C. Linn is supported by an NIH grant (NIH NEI EY022795).

使用受试动物的所有程序已经按照动物护理和使用委员会（IACUC）在西密歇根大学研究所的标准。 1.动物<ol><li>使用雄性和雌性大鼠3个月大了这项研究。 </li><li>保持12小时光/暗周期动物自由获取食物和水。 </li></ol> 2. KAX鸡尾酒动物麻醉的制备<ol><li>在5ml氯胺酮（100毫克/毫升）与1ml乙酰丙嗪（10毫克/毫升）和3毫升蒸馏水溶解50mg的甲苯噻嗪（20毫克/毫升）的。调匀。 </li><li>用注射器过滤消毒并存储该溶液到10ml血清瓶中。 </li></ol> 3.注射KAX <ol><li>称取动物（G），并返回到笼子里，直到准备注射。 </li><li>注入0.1毫升KAX / 100g的动物体重腹膜内，使用具有28克针1毫升胰岛素注射器。 </li><li>允许动物变得不省人事。捏脚和尾巴检查见长。 </li><li>保留所有动物安全实验室进行手术的时间。 </li><li>手术后，替代动物进入它们的笼子，并在RT保持舒适的意识，直到重新获得。只有回到动物动物设施当动物苏醒并恢复正常的行为。 </li></ol> 4.准备手术以及微大会<ol><li>使无菌2M的NaCl溶液。 </li><li>使用微电极拉出器（ 图1C）到拉一0.86毫米内径重抛光标准和薄壁的硼硅管成两个细的锥形玻璃微针（ 图1D，图1E）。 </li><li>从与用回填注射器针和1ml注射器（ 图1B）的2M食盐先前步骤回填一个微型针。从电极的尖端挖掘出气泡。 </li><li>填写第二1ml注射器用2M氯化钠。连接一个18G的针头，然后安装聚乙烯管（ 图1A）的长度（约10英寸）。使用针筒柱塞通过针来填充用生理盐水聚乙烯管。 </li><li>当两个微针和导管填充有盐水，仔细连接两个。消除任何空气中（ 图2）在它们之间的连接。 </li><li>刮得很轻轻地靠着一门课程纸巾的晶粒细锥微针的尖端。 </li><li>轻轻推动注射器柱塞，直到液体的微细流可以在纸巾中可以看出检查微针的电阻。的液体流应不大于0.5mm宽。 </li></ol> 5.编写动物<ol><li>应用1 - 2滴局部麻醉剂至角膜（丙美卡因盐酸眼用溶液，USP，0.5％）。等到没有眼反射发生。 </li><li>修剪用剪刀晶须。 </li><li>小号aturate与优碘溶液和周围的实验眼拭子区棉尖涂药。 </li><li>使用显微镜，重视止血钳将上下眼睑鼓起眼睛，露出巩膜静脉和限制眼球运动。 （ 图3，箭头） </li></ol> 6.青光眼诱导盐水注射<ol><li>当制备微针组装和动物，开始注射。 </li><li>当动物被证实是没有反应英尺/尾捏，小心地在到静脉（ 图3，白色箭头）10和20度之间的低角度来刺破用微针的巩膜静脉。一个成功的穿刺到静脉可知当血液进入微针（ 图3，黑色箭头）的前端。 </li><li>慢慢地，手动注入约50微升生理盐水静脉。这大约需要10秒。静脉将灼白盐循环救援人员到场啊脉管（ 图4，箭头）。一些地区可能维持一个血红色的外观（ 图4，箭头）。 <ol><li>执行第二注射到静脉，相对的第一部位，以确保对完整视网膜神经节细胞层彻底视网膜损伤。 注意:在几分钟之内，应该看到，通过眼睛的虹膜独特混浊外观的盐循环通过血管系统。 </li></ol></li><li>离开对面眼未处理用作内部对照。 </li></ol> 7.动物恢复<ol><li>取出止血。 </li><li>用棉签三联抗生素软膏（杆菌肽锌，硫酸新霉素，polymysin乙硫酸盐）适用于止血和注射部位夹住的部位。眼部周围组织损伤不会出现使用止血。 </li><li> &gt;将麻醉动物在笼子上的循环温水毯上一个耳鼻喉科体温过低。不时观察动物直至意识和正常行为的恢复。运输清醒动物回到动物殖民地。动物留在殖民地，直到牺牲的时间。 </li></ol> 8.动物牺牲和视网膜去除<ol><li>一个月之后，以诱发青光眼的过程中，动物用CO 2窒息和二次胸穿安乐死。 <ol><li>放置在腔室中的动物，并把盖子上的安全。 </li><li>打开的 CO 2和气体调节阀，以允许20％体积/分钟的 CO 2的位移 氧气在该腔室。 </li><li>允许四到5分钟的动物过期。 </li><li>关闭两个阀门。 </li><li>从腔室中取出的动物和用无菌解剖刀执行辅助胸椎穿刺。 </li></ol></li><li>安乐死后，用手术刀切在眼眶周围的眼睛，贝结缔组织NG小心不要切入眼球本身。 </li><li>小心使用曲边剪刀剪断视神经和任何剩余的组织中提取完整的眼球。将提取的眼球无菌60毫米×15毫米培养皿一次性含有新鲜的PBS菜。 </li><li>使从眼球的眼罩。要做到这一点，请用手术刀小切口正好后方虹膜和巩膜之间的边界。按照眼部周围的小弹簧剪刀圆周此切口从眼球取出角膜半球。连接到视神经半球保持。 </li><li>查找安乐死的动物眼罩内的非常薄的粉红/米色视网膜。握住钝镊子视网膜的色素层，以稳定眼罩。使用另一对闭合钳非常轻轻挑逗整个完整的视网膜落睛的背面。避免捏，拉，或揪着直接视网膜。 </li><li>用小弹簧剪刀剪开区域，视神经仍连接到视网膜。 </li><li>一定要切掉从视网膜任何残留色素上皮或巩膜组织。 </li><li>使用移液管，分离的视网膜轻轻转移到涂覆的含有新鲜的PBS35毫米×10mm的皮氏培养皿一干净SYLGARD。 </li></ol> 9.整个安装视网膜准备<ol><li>一旦在Sylgard菜，使用镊子和一个仙人掌针引脚视网膜到位。保持视网膜神经节细胞层朝上和视神经下来。视网膜的半球形状，即使固定后显着。视网膜的曲率将卷曲朝向天花板时视网膜神经节细胞层是在所希望的方向。 </li><li>用小剪刀到视网膜切成四个象限，使得四叶草从视神经头散热的形状。 </li><li>针附加仙人掌针的视网膜象限使视网膜尽可能平坦无覆盖UT拉伸（ 图5）。 </li><li>修复在Sylgard菜的固定视网膜用3毫升的4％多聚甲醛O / N在室温。 </li></ol>视网膜10.抗体染色注意:与第一和第二抗体染色的固定视网膜中视网膜观看神经元（ 图6）。 <ol><li>漂洗固定的，平面安装视网膜2分钟3次，每次在PBS中。 </li><li>透视网膜，用1％的Triton X-100用在PBS中的1％胎牛血清60分钟。 </li><li>漂洗视网膜三次，每次2分钟，在PBS中。 </li><li>用0.1％的Triton X-100的PBS，每次洗涤5分钟漂洗两次。 </li><li>用PBS，每洗5分钟，冲洗两次。 </li><li>与在RT 1％的Triton X-100和1％胎牛血清的PBS中孵育45分钟。 </li><li>用0.1％的Triton X-100的PBS，每次洗涤5分钟漂洗两次。 </li><li>用PBS，每洗5分钟，冲洗两次。 </li><li>孵育在PBS中的3毫升1-％胎牛血清每个视网膜用纯化的小鼠抗大鼠CD90 /小鼠CD90.1（1:300稀释）O / N在室温。 </li><li>用0.1％的Triton X-100冲洗视网膜一旦在PBS 5分钟。 </li><li>用PBS，每洗5分钟，冲洗两次。 </li><li>继发的Alexa Fluor 594山羊抗小鼠IgG孵育在3ml的PBS（无FBS）的各视网膜（1:300）O / N在室温。 </li><li>用PBS洗涤宽松视网膜。 </li><li>使用解剖显微镜，小心地从视网膜移除仙人掌针。 </li><li>轻轻地用移液管转移到视网膜显微镜载玻片。一定要保持朝向天花板视网膜神经节细胞层的方向。视网膜的半球形状，即使固定后显着。视网膜的曲率将卷曲朝向天花板时视网膜神经节细胞层是在所希望的方向。 </li><li>吸收任何过量的PBS与的Kimwipe或其它这类吸湿材料。要小心，不要吸收视网膜。 </li><li>加入5滴½甘油和重量上午½PBSounting媒体。 </li><li>盖上盖玻片视网膜，避免产生气泡。 </li><li>安全使用盖玻片透明指甲油，胶水或其他粘合剂。 </li></ol>

The authors have no conflicting or competing interests to disclose.

这个协议描述诱导在体内大鼠模型青光眼样病症的方法。此过程使用高渗盐水的注射诱导疤痕在小梁网29，32。发展疤痕组织闭塞房水的流出这增加前房压力。随着流出和压力降低建立，通过弹性悬挂韧带镜头推回到玻璃体腔。玻璃体然后施加压力到视网膜损害脆弱的视网膜神经细胞。使用此过程我们的结果表明，视网膜神经节细胞数开始以2周1个月后的过程神经节细胞损失显著损?...

青光眼是一组影响的神经元中的视网膜，具体而言，视网膜神经节细胞1-2眼疾。这些细胞的轴突收敛到成为视神经承载可视信息，其中视觉感知的大脑。因此损害视网膜神经节细胞和其轴突导致视觉缺陷。 与青光眼疾病相关的主要特征是RGC变性和死亡，增加的眼内压（IOP），并视盘拔罐和萎缩。这些特点导致视野丧失或完全，不可逆转的失明。目前，青光眼全球7000万人造成3失明。因此，它是世界上第三大致盲4的原因。 RGC死亡的青光眼的确切机制尚不清楚。许多研究已经完成，以解开这个谜。然而，已经知道的是，青光眼的主要危险因素之一是增加我Ñ​​眼压由于房水（AH）在眼睛的前房的不规则循环。 AH充当眼睛的缺血性前房无色透明的替代血液。它滋养周围的细胞，分泌删除废物从代谢过程，运输的神经递质，并允许在病理状态1的药物和眼内炎症细胞的循环。 房水流通的维护包括睫状体和小梁网。房水由睫状体产生的。然后，它流入前房维持眼组织的总体健康状况。 75 - 房水外流的80％时，流体通过在睫状肌海绵组织的三层过滤正在积极通过非颜料睫状体上皮分泌的。穿过小梁网并通过施累姆氏管其制止所述流体排出独立实体进入血液系统5。其余20 -流出的25％绕过小梁网和通过超滤和扩散通过uveo巩膜途径被动分泌。这一途径似乎是相对独立的眼压1。 当房水的生产和流出都失去平衡，压力增大眼内。如上所述，这种增加的眼内压是在青光眼的发展的主要危险因素。这种压力导致在眼睛的回视网膜神经元的复杂的层的损伤。损伤视神经的视网膜神经节细胞轴突导致大脑不再接收准确的视觉信息。其结果是，视觉的知觉丧失，并且可以发生完全失明。 到目前为止，还没有治愈青光眼。不同的治疗方法存在的主要目的是降低眼压。这些措施包括外用药物类，如β1的肾上腺素能受体阻断剂，或局部前列腺素类似物。 β阻滞剂通过减少房水的产生7的降低眼内压。前列腺素的作用，通过增加房水8-14外流以减少眼压。 α-肾上腺素能激动剂和碳酸酐酶抑制剂也可作为治疗的辅助方法。阿尔法肾上腺素受体 ​​激动剂通过葡萄膜巩膜途径15-17加大流出。碳酸酐酶抑制剂，通过抑制酶的18降低生产AH的。更侵入性程序也被用于治疗青光眼。激光小梁成形术是用于增加房水19的流出。另一个手术疗法，称为小梁切除术，创建了一个另类的排水现场时，传统的小梁途径被阻断20-21过滤啊。 这些治疗方案已经知道EFFectively眼压降低。然而，青光眼患者的高达40％的正常显示眼压水平表明需要更完整的治疗方法。22,23此外，在青光眼看到视网膜神经节细胞的死亡是不可逆的，一旦开始，目前的治疗不停止疾病的进展24-28。这强调了靶向神经元本身的生存有效的神经保护治疗的需要。青光眼模型的发展是这一发展的关键。 在这项研究中，我们证明使用最初由莫里森29中概述的改性过程诱导成年龙Evans大鼠青光眼样作用的方法。在此过程中，2M的高渗盐水注射到巩膜静脉丛被瘢痕组织引起青光眼样的条件，以减少在小梁网，导致增加的眼内压和视网膜神经节细胞的w的显著损失水状液流出ithin程序30-31的一个月。青光眼诱导程序，如这里描述的，可能是关键解锁在青光眼治疗的新发展。

<table border="0" cellpadding="0" cellspacing="0" width="982">
	<tbody>
		<tr height="20">
			<td height="20" style="height:21px;width:299px;">Xylazine hydrochloride, Minimum 99%</td>
			<td style="width:156px;">Sigma, Life Science</td>
			<td style="width:200px;">X1251-1G</td>
			<td style="width:327px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Ketamine hydrochloride injection, USP, 100mg/mL&#160;</td>
			<td>Putney, Inc</td>
			<td>NDC 26637-411-01</td>
			<td>10 mL bottle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Acepromazine Maleate, 10mg/mL</td>
			<td>Phoenix Pharmaceutical, Inc</td>
			<td>NDC 57319-447-04, 670008L-03-0408</td>
			<td>50 mL bottle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Serum bottle, 10 mL</td>
			<td>VWR</td>
			<td>16171319</td>
			<td>Borosilicate glass</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">1 mL insulin syringe&#160;</td>
			<td>VWR</td>
			<td>BD329410</td>
			<td>28 gauge needle&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Sodium chloride</td>
			<td>Sigma&#160;</td>
			<td>S7653</td>
			<td>2 M Solution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Microelectrode Puller&#160;</td>
			<td>Narishige Group</td>
			<td>PP-830</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Heavy Polished Standard and Thin Walled Borosilicate Tubing&#160;</td>
			<td>Sutter Instruments</td>
			<td>B150-86-10HP</td>
			<td>without filament, 0.86 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Microfil syringe needle for filling micropipettes</td>
			<td>World Precision Instruments, Inc</td>
			<td>MF28G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">18 gauge Luer-Lock needle</td>
			<td>Fisher Scientific</td>
			<td>1130421</td>
			<td>Syringe needle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Flexible Polyethylene Tubing</td>
			<td>Fisher Scientific</td>
			<td>22046941</td>
			<td>0.034 inch diameter, approximately 10 inches&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Proparacaine Hydrochloride Opthalmic Solution, USP, 0.5%</td>
			<td>Akorn, Inc</td>
			<td>NDC 17478-263-12</td>
			<td>15 mL&#160; sterile bottle&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Curved Scissors</td>
			<td>Fine Science Tools</td>
			<td>14061-11</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Microscope</td>
			<td>Leica&#160;</td>
			<td>StereoZoom 4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Hemostat Clamp&#160;</td>
			<td>Fine Science Tools</td>
			<td>1310912</td>
			<td>curved edge</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Triple Antibiotic Ointment&#160;</td>
			<td>Fisher Scientific</td>
			<td>NC0664481</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Scalpel handle</td>
			<td>Fine Science Tools&#160;</td>
			<td>10004-13</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Scalpel blade # 11</td>
			<td>Fine Science Tools&#160;</td>
			<td>10011-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">60 mm x 15 mm Disposable Petri Dish</td>
			<td>VWR</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Phosphate Buffered Saline 10x Concentrate</td>
			<td>Sigma, Life Science&#160;</td>
			<td>P7059-1L</td>
			<td>1x dilution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Spring Scissors</td>
			<td>Fine Science Tools&#160;</td>
			<td>15009-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Forceps (2), Dumont # 5</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">3 mL Transfer Pipets, polyethylene, non sterile</td>
			<td>BD Biosciences</td>
			<td>357524 or 52947-948</td>
			<td>1 and 2 mL graduations</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">35 mm x 10 mm Easy Grip Petri Dish&#160;</td>
			<td>BD Biosciences</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Sylgard 184</td>
			<td>VWR</td>
			<td>102092-312</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Cactus Needles</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Paraformaldehyde</td>
			<td>EMD Millipore&#160;</td>
			<td>PX0055-3 or 818715.0100</td>
			<td>Made into a 4% solution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Triton X-100</td>
			<td>Sigma&#160;</td>
			<td>T9284-100 mL</td>
			<td>Made into both a 1% and 0.1% solution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Fetal Bovine Serum&#160;</td>
			<td>Atlanta Biological</td>
			<td>S11150</td>
			<td>500 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Purified Mouse Anti-Rat CD90/mouse CD90.1</td>
			<td>BD Pharmingen</td>
			<td>Cat 554892</td>
			<td>1:300 dilution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Alexa Fluor 594 goat anti-mouse&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>A11005</td>
			<td>1:300 dilution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Microscope Slides</td>
			<td>Corning&#160;</td>
			<td>2948-75x25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Glycerol&#160;</td>
			<td>Sigma&#160;</td>
			<td>G5516-100 mL&#160;</td>
			<td>50% glycerol to 50% PBS, by weight&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Coverglass&#160;</td>
			<td>Corning&#160;</td>
			<td>2975-225</td>
			<td>Thickness 1 22 x 50 mm&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Confocal Microscope</td>
			<td>Nikon&#160;</td>
			<td>C2 Eclipse Ti</td>
			<td></td>
		</tr>
	</tbody>
</table>

glaucoma-inducing procedure in an in vivo rat model and whole-mount retina preparation

青光眼是一种影响视网膜神经节细胞（RGC）的中枢神经系统的疾病。研资局轴突组成视神经进行视觉输入到大脑的视觉感受。损害视网膜神经节细胞和它们的轴突导致视力丧失和/或失明。尽管青光眼的具体原因不明，为疾病的主要危险因素之一是升高的眼内压。在动物模型青光眼诱导过程是一个有价值的工具，研究人员在研究RGC死亡的机制。这样的信息可导致可能在预防视力丧失的有助于有效的神经保护治疗的发展。本文中的协议描述诱导的青光眼的方法-像，其中50微升2M的高渗盐水注入巩膜静脉丛在体内大鼠模型的条件。血管的热烫表示成功注入。此过程会导致视网膜神经节细胞的损失来模拟青光眼。一个月以下注射后，将动物处死，眼睛被除去。接着，角膜，晶状体和玻璃体被除去以使一个眼罩。视网膜然后从眼睛的后部剥离和钉扎到使用仙人掌针SYLGARD菜肴。在这一点上，在视网膜的神经元可染色进行分析。从这个实验结果表明，相对于内部控制时，视网膜神经节细胞的约25％是在过程的一个月内丢失。这个过程允许在体内大鼠青光眼模型的视网膜神经节细胞死亡的定量分析。

本节说明用于诱导在体内大鼠青光眼模型青光眼样条件的装置组件和过程。我们表明用于执行高渗盐水注射导致眼内压增加的个体的工具和设备。我们展示了注入以其特有的漂白效果和前房导致的浑浊外观巩膜静脉丛。我们还描述了视网膜去除和平面安装丢失RGC的分析过程。最后，我们显示在视网膜神经节细胞存活的注射的效果。作为视网膜神经节细胞的分布是在?...

Watch this Scientific Journal Video about Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation at JoVE.com

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

青光眼诱导在程序<em&gt;在体内</em&gt;大鼠模型和全安装视网膜准备

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

glaucoma-inducing-procedure-an-vivo-rat-model-whole-mount-retina

Research

JoVE Journal

Medicine

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

12.9K 次观看.  Western Michigan University. Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis. 

视频: Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

利用磁共振成像，容积电脑断层扫描及超音波的裸鼠乳腺癌骨转移大鼠模型血管生成的多模态成像

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

科研

医学

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

通过40W的Elvax缓释给药的大鼠视网膜:对慢性疾病的治疗

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

增殖性糖尿病视网膜病变纤维血管并发症的体外组织培养模型

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

古树康 (GSK) 颗粒的制备,用于 Vivo 和体外实验

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

毛利精密切肝片作为肝脏生物学的Ex Vivo模型

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Standardized Rat Coronary Ring Preparation and Real-Time Recording of Dynamic Tension Changes Along Vessel Diameter

As a key event of cardiovascular system diseases, coronary artery disease (CAD) has been widely regarded as the main culprit of atherosclerosis, myocardial infarction, and angina pectoris, which seriously threaten the life and health of people all over the world. However, how to record the dynamic biomechanical characteristics of isolated blood vessels has long puzzled people. Meanwhile, precise positioning and isolation of coronary arteries to measure in vitro dynamic vascular tension changes have become a trend in CAD drug development. The present protocol describes the macroscopic identification and microscopic separation of rat coronary arteries. The contraction and dilation function of the coronary artery ring along the vessel diameter was monitored using the established multi myograph system. The standardized and programmed protocols of coronary ring tension measurement, from sampling to data acquisition, tremendously improve the repeatability of the experimental data, which ensures the authenticity of vascular tension records after physiological, pathological, and drug intervention.

The present protocol describes the wire myograph technique for measuring vascular reactivity of the rat coronary artery.