小鼠透明血管的评估与表征
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| AK-Fluor (fluorescein injection, USP) | Akorn | 17478-253-10 | |
| Anti-CD31 antibody | Abcam | ab28364 | |
| Antifade mounting medium | Thermo Fisher | S2828 | |
| Antifade Mounting Medium with DAPI | Vector Laboratories | H-1200 | |
| Artificial tear eyedrop | Systane | N/A | |
| Bovine serum albumin (BSA) | Sigma-Aldrich | A2058 | |
| C57BL/6J mice | The Jackson Laboratory | Stock NO: 000664 | |
| Calcium chloride (CaCl2) | Sigma-Aldrich | C1016 | |
| Cryostat | Leica | CM3050S | |
| Cryostat | Leica | CM3050 S | |
| Cyclopentolate hydrochloride and phenylephrine hydrochloride eyedrop | Cyclomydril | N/A | |
| Gelatin | Sigma-Aldrich | G9382 | |
| Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | ThermoFisher Scientific | A-11008 | |
| Heating board | Lab-Line Instruments Inc. | N/A | |
| Isolectin GS-IB4, 594 conjugate | ThermoFisher Scientific | I21413 | |
| Ketamine hydrochloride injection | KetaVed | NDC 50989-996-06 | |
| Lrp5-/- mice | The Jackson Laboratory | Stock NO. 005823 | Developed by Deltagen Inc., San Mateo, CA |
| Micron IV and OCT | Phoenix Research Labs | N/A | Imaging software: InSight |
| Microscope | Zeiss | discovery v8 | |
| Microsurgery forceps | Scanlan International | 4004-05 | |
| Microsurgery scissors | Scanlan International | 6006-44 | |
| Optimal cutting temperature compound | Tissue-Tek | 4583 | |
| Optimal cutting temperature compound | Agar Scientific | AGR1180 | |
| Paraformaldehyde (16%) | Electron Microscopy Sciences | 15710 | |
| Peel-A-Way disposable embedding molds (tissue molds) | Fisher Scientific | 12-20 | |
| Phosphate-buffered saline (PBS) buffer (10X) | Teknova | P0496 | |
| Slide cover glass | Premiere | 94-2222-10 | |
| Superfrost microscope slides | Fisherbrand | 12-550-15 | |
| Triton X-100 | Sigma-Aldrich | X100 | |
| Xylazine sterile solution | Akorn: AnaSed | NDC: 59399-110-20 |
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