我们感谢克里斯汀·约翰逊的图形工作。

所有涉及动物的协议是在波士顿儿童医院提交并经实验动物管理和使用委员会，并按照协会评估实验动物照护及认可委员会（AAALAC）的建议进行的。请务必使用消毒仪器和无菌techniqure在执行的过程。 1，制备的颗粒<ol><li>称取硫糖铝10毫克，并用无菌展平铲60毫克海昌的。发生在不同的离心管。 </li><li>将1厘米见方的块网格进入无菌10cm皿。反转所以目休息的浅盖。抛开。 </li><li>加入500μl的乙醇，以在昌，涡旋至少10分钟。 </li><li>添加生长因子（标准：20微克的bFGF和50微克的血管内皮生长因子），适量的硫糖铝和涡简要介绍。在商店里生长因子-80℃的冰箱中以1毫克/毫升的浓度。补充液体量应至少为20微升，以确保硫糖铝完全润湿，但不得超过50微升。更大的体积，使液体中的下一个步骤的蒸发困难。 </li><li>将硫糖铝混合物倒入离心蒸发器在低设置，直到混合物完全干燥，通常30-50分钟取决于液体用的卷上。 <ol><li>检查残留水分的混合物，用无菌刮刀的锥形端。该混合物应该感到“脆脆”。 </li></ol></li><li>使用刮刀的锥形端，以在管内打破了硫糖铝混合物。 </li><li>加入10微升昌与胃溃宁。 <ol><li>由于海昌的粘稠性，降低从200微升枪头年底再画，再吹打起来，加入到硫糖铝之前释放10微升。 </li></ol></li><li>赶紧使用弯曲的口水战乌拉混合的海昌和硫糖铝和管取出。 <ol><li>收集沿着弯曲刮铲尖的底侧的混合物，然后旋转该管的同时绘制抹刀沿所述管的壁。该混合物将迅速干燥所以这一步必须高效地完成。 </li></ol></li><li>用钳子将其固定到位混合物涂抹在准备网格。该混合物应为偶数层，填充到网格的孔中。 </li><li>沉浸在弯曲刮铲入含管昌和使用目的是外套两侧。 </li><li>将涂目靠在一边对菜的边缘，并允许在室温下干燥30-45分钟。 </li><li>放置在-20℃冰箱网格或立即进行下一个步骤。保持网内盘。 </li><li>将35毫米的菜大培养皿中，盖子里面。用钳子小心地拉网的纤维间隔超过35毫米的菜。流浪小球ç一个由在年底较大盘收集。 <ol><li>检查颗粒的均匀性下的范围。这个过程会产生约250颗粒因而每个块的标准剂量bFGF和VEGF是80纳克和200纳克，分别为。 </li></ol></li><li>商店粒料在35mm培养皿中的-20℃的冰箱中长达3个月。 </li></ol>球团2手术植入<ol><li>设立手术显微镜下手术区域。 </li><li>麻醉小鼠用阿佛丁（400-500毫克/千克，腹膜内），并在显微镜下将鼠标所以眼是可见的。执行脚趾捏，以确保麻醉足够的水平。 </li><li>通过将1滴眼球之上的解决方案丙美卡因麻​​醉小鼠眼球。等待20-30秒，轻拍用纱布垫。 </li><li> Proptose鼠标眼睛的钝＃1珠宝商镊子是一定要离开钳和眼睛之间的皮肤。它来判断正常施加的压力是很重要的。60;拿着眼睛太用力会导致它变成红色并有点烦，而太松一抱让眼睛在手术过程中移动。 </li><li>使用30°micr​​oknife使切口入角膜的角膜缘约1毫米。切口应是1-2毫米长。切口应足够深，以穿透外上皮细胞层到midstroma但没有那么深破裂的眼睛。 </li><li>用冯格赖夫刀做一个口袋垂直切口。 <ol><li>滑动角膜层下方的刀在切口部位并在轻轻地刀和移动它沿切口扩大的空间中，从而形成口袋。它有助于移动鼠标，以及能够与眼球的曲率工作。要小心，不要用刀尖向下推，以避免破裂的眼球。 </li></ol></li><li>湿＃5珠宝商的镊子与丙美卡因和挑球团起来了菜。放置在眼睛的颗粒。 </lI&gt; <li>滋润冯格赖夫刀丙美卡因和转移一些液体颗粒上，使橡胶和柔韧。去除多余的水分用纱布垫或棉签。 </li><li>将沉淀到使用冯格赖夫刀到它里面推角膜层下方的口袋。一旦整个粒料是内部运行的冯格赖夫刀的平面侧上的网站，以检查该粒料是安全的。 </li><li>涂眼三重抗生素软膏。 </li><li>把鼠标和在相反的眼睛重复手术。 </li></ol> 3，定量角膜新生血管。 让动物发展船舶在设定的时间段。容器等级的天取决于所用的生长因子。级bFGF对第6天5天，较弱的血管内皮生长因子植入的当天为0天。 <ol><li>麻醉用阿佛丁鼠标（400-500毫克/千克，腹膜内）。 </li><li>用裂隙灯显微镜GRA丁。一个眼有作为测量辅助量化血管长度和距离的眼部周围的周长，该船只已萌生目镜内的手提袋，行。 <ol><li>按住鼠标的范围前，定位鼠标指针的眼睛被认为是在眼部与球团正前方。稳定的拇指和食指之间的头，使皮肤收紧面部和眼部轻微proptosed。 </li></ol></li><li>放置在手提包的y轴沿角膜缘容器直接沉淀的下方。测量血管分支向上朝向丸粒的长度。记录此测量以毫米的十分之一，并指定容器长度（VL）（ 图1A）。 </li><li>转动鼠标或分划板，使眼部周围的距离，这些船只如雨后春笋变得清晰。它是最容易想到的眼睛作为时钟面具有1间隔的通过12候这个数目时钟小时（CH）（ 图1中B）。它可以是一个整数或为0.25（ 例如，2，2.25，2.5或2.75）的级分。注：CH是主观的。例如，一些可以测量眼部周围的距离，包括来自角膜缘萌生，而其他人可能会停止在该点所在船只长度为半最大测量所有船只。的重要因素，是要与一个如何选择等级相一致。 </li></ol><img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51375/51375fig1highres.jpg" src="/files/ftp_upload/51375/51375fig1.jpg" /> 图1：定量角膜新生血管（A）80纳克的bFGF颗粒植入C57BL / 6J小鼠显示出裂隙灯手提袋导向来衡量血管长度。 VL = 0.9毫米。 （二）同一只眼睛的表现裂隙灯手提袋旋转测量时钟小时。 CH = 3.25。计算出的血管面积= 1.84毫米2。这些图像被数字提高到允许添加划板原理图， <a href="https://www.jove.com/files/ftp_upload/51375/51375fig1highres.jpg" target="_blank">请点击这里查看大图。</a> <ol start="5"><li>计算血管面积（VA）使用下面的公式，它是基于椭圆的面积（注意，这是有帮助的分级时，记住这一点。）特快VA在平方毫米。其计算公式如下： Vessel长度x时钟小时×0.2 =π血管面积</li></ol>

<ol>
	<li>Gospodarowicz, D., Moran, J., Braun, D., Birdwell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+growth+of+bovine+vascular+endothelial+cells:+fibroblast+growth+factor+as+a+survival+agent.">Clonal growth of bovine vascular endothelial cells: fibroblast growth factor as a survival agent.</a> Proc. Natl. Acad. Sci. USA. 73, 4120-4124 (1976).</li><li>Glaser, B. M., D'Amore, P. A., Seppa, H., Seppa, S., Schiffmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+tissues+contain+chemoattractants+for+vascular+endothelial+cells.">Adult tissues contain chemoattractants for vascular endothelial cells.</a> Nature. 288, 483-484 (1980).</li><li>Kubota, Y., Kleinman, H. K., Martin, G. R., Lawley, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+laminin+and+basement+membrane+in+the+morphological+differentiation+of+human+endothelial+cells+into+capillary-like+structures.">Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures.</a> J. Cell Biol. 107, 1589-1598 (1988).</li><li>Montesano, R., Orci, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-promoting+phorbol+esters+induce+angiogenesis+in+vitro.">Tumor-promoting phorbol esters induce angiogenesis in vitro.</a> Cell. 42, 469-477 (1985).</li><li>Korff, T., Augustin, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+endothelial+cells+in+multicellular+spheroids+prevents+apoptosis+and+induces+differentiation.">Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation.</a> J. Cell Biol. 143, 1341-1352 (1998).</li><li>Nicosia, R. F., Tchao, R., Leighton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histotypic+angiogenesis+in+vitro:+light+microscopic,+ultrastructural,+and+radioautographic+studies.">Histotypic angiogenesis in vitro: light microscopic, ultrastructural, and radioautographic studies.</a> In Vitro. 18, 538-549 (1982).</li><li>Wong, K. H., Chan, J. M., Kamm, R. D., Tien, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+models+of+vascular+functions.">Microfluidic models of vascular functions.</a> Annu Rev Biomed Eng. 14, 205-230 (2012).</li><li>Sandison, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+microscopic+study+of+living+growing+tissues+by+the+introduction+of+a+transparent+chamber+in+the+rabbit's+ear.">A new method for the microscopic study of living growing tissues by the introduction of a transparent chamber in the rabbit's ear.</a> Anat. Rec. 28, 281-287 (1924).</li><li>Jain, R. K., Schlenger, K., Hockel, M., Yuan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+angiogenesis+assays:+progress+and+problems.">Quantitative angiogenesis assays: progress and problems.</a> Nat. Med. 3, 1203-1208 (1997).</li><li>Rogers, M. S., Birsner, A. E., D'Amato, R. J. <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+cornea+micropocket+angiogenesis+assay.">The mouse cornea micropocket angiogenesis assay.</a> Nat. Protoc. 2 (10), 2545-2550 (2007).</li><li>Gimbrone, M. A., Leapman, S. B., Cotran, R. S., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+dormancy+in+vivo+by+prevention+of+neovascularization.">Tumor dormancy in vivo by prevention of neovascularization.</a> J. Exp. Med. 136, 261-276 (1972).</li><li>Gimbrone, M. A., Cotran, R. S., Leapman, S. B., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+growth+and+neovascularization:+an+experimental+model+using+the+rabbit+cornea.">Tumor growth and neovascularization: an experimental model using the rabbit cornea.</a> J. Natl. Cancer Inst. 52, 413-427 (1974).</li><li>Rohan, R. M., Fernandez, A., Udagawa, T., Yuan, J., D'Amato, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+heterogeneity+of+angiogenesis+in+mice.">Genetic heterogeneity of angiogenesis in mice.</a> FASEB J. 14 (7), 871-876 (2000).</li><li>Chang, L. K., 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=Dose-dependent+response+of+FGF-2+for+lymphangiogenesis.">Dose-dependent response of FGF-2 for lymphangiogenesis.</a> Proc Natl Acad Sci U S A. 101 (32), 11658-11663 (2004).</li><li>Udagawa, T., Birsner, A. E., Wood, M., D'Amato, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+suppression+of+angiogenesis+following+radiation+exposure+is+independent+of+hematopoietic+reconstitution.">Chronic suppression of angiogenesis following radiation exposure is independent of hematopoietic reconstitution.</a> Cancer Res. 67 (5), 2040-2045 (2007).</li><li>Turner, P. V., Albassam, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+rats+to+corneal+lesions+after+injectable+anesthesia.">Susceptibility of rats to corneal lesions after injectable anesthesia.</a> Med Comp. 55 (2), 175-182 (2005).</li><li>Calderone, L., Grimes, P., Shalev, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+reversible+cataract+induced+by+xylazine+and+by+ketamine-xylazine+anesthesia+in+rats+and+mice.">Acute reversible cataract induced by xylazine and by ketamine-xylazine anesthesia in rats and mice.</a> Exp. Eye Res. 42, 331-337 (1986).</li></ol>

作者什么都没有透露。

有在执行一个成功的角膜检测的几个关键步骤。首先是制作能够被植入并刺激血管均匀的颗粒。粒料制备中最重要的部件是：1）使用无载体的生长因子; 2）确保生长因子与硫糖铝和海昌和3个好混合物）迅速但仔细一动最终混合物从Eppendorf管到的颗粒投网。我们建议进行“虚拟”颗粒无生长因子练技术。球团应该会出现白色和粉化，并大致呈正方形。这包括明确的区域的任何颗粒，应排除在外，?...

血管生成的过程中，新血管的形成，形成预先存在的那些，是非常复杂的，并通过控制不同的步骤血管出芽和形态发生的几内源性因子的调节。血管生成是由于亲和抗血管生成因子之间的转变，平衡，通常保持在静止状态下的脉管系统的平衡被触发。血管发生在成人中发生一定的生理条件，如女性卵巢周期期间或在维修过程中如伤口愈合和组织再生。然而，它也是几种病状，包括恶性肿瘤，自身免疫性疾病和炎症性疾病的标志。血管生成的这些生理和病理条件下参与使得它对于研究和治疗的一个有吸引力的目标的一个重要课题。 由于血管发生的复杂性和几种策的参与LLS和因素的过程中，包括内皮细胞，周细胞，循环细胞和基质细胞， 在体外模型中仍是有限的，不能概括的唯一的体内微环境。 在血管生成的体外测定法的主要在很大程度上集中于观察对内皮细胞的直接影响和控制的条件下测量在血管生成过程中的某些步骤。这些测定法包括内皮细胞的增殖1，迁移2，网络形成三管形成4的量化，并从球状体5发芽。 体外模型，不同的是在体外的人，都比较复杂并包含多种组织类型的细胞，一个例子是主动脉环测定法6。然而，像其他系统，它不能在体内存在的捕获循环细胞和贡献内皮细胞的天然基质。尝试研究根据流量的血管生成模拟体内环境使用微流体系统7中执行，但即使是这些试验中，虽然大大改善，仍无法考虑到所有存在于体内的车厢。 由于在体外和离体血管生成模型中，限制在体内模型仍然是血管生成的研究更可靠的选择。这些模型的实例包括透明腔室，“窗口”，即允许的生长血管显微镜8下的可视化的植入剂，注射皮下植入物，如基质胶和血管形成中的正常组织，如小鼠耳和鸡胚绒毛尿囊膜（CAM ）。然而，最可接受的和定量的体内血管生成的模型之一是在此描述的角膜微囊新血管形成测定法，其利用了自然股骨头缺血性角膜作为一个“屏”的可视化和评估新的血管生长9。 在这里，我们描述了角膜微囊测定作为开发中的小鼠。最初的模型被用来测量在兔角膜的非特异性血管生成的刺激。这是通过引入肿瘤块放入兔眼的前房的前房进行测定和肿瘤诱导的血管生成11。 然而，该法后来演变来研究特定生长因子的影响10，以更好地说明和规范血管生成作用。为了释放生长因子在眼内，来代替组织中含有已知量的血管生成生长因子的缓释颗粒。纯化的重组血管生成蛋白如的bFGF或VEGF的可用性使他们的血管生成modulaters 12的具体目标使用。最初，该测定是主要用于在兔，这是更容易使用，因为它们的大小，但后来的模型被翻译成小鼠正常工作;更小和更便宜的动物模型。从兔转移到小鼠的效果是：能够使用遗传操作的动物，从而产生一个新的研究领域进入遗传组分影响血管13的一个重要优点。除了角膜法的研究血管生成的多种可接受的使用，其他的生物学过程，也被用改性试验研究。例如，淋巴管生成的研究，通过低剂量的bFGF球团这使淋巴管，通过特定的分子标记14的可视化的植入能够成为可能。此外，该测定中提供了评估血管生成15辐射的影响的装置。 在总结中，角膜微囊血管生成分析是定量的，代表roducible，血管生成的体内灵活的评估。该测定的主要优点是，背景血管测量是不必要的，因为血管生长在天然无血管组织。我们在这里描述这个实验的详细协议，并讨论可能出现不同的情况。该试验由3离散区段。在这里，我们将描述的生长因子包括粒料用于量化所得到的新血管生长的制备，随后的外科手术植入，最后的方法。

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			<td height="21" style="height:21px;width:216px;">Section 1: Pellet preparation</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:216px;">Sucralfate</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">#S0652</td>
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			<td height="21" style="height:21px;width:216px;">Hydron (aka Poly(2-Hema))</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">#P3932-10G</td>
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			<td height="42" style="height:42px;width:216px;">Ethanol</td>
			<td style="width:111px;">Pharmco Products Inc</td>
			<td style="width:119px;">#111000200CSGL</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Growth factors:</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Must be carrier-free (no bovine serum albumin(BSA))</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Fibroblast growth factor (FGF)</td>
			<td style="width:111px;">PeproTech</td>
			<td style="width:119px;">#AF-100-18B</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vascular endothelial growth factor (VEGF)</td>
			<td style="width:111px;">R &#38; D Systems</td>
			<td style="width:119px;">#293-VE-050/CF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm dish</td>
			<td style="width:111px;">Becton-Dickson</td>
			<td style="width:119px;">#353001</td>
			<td>Used for storage of pellets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 cm petri dish</td>
			<td style="width:111px;">VWR</td>
			<td style="width:119px;">#25384-342</td>
			<td>Used as work surface for preparing pellets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mesh</td>
			<td style="width:111px;">Sefar America</td>
			<td style="width:119px;">#03--300/51</td>
			<td>300 um nitex nylon, cut into cm square pieces and sterilzed in autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Spatulas</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:119px;">#21-401-10</td>
			<td>Use tapered end of one to break up pellet mixture. Bend tapered end of other to help remove mixture from microcentrifuge tube.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge tubes</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:119px;">#05-408-146</td>
			<td>One for hydron, one for sucralfate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jewelers forceps, #5</td>
			<td style="width:111px;">Ambler Surgical</td>
			<td style="width:119px;">#2315E</td>
			<td>Need 2 for pulling mesh apart</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifugal evaporator</td>
			<td style="width:111px;">ThermoSavant</td>
			<td style="width:119px;">DNA110 SpeedVac</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Section 2: Surgical implantation of pellets</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Operating microscope</td>
			<td style="width:111px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2.5% Avertin</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>General anesthetic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Proparacaine hydrochloride ophthalmic solution 0.5%</td>
			<td style="width:111px;">Falcon</td>
			<td>NDC# 6131401601</td>
			<td>Eye anesthetic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triple Antibiotic Ophthalmic Ointment</td>
			<td style="width:111px;">Bausch &#38; Lomb</td>
			<td style="width:119px;">NDC# 2420878055</td>
			<td>Contains neomycin, polymixin and bacitracin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ophthalmic microknife, 5 mm</td>
			<td style="width:111px;">Surgistar</td>
			<td style="width:119px;">#924501</td>
			<td>30 degree angle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">von Graef knife</td>
			<td style="width:111px;">Ambler Surgical</td>
			<td style="width:119px;">#3401E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jewelers forceps, #1</td>
			<td style="width:111px;">Ambler Surgical</td>
			<td style="width:119px;">#2301E</td>
			<td>Must be blunted with sharpening stone for proptosing eye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jewelers forceps, #5</td>
			<td style="width:111px;">Ambler Surgical</td>
			<td style="width:119px;">#2305E</td>
			<td>For picking up pellets and placing on eye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small curved scissors</td>
			<td style="width:111px;">Ambler Surgical</td>
			<td style="width:119px;">#5636E</td>
			<td>For trimming whiskers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gauze</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>For blotting eye after proparacaine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Section 3: Grading of Corneal Neovascularization</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2.5% Avertin</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>General anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Slit lamp</td>
			<td style="width:111px;">Nikon</td>
			<td style="width:119px;">FS-2</td>
			<td>Needs an ocular with a reticule to assist in measuring</td>
		</tr>
	</tbody>
</table>

the corneal micropocket assay: a model of angiogenesis in the mouse eye

小鼠角膜微囊法是一个强大的和定量的体内试验，用于评估血管生成。通过使用含有该触发血管生长的整个自然股骨头缺血性角膜特异性生长因子标准化的缓释小球，血管生成可被测量和量化。在该测定中的血管生成反应是在数天的过程中产生的，这取决于所用的生长因子的类型和剂量。新血管形成的诱导通常由任一碱性成纤维细胞生长因子（bFGF）或血管内皮生长因子（VEGF）引起的。通过组合这些生长因子与硫糖铝和昌（聚-HEMA（聚（2 - 羟乙基甲基丙烯酸酯）））和铸造的混合物制成丸粒，它们可以通过外科手术植入在小鼠眼。这些颗粒均匀缓慢释放的生长因子在五，六天（bFGF的或VEGF分别为），使所需的船只区域Q足够的血管生成反应uantification用裂隙灯。此测定法可用于不同的应用，包括血管生成调节剂的药物或治疗方法，以及影响血管生成不同的遗传背景之间的比较进行评价。练此法后，一个熟练的研究者可以在每眼不到5分钟植入颗粒。

典型的结果为bFGF和VEGF的小球在正常低血管C57BL / 6J小鼠示于图2A和B中 。 图2E示出了血管区域的正态分布（VA）在C57BL / 6J应变与不同剂量的这些生长因素。 80纳克的bFGF粒料通常导致的约为2.0 mm 2的VA，虽然在一系列1.8-2.4毫米2的值是可以接受的。 200 ng的血管内皮生长因子颗粒通常导致0.7mm左右2的响应，范围可以接受的0.6-0.9毫米2...

Watch this Scientific Journal Video about The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye at JoVE.com

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

该协议描述为开发在小鼠角膜微囊测定。

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 ...

the-corneal-micropocket-assay-a-model-of-angiogenesis-in-the-mouse-eye

Research

JoVE Journal

Neuroscience

16.6K 次观看.  Boston Children's Hospital. 该协议描述为开发在小鼠角膜微囊测定。 

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

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

科研

神经科学

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

一个视神经挤压伤的小鼠模型，研究视网膜神经节细胞生存

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

Detection of Neuritic Plaques in Alzheimer&#039;s Disease Mouse Model

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

在阿尔茨海默氏症小鼠模型神经炎斑检测

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

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. ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

一个简单的行为分析在检测视觉功能<em&gt;非洲爪蟾</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

在小鼠大脑的各个区域端粒酶活性：非放射性端粒酶重复序列扩增法（TRAP）分析

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

小鼠蛛网膜下腔出血模型脑血管痉挛量化的容积法

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...