Name: assessment and characterization of hyaloid vessels in mice
Uploaded: 2019-05-15T13:30:00
Description: Watch this Scientific Journal Video about Assessment and Characterization of Hyaloid Vessels in Mice Video at JoVE.com

这项工作得到了国家卫生研究院(NIH)对J.C.Z.W.的助学金(R01 EY024963和EY028100)的支持,并得到了圣殿骑士眼基金会职业启动补助金的支持。本研究中描述的透明体隔离程序,经过修改,修改了理查德·朗博士、东史德·库里哈拉博士和洛伊斯·史密斯博士慷慨分享的协议,作者对此表示感谢。

所有动物都按照美国国家卫生研究院的《眼科和视觉研究协会(ARVO)关于在眼科和视觉研究中使用动物进行动物实验的声明》进行治疗(NIH)关于照料和使用动物进行实验程序以及波士顿儿童医院机构动物护理和使用委员会(IACUC)制定的法规。Lrp5-/-小鼠(库存号005823;杰克逊实验室)及其野生型(WT)控制C57BL/6J小鼠(库存号000664;杰克逊实验室)用于这项研究。
1. 第一部分:使用...

<ol>
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评估和描述透明血管的技术是观察动物模型中的透明血管回归的直观和必要的程序,以便研究发育过程中血管回归背后的机制。虽然体内视网膜成像允许对同一动物的透明体回归进行纵向观察,但获得OCT和FFA的啮齿动物基质成像系统可能是一个限制因素。此外,活小鼠在睁开眼睛之前进行体内成像是不可行的。因此,这种方法不适用于新生儿期眼部发育。另一方面,虽然孤立眼睛的成像横截面可用于小?...

眼睛的血液供应对于确保视网膜和周围眼部组织的正常发育以及适当的视觉功能至关重要。眼睛有三个血管病床:视网膜血管、丘风和透明血管的瞬态胚胎循环网络。眼血管的发展需要整个胚胎生成和组织成熟的空间和时间协调。在三个血管病床上,透明血管是第一个功能性的血液供应系统,为新形成的胚胎透镜和发育的视网膜提供营养和氧气。透明体血管在视网膜血管发育和成熟的同时退步1。透明血管的回归对于为视觉功能的发展提供清晰的视觉途径至关重要;因此,这种血管回归过程与视网膜血管的生长一样重要。受损的透明体回归可能导致眼睛疾病。此外,透明体血管的回归提供了一个模型系统,以研究血管回归调节所涉及的细胞和分子机制,这可能对其他器官的血管生成调节产生影响。
从透明动脉 (HA) 衍生的透明血管,由瓦萨 hyaloidea 扩张 (VHP)、 图尼卡血管纤维素 (TVL) 和阴孔膜 (PM) 组成。它在胚胎发育期间为发育的视网膜、原发性视网膜和透镜提供营养。VHP 从 HA 产生,通过透镜前向镜头分支。TVL 将透镜胶囊的后表面和麻醉剂与PM连接在一起,并连接到前动脉,覆盖透镜2、3的前表面,从而在PM形成血管网络3,4,5.有趣的是,在透明血管中没有静脉,系统利用胆囊静脉来完成静脉排水。
在人类胚胎中,在妊娠第九周时,透明血管几乎完成,并在妊娠2的第四个月出现第一视网膜血管时开始退步。从 VHP 萎缩开始,TVL、PM 的毛细管网络回归,最后,HA 随后出现2、3 。同时,原发性葡萄缩回和次生性葡萄开始形成,由细胞外基质成分组成,包括胶原纤维。到妊娠第六个月,原发性葡萄球体被简化为一条从视神经盘延伸到透镜的小透明管道,称为Cloquet的运河或透明管,次生性乙光成为后段的主要组成部分2,3.透明环流大多在妊娠35至36周消失,就在出生3周前。
与人类不同,在出生时,透明血管完全倒退,小鼠透明血管系统在出生后开始回归。由于小鼠视网膜出生时血管和视网膜在产后发育,透明血管从产后(P)4同时退步,并且大多完全由P216(图1)退位。PM首先在P10和P12之间消失,VHP在P12和P16之间消失,而少量的TVL和HA细胞甚至停留在P16,而到P21,透明血管系统回归几乎完成6。同时,视网膜血管在出生后开始发展。血管丛的表面层完全延伸到P7~P8处的外周视网膜,深层层(位于外层丛形层)从P7~P12发展,最后,内丛层中的中间丛在P12和P15 7之间发展.随着视网膜血管的发展,它逐渐取代伴随回退的透明血管的功能,为发育的眼睛提供营养和氧气。小鼠的产后透明血管回归为观察和研究透明血管血管的实验性模型提供了一个易于获取的实验模型,以及控制生理和血管回归过程的分子基础。病理条件8.
透明体回归的失败可以在诸如PHPV等疾病中看到,PHPV是一种罕见的先天性发育异常,由胚胎、原发性性视网膜和透明质血管衰竭的失败或不完全回归导致眼睛的先天性发育异常。调节透明血管回归过程的机制复杂,研究广泛。对透明体血管正常回归至关重要的一个主要分子通路是Wnt信号通路10,因为影响Wnt配体和受体的这个途径的基因突变与人类的PHPV9有关。实验研究鉴定了一种Wnt配体,Wnt7b,这是由发育眼部的透明血管周围的巨噬细胞产生的,以调解这种回归过程。Wnt7b激活Wnt信号,通过结合在相邻的内皮细胞中卷曲4(FZD4)/LRP5的受体,启动细胞凋亡,导致透明血管10的回归。结果,Wnt7b缺乏的小鼠表现出持续性透明血管10。同样,一种非常规的Wnt配体,Norrin(由Ndp基因编码),也与FZD4/LRP5结合,在发育过程中诱导透明体血管回归。Ndpy/-, Lrp5---和Fzd4-/-小鼠都显示延迟的透明血管回归, 支持 Wnt 信号11、12的临界调节作用, 13,14,15,16.此外,另一个Wnt受体LRP6与LRP5在其功能调节在透明体血管内皮细胞的Wnt信号通路17。可能也导致透明质回归的其他因素包括缺氧诱导因子18,19,血管内皮生长因子20,21,胶原蛋白-18-22, 23,Arf24,血管蛋白-225,和骨形态遗传蛋白-426。在本文中,我们使用Lrp5-/-小鼠作为持久性透明血管的模型,通过体内和体外方法来演示评估和描述透明体血管的技术。
体内和体外透明血管的可视化对于研究透明血管回归机制至关重要。目前观察透明血管的方法主要侧重于通过OCT和FFA图像、眼横截面和透明体平安装来可视化和分析VHP和HA。OCT 和 FFA 是强大的体内成像工具,允许在活体动物睁开眼睛后进行纵向观察。此外,孤立的透明体扁平安装提供了整个透明血管的可视化,并实现了容器数的精确量化。然而,透明血管的微妙和脆弱性质及其隔离造成的技术困难,可能限制了它在眼科研究中的使用。在本文中,我们提供了一个详细的方案,以显着血管的可视化,结合体内活体视网膜成像和前体分离的透明体平装,以提高这些技术的可行性。该协议已经适应了修改和扩展,从以前的出版物的活体基底和OCT成像28和分离的透明质平装11的体外方法的体内方法。

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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Antifade mounting medium</td>
			<td style="width:191px;">Thermo Fisher</td>
			<td style="width:156px;">S2828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antifade Mounting Medium with DAPI</td>
			<td style="width:191px;">Vector Laboratories</td>
			<td style="width:156px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Artificial tear eyedrop</td>
			<td style="width:191px;">Systane</td>
			<td style="width:156px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Bovine serum albumin (BSA)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:156px;">A2058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">C57BL/6J mice</td>
			<td style="width:191px;">The Jackson Laboratory</td>
			<td style="width:156px;">Stock NO: 000664</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Calcium chloride (CaCl2)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:156px;">C1016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Cryostat</td>
			<td style="width:191px;">Leica</td>
			<td style="width:156px;">CM3050S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Cryostat</td>
			<td style="width:191px;">Leica</td>
			<td style="width:156px;">CM3050 S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Cyclopentolate hydrochloride and phenylephrine hydrochloride eyedrop</td>
			<td>Cyclomydril</td>
			<td style="width:156px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin&#160;</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:156px;">G9382</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:156px;">A-11008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating board</td>
			<td style="width:191px;">Lab-Line Instruments Inc.</td>
			<td style="width:156px;">N/A</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:355px;">Isolectin GS-IB4, 594 conjugate</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:156px;">I21413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Ketamine hydrochloride injection</td>
			<td style="width:191px;">KetaVed</td>
			<td style="width:156px;">NDC 50989-996-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Lrp5-/- mice</td>
			<td style="width:191px;">The Jackson Laboratory</td>
			<td style="width:156px;">Stock NO. 005823</td>
			<td>Developed by Deltagen Inc., San Mateo, CA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Micron IV and OCT</td>
			<td style="width:191px;">Phoenix Research Labs</td>
			<td style="width:156px;">N/A</td>
			<td>Imaging software: InSight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Microscope</td>
			<td style="width:191px;">Zeiss</td>
			<td style="width:156px;">discovery v8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Microsurgery forceps</td>
			<td style="width:191px;">Scanlan International</td>
			<td style="width:156px;">4004-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Microsurgery scissors</td>
			<td style="width:191px;">Scanlan International</td>
			<td style="width:156px;">6006-44</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Optimal cutting temperature compound</td>
			<td style="width:191px;">Tissue-Tek</td>
			<td style="width:156px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Optimal cutting temperature compound</td>
			<td style="width:191px;">Agar Scientific</td>
			<td style="width:156px;">AGR1180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde (16%)</td>
			<td>Electron&#160;Microscopy&#160;Sciences</td>
			<td style="width:156px;">15710</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Peel-A-Way disposable embedding molds (tissue molds)</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:156px;">12-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-buffered saline (PBS) buffer (10X)</td>
			<td style="width:191px;">Teknova</td>
			<td style="width:156px;">P0496</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Slide cover glass</td>
			<td style="width:191px;">Premiere</td>
			<td style="width:156px;">94-2222-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost microscope slides&#160;</td>
			<td style="width:191px;">Fisherbrand</td>
			<td style="width:156px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Triton X-100</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:156px;">X100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Xylazine sterile solution</td>
			<td style="width:191px;">Akorn: AnaSed</td>
			<td style="width:156px;">NDC: 59399-110-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment and characterization of hyaloid vessels in mice

在眼睛中,胚胎透明体血管滋养发育的透镜和视网膜,并在视网膜血管发育时退步。在持续性超塑性原发性性性性(PHPV)等疾病中,可看到透明体血管的持续或失败回归,导致光道受阻和视觉功能受损。了解透明体血管回归背后的机制可能导致对血管回归过程的新分子见解,以及利用持久性透明血管管理疾病的潜在新方法。在这里,我们描述了用光学相干断层扫描(OCT)和荧光素血管造影(FFA)对活小鼠的透明质成像程序,以及用于定量分析的隔离和扁平安装透明质外体的详细技术方案。低密度脂蛋白受体相关蛋白5(LRP5)敲除小鼠被用作持久性透明质血管的实验模型,以说明该技术。总之,这些技术可促进对透明体血管进行彻底评估,作为血管回归的实验模型,并研究持久性透明血管的机理。

活小鼠子宫体血管的体内成像 图 3A揭示了 3 个月大的 WT 和Lrp5-/-小鼠(具有持久性透明质动物模型)中视网膜和透明体组织的 OCT 图像的横截面视图。WT 眼睛显示没有透明体组织,而Lrp5-/-眼睛显示两个从视神经头衍生的持久性透明血管。图 3B显示 6 周长Lrp5-/-小鼠在荧光场...

Watch this Scientific Journal Video about Assessment and Characterization of Hyaloid Vessels in Mice Video at JoVE.com

Assessment and Characterization of Hyaloid Vessels in Mice Video (Video) | JoVE

该协议描述了体内和体外方法,以完全可视化和表征透明血管,小鼠眼睛血管回归的模型,使用光学相干断层扫描和荧光素血管造影进行活成像和外体成像隔离和随后的透明质的扁平安装,用于定量分析。

小鼠透明血管的评估与表征

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...

我们的协议提供体内和外体测量，以可视化和描述眼睛内的海合体血管，作为研究血管回归的有用实验模型。该技术结合了活鼠体内对海洛类血管的体内纵向观察，以及活体体可视化和解剖全海样血管的精确定量。本文技术为研究持续性胎儿血管的发病机制和基本机制提供了一种实用可行的方法。这种方法还提供了对细胞和分子机制参与调节眼血管回归的见解，这可能与其他器官的血管生成研究相关。海兰隔离步骤对初学者来说在技术上可能具有挑战性。大量的实践和耐心将有助于确保成功的解剖。可视化此方法将提供有关该技术的详细动手信息和提示，以帮助观看者更有效地掌握技能。对于光学相干断层扫描或 OCT 成像，调整鼠标和探针，使光学神经头位于基金图像中视场的中心，并调整 OCT 成像软件中指示线的焦点和角度，在获取图像之前显示持久性海卢体血管。对于海样血管的荧光血管造影成像，稍微调整对齐方式，将视神经头置于视场中心，将显微镜滤镜更改为绿色荧光通道。然后专注于持久性海兰类血管，并在输入后一、三、五和十分钟内获取多个图像，以确定观察容器的信号与背景比率的最佳时间点。对于外活体子宫体体解剖和可视化使用显微外科钳打开产后八天小鼠的眼睑，并在地球轨道下放置弯曲的显微手术钳，以抓住视神经，而不会挤压眼球。用钳子轻轻拉取和取出眼球，并在室温下将眼睛固定为4%的甲醛。30分钟后，在解剖显微镜下将固定的眼球转移到冰冷的PBS上，并在四个均匀的部位将50微升明胶溶液注入眼球的玻璃体中。然后在冰上孵育眼球30分钟，在玻璃空间内凝固注射的明胶。当明胶治愈后，将眼球转移到解剖显微镜下的 PBS 培养皿上，并使用显微手术剪刀在边缘做切口以切除角膜。在解剖显微镜下狙击并去除视神经。使用两对钳子剥离并丢弃硬膜、胆小球和视网膜色素上皮层，然后去除虹膜。视网膜的视神经侧朝上，镜片朝下，在视网膜杯下方注入50微升PBS，使PBS在明化玻璃体和视网膜之间积累。使用显微外科钳从玻璃体中轻轻剥下视网膜杯和硅体。使用转移移液将含有浸入 PBS 中的海体组织的明胶杯转移到显微镜幻灯片。将剩余的组织翻过来，使镜头朝上。然后抬起镜头，轻轻松开透镜图尼卡血管和血管葫风机之间的连接，然后用显微外科剪刀切割海洛外动脉，以去除镜片图尼卡血管松烷。保持平装的葫二烯杯的瓦萨海洛伊达丙烯丙酮区域。用于平装的透明体容器样品用新鲜 PBS 轻轻冲洗海轮杯，以清除任何解剖碎屑。在 PBS 中浮动的组织使用显微外科钳来轻轻地排列和调整明胶化海体杯的位置，使杯的边缘朝下。将浸入 PBS 中的葫二度杯的幻灯片放在 37 摄氏度的滑动加热器上。明胶熔化后，葫风板扁平，在滑梯几乎干燥时将其去除，并加入一滴防褪色安装介质，用 DAPI 染色海合体中的核。然后轻轻地将盖玻片放在葫风板上，以完成扁平安装。要成像海体容器的 DAPI 染色，请将安装的样品转移到荧光显微镜的舞台上，并确认中央海样动脉是可见的。然后识别直接从海轮动脉派生的容器分支，并手动计算容器分支的数量进行定量。在这里，显示了三个月大的野生类型和低密度脂蛋白受体相关蛋白5或LRP5敲除小鼠（具有持久性海洛类血管的动物模型）视网膜和海合体组织的MACD图像的横截面视图。在这些基金荧光素血管造影图像的持久性催眠体血管八分支的催眠体观察在六周大的淘汰小鼠的玻璃体，而没有残余的葫风血管在野生动物类型动物的年龄。在这些透明体中扁平安装血管细胞和相关巨噬细胞可以通过其核DAPI染色来识别。每行细胞从中央海洛氏动脉分支代表一个血管的血管。野生型小鼠在产后第8天平均有12个海兰类血管分支，而年龄匹配的LRP5敲除幼幼表现出大约25个海兰类血管分支，显示出海兰类血管的显著受损回归。此外，在LRP5敲除幼崽中也观察到延迟和不完全的视网膜血管发育。事实上，在产后第8天，在LRP5敲除眼组织部分的横截面中仍然可以识别海样血管。而野生型的眼睛不再显示这些船只。要记住的最重要的事情是温柔和耐心时，从视网膜和其他眼组织隔离的海样系统。隔离与不同细胞标记的海洛类血管免疫覆盖后，可以进行，以发现调节海洛体回归的细胞相互作用。该技术探讨了在眼血管生成研究中持续胎儿血管的基础细胞和分子机制。甲状甲醛、氯胺酮和西拉辛是危险的。请使用适当的个人防护设备在化学烟罩中准备这些试剂。

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Assessment and Characterization of Hyaloid Vessels in Mice

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