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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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 border="0" cellpadding="0" cellspacing="0" width="1224">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">C57Bl/6 mice ((Other strains may be used; angiogenic response varies from one strain to the other)</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:639px;">CD1 nursing mothers</td>
			<td style="width:181px;">Vendor of choice</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Operating Scissors straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">14192</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Dissecting Scissors straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">14393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Vannas Eye Scissors</td>
			<td style="width:181px;">Harvard Apparatus</td>
			<td style="width:175px;">72-8483</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Iris Forceps, curved, serrated</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">15915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Brushes 362R size 0</td>
			<td style="width:181px;">Dynasty</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Dumont Forceps #3; straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">500338</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Surgical Blade, size 10</td>
			<td style="width:181px;">Bard-Parker</td>
			<td style="width:175px;">371110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Rhodamine Griffonia (Bandeiraea) Simplicifolia Lectin I</td>
			<td style="width:181px;">Vector Laboratories, Inc</td>
			<td style="width:175px;">RL-1102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Microscope slides</td>
			<td style="width:181px;">VWR</td>
			<td style="width:175px;">16004-368</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Fluoromount G</td>
			<td style="width:181px;">Electron Microscopy Sciences</td>
			<td style="width:175px;">17984-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Zeiss Axio Observer Z1 Inverted Phase and Fluorescence Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:639px;">Leica MZ9.5 Stereomicroscope</td>
			<td rowspan="2" style="width:181px;">Leica</td>
			<td rowspan="2" style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Fluorescein isothicyanate-dextran, 70000</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:175px;">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). ...

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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

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

量化的活动顺</em在小鼠视网膜>调控元件外植体电穿孔

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

科研

神经科学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

角膜微囊含量：血管生成在小鼠眼模型

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

成像钙<sup&gt; 2+</sup&gt;在小鼠视网膜锥光感受器轴突终末动态

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

鼠标显微注水技术进行有针对性的物质传递到中枢神经系统<i&gt;通过</i&gt;的颈内动脉

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

哺乳动物轴突再生: 成年小鼠背根神经节体内电穿孔的研究

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

间刺激间隔对啮齿类动物时间处理评估的力量

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

鼠脑水平海马片

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

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 ...