进幼犬室内环境氧张力（ 图中1）。这一模式松散模仿一个场景:一个早产儿通风与O 2。鼠标幼仔高氧暴露引发视网膜毛细血管和微血管病变，并产生血管闭塞的可重复的区域（VO）通常在评估从O 2的出口在P12，虽然最大VO面积达到48小时（P9）后接触到O 2 28。在小鼠中，股骨头缺血性VO区自发地重新在一周的过程中下面再介绍了室内空气，并最终VO区域被完全重新血管化（ 图2）。重新引入到经受OIR小鼠的室内空气也引发视网膜前新生血管形成（NV）（最多为P17），它通常评估，以确定抗血管生成治疗模式的疗效。在其最纯粹的形式，OIR模型提供了一个高度可重复的，可量化的工具来评估氧 ​​诱导的血管退化，并确定破坏性预视网膜新生血管形成29-31的程度。 30,31被彻底说明。在这里，我们提供了一个简单的一步一步的过程由药物化合物，有意治疗，病毒载体进行调查生理血运重建的调节神经视网膜内或研究的候选基因在转基因或基因敲除小鼠的影响。

<table><tbody><tr><td>C57Bl/6 mice (Other strains may be used; angiogenic response varies from one strain to the other)</td><td></td><td></td><td></td></tr><tr><td>CD1 nursing mothers</td><td>Vendor of choice</td><td></td><td></td></tr><tr><td>Operating Scissors straight</td><td>World Precision Instruments</td><td>14192</td><td></td></tr><tr><td>Dissecting Scissors straight</td><td>World Precision Instruments</td><td>14393</td><td></td></tr><tr><td>Vannas Eye Scissors</td><td>Harvard Apparatus</td><td>72-8483</td><td></td></tr><tr><td>Iris Forceps, curved, serrated</td><td>World Precision Instruments</td><td>15915</td><td></td></tr><tr><td>Brushes 362R size 0</td><td>Dynasty</td><td></td><td></td></tr><tr><td>Dumont Forceps #3; straight</td><td>World Precision Instruments</td><td>500338</td><td></td></tr><tr><td>Surgical Blade, size 10</td><td>Bard-Parker</td><td>371110</td><td></td></tr><tr><td>Rhodamine Griffonia (Bandeiraea) Simplicifolia Lectin I</td><td>Vector Laboratories, Inc</td><td>RL-1102</td><td></td></tr><tr><td>Microscope slides</td><td>VWR</td><td>16004-368</td><td></td></tr><tr><td>Fluoromount G</td><td>Electron Microscopy Sciences</td><td>17984-25</td><td></td></tr><tr><td>Zeiss Axio Observer Z1 Inverted Phase and Fluorescence Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Leica MZ9.5 Stereomicroscope</td><td>Leica</td><td></td><td></td></tr><tr><td>Fluorescein isothicyanate-dextran, 70000</td><td>Sigma-Aldrich</td><td>46945</td><td></td></tr></tbody></table>

assessment of vascular regeneration in the cns using the mouse retina

啮齿动物视网膜也许是最方便的哺乳动物系统中，中枢神经系统（CNS）内神经血管调查的相互作用。它被越来越多地认识到，一些神经变性疾病如阿尔茨海默氏症，多发性硬化症，与血管妥协肌萎缩性侧索硬化症本元素。此外，失明在儿科和工作年龄人口（早产儿视网膜病变和糖尿病性视网膜病变，分别）的最突出的原因是其特征在于，生理血管再生长的血管退化和故障。本技术文件的目的是提供一个详细的协议来研究中枢神经系统血管再生的视网膜。该方法可以用来阐明导致血管生长的故障缺血性损伤后的分子机制。此外，潜在的治疗方式，以加速和恢复健康的血管丛可以探讨。结果obtaineD使用所描述的方法可以提供的治疗途径，缺血性视网膜病变，如糖尿病或早产儿，可能有利于中枢神经系统的其他血管疾病。

的OIR模型被广泛用于研究氧诱导血管变性和局部缺血引起的病理性新生血管在视网膜中和一直在为眼部疾病27，29，30目前使用的抗血管生成治疗的发展。利用该模型得到的结果可以大致推断缺血性视网膜病变，如增殖性糖尿病视网膜病变和30早产儿视网膜病变。 在这里，我们提出一个替代使用这个模型来研究血管再生。我们将描述一个策略的例子来再生，这?...

Watch this Scientific Journal Video about Assessment of Vascular Regeneration in the CNS Using the Mouse Retina at JoVE.com

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

啮齿动物视网膜长期以来被认为是可访问的窗口到大脑。在此技术论文中，我们提供了一个协议，它采用的氧诱导视网膜病变研究，导致血管再生的失败缺血性损伤后的中枢神经系统内的机制的小鼠模型。所描述的系统也可以利用，以探讨策略，视网膜和中枢神经系统内促进功能性血管再生。

血管再生在中枢神经系统中使用的小鼠视网膜评估

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

assessment-of-vascular-regeneration-in-the-cns-using-the-mouse-retina

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

14.3K 次观看.  McGill University. 啮齿动物视网膜长期以来被认为是可访问的窗口到大脑。在此技术论文中，我们提供了一个协议，它采用的氧诱导视网膜病变研究，导致血管再生的失败缺血性损伤后的中枢神经系统内的机制的小鼠模型。所描述的系统也可以利用，以探讨策略，视网膜和中枢神经系统内促进功能性血管再生。 

视频: Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

科研

神经科学

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

在视神经纤维通过诚信小鼠对比增强MRI 活体成像

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

之后，正常小鼠70％部分肝切除血管和实质再生的可视化

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

医学

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

罗登斯缺血性视网膜疾病的氧诱导视网膜病变模型

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

非增殖性和增殖性视网膜病变小鼠整个视网膜血管参数的定量

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

体内 小鼠视网膜血管损伤读数以促进可重复性

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

眼部微血管血管生成前维沃胆球状芽分析

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

血管再生在中枢神经系统中使用的小鼠视网膜评估

摘要

探索更多视频

此视频中的章节

在中央小鼠中枢神经系统的干细胞移植的多模态成像

整装免疫荧光染色新生小鼠视网膜血管生成调查

在视神经纤维通过诚信小鼠对比增强MRI 活体成像

之后，正常小鼠70％部分肝切除血管和实质再生的可视化

小鼠视网膜的经瞳双光子体内成像

罗登斯缺血性视网膜疾病的氧诱导视网膜病变模型

非增殖性和增殖性视网膜病变小鼠整个视网膜血管参数的定量

体内小鼠视网膜血管损伤读数以促进可重复性

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

眼部微血管血管生成前维沃胆球状芽分析

血管再生在中枢神经系统中使用的小鼠视网膜评估

摘要

探索更多视频

此视频中的章节

在中央小鼠中枢神经系统的干细胞移植的多模态成像

整装免疫荧光染色新生小鼠视网膜血管生成调查

在视神经纤维通过诚信小鼠对比增强MRI 活体成像

之后，正常小鼠70％部分肝切除血管和实质再生的可视化

小鼠视网膜的经瞳双光子体内成像

罗登斯缺血性视网膜疾病的氧诱导视网膜病变模型

非增殖性和增殖性视网膜病变小鼠整个视网膜血管参数的定量

体内 小鼠视网膜血管损伤读数以促进可重复性

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

眼部微血管血管生成前维沃胆球状芽分析

体内小鼠视网膜血管损伤读数以促进可重复性