穿刺诱导的虹膜新生血管虹膜虹膜小鼠模型的建立
Summary
虹膜新生血管是缺血性视网膜疾病的常见并发症, 可能导致视力威胁的新生血管性青光眼。在这里, 我们描述了诱导实验性虹膜新生血管的小鼠协议, 可用于无创性评价血管生成调节物质。
Abstract
我们描述了穿刺诱导的虹膜新生血管模型作为无创性血管生成评价的一般模型。该模型还与靶向新生血管性青光眼有关, 这是糖尿病视网膜病变的一种视力威胁并发症。该方法以 BALB 小鼠的一系列自密封葡萄膜黑色素穿刺诱导虹膜血管反应为基础, 利用小鼠眼血管的产后成熟。老鼠幼崽从产后12.5 葡萄膜黑色素穿刺, 当幼崽自然睁开眼睛, 直到产后24.5 天。由于角膜的透明度, 虹膜血管可以通过无创的体内方法很容易地通过时间进行分析。此外, BALB 小鼠的半透明虹膜可 flatmounted 为详细的 immunohistologic 分析和最小的非特异背景染色。在该模型中, 血管生成主要由炎症和纤溶酶激活系统驱动。穿刺诱导模型是诱发小鼠虹膜新生血管的第一种, 具有直接无创性在体内分析血管生成过程的优势。此外, 该模型可与血管生成调节物质结合, 突出了其在血管新生研究中的潜力, 其中以体内为视角。
Introduction
虹膜, 连同睫状体和脉络膜, 包括 uvea, 这是最具血管的眼睛组织。虹膜血管是维持眼睛前腔内稳态的必要条件。由于动脉和静脉之间有大量的吻合口连接, 虹膜血管不仅为虹膜本身提供养分和氧气供应, 而且对眼睛的整个前段1。
新血管的形成, 或预先存在的血管生成, 是生理过程的基础, 如发展和伤口愈合2。血管内皮生长因子 (VEGF) 和纤溶酶原激活剂抑制剂 (排) 等多种典型因素对血管生成有很好的调节作用, 并有多个炎症因子, 这些因素的不平衡会导致病理血管生成3。
在眼中, 新生血管是引起视力威胁的疾病的原因, 例如增生性糖尿病视网膜病变 (NVG) 和新生血管性青光眼。在这些眼部疾病中, 病灶新生血管通常位于视网膜组织中, 但眼后和前眼室炎症和血管生成因子的不平衡与虹膜虹膜有关,临床术语虹膜病理血管生成 4.这些病理表明, 成人虹膜的能力, 以接受血管生成。小鼠出生后眼血管发育不成熟, 产后持续成熟。这种发展的特点被利用在老鼠模型的氧诱导视网膜病变, 一个模型, 密切模仿的临床条件的早产儿视网膜病变5。此外, 血管生成和炎症在创伤愈合机制中起关键作用6, 伤口愈合本身也与血管生成模型7有关。
在本研究中, 我们描述了穿刺诱导的虹膜新生血管模型。葡萄膜黑色素穿刺在边缘附近进行, 通过触发创面愈合系统诱导虹膜新生血管。由于角膜的透明性, 虹膜血管可以通过无创方法在体内进行简单的活体分析。穿孔的眼睛呈现增加的血管床在虹膜, 这是与增加的纤溶酶激活和炎症标记8。该模型具有很大的潜力, 作为一种新的工具来研究血管新生和筛选血管生成化合物, 并允许直接在体内可视化血管形成过程。
Protocol
Representative Results
Discussion
在本协议中, 提出了一种新的葡萄膜黑色素穿刺诱导虹膜血管反应的方法。穿刺触发伤口愈合机制和促进血管反应在虹膜10,11。这与眼部病理一致, 如民主共和国和 NVG, 在虹膜虹膜的临床情况下, 眼睛后段视网膜血管生成反应加剧, 虹膜的血管化增加12 ,13。
角膜是缺血性的, 并且具有特殊的?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢 Linnea Tankred 和戴安娜 Rydholm 的畜牧业。
Materials
| Bonn eye scissors | Bausch & Lomb | 23060 | |
| Clayman-Vannas curved scissors | Bausch & Lomb | E3383 C | |
| Clayman-Vannas straight scissors | Bausch & Lomb | E3383 S | |
| Objective adapter for camera | Handcrafted | N/A | Or any system that allows adapting a camera to the microscope |
| Heating Pad 100-110 watts | Non Applicable | N/A | Available in pet/veterinarian stores |
| Hypodermic 30g beveled needle | KDM GmBH germany | 911914 | |
| Iphone 4S | Apple | Non Applicable | Or other high resolution image acquistion device |
| Isoflurane | Baxter | KDG 9623 | |
| McPherson tying forceps | Bausch & Lomb | E1815 S | |
| Micro tying forceps | Bausch & Lomb | 63140 | |
| Minims tetracaine hydrochloride | Bausch & Lomb | N/A | 1 % (w/v) Eye Drops |
| Neutral-buffered formalin | Bioreagens | 0018-40 | |
| Normal saline solution | Fresenius Kabi | 210352 | 0.9 % (w/v) NaCl in injectable water |
| Phosphate-buffered saline | ThermoFisher Scientific | 10010023 | Balanced and buffered PBS pH 7.4 |
| Petri dish 10 cm | Starstedt | 83.3902 | |
| Petri dish 3 cm | Starstedt | 83.3900 | |
| Safe Seal Tube 2.0 mL | Starstedt | 72.685.200 | Or any eppendorf style tubes |
| TC plate 96-well | Starstedt | 83.3924 | |
| Transfer pipette 3.5 mL | Starstedt | 86.1171 | Or any other Pasteur pipette style |
| Univentor 400 anesthesia unit | Univentor Limited | N/A | Or equivalent flow regulator with induction chamber and mask for volatile anesthesia |
| Wild M650 surgical microscope | Wild Heerbrugg | N/A | Or other surgical or magnifying stereoscope |
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