监测氧诱导视网膜病变小鼠模型中视网膜血管的动态生长
Summary
该协议描述了用于制备和免疫荧光染色小鼠视网膜平板支架和分析的详细方法。还详细描述了荧光素眼底血管造影（FFA）对小鼠幼崽和图像处理的使用。
Abstract
氧诱导性视网膜病变 （OIR） 广泛用于研究缺血性视网膜疾病的异常血管生长，包括早产儿视网膜病变 （ROP）、增殖性糖尿病视网膜病变 （PDR） 和视网膜静脉阻塞 （RVO）。大多数OIR研究在特定时间点观察到视网膜新生血管形成;然而，活小鼠在时间过程中的动态血管生长对于理解OIR相关的血管疾病至关重要，但尚未得到充分研究。在这里，我们描述了诱导OIR小鼠模型的分步方案，强调了潜在的陷阱，并提供了一种改进的方法，以使用免疫荧光染色快速量化血管闭塞（VO）和新生血管形成（NV）区域。更重要的是，我们通过在OIR小鼠模型中进行荧光素眼底血管造影（FFA）来监测活小鼠从P15到P25的血管再生。将FFA应用于OIR小鼠模型使我们能够观察血管再生过程中的重塑过程。
Introduction
视网膜新生血管形成（RNV）被定义为新的病理血管起源于现有视网膜静脉的状态，通常沿着视网膜内表面延伸并生长到玻璃体（或在某些情况下的视网膜下空间）1。它是许多缺血性视网膜病的标志和共同特征，包括早产儿视网膜病变 （ROP）、视网膜静脉阻塞 （RVO） 和增殖性糖尿病视网膜病变 （PDR）2。
大量临床和实验观察表明，缺血是视网膜新生血管形成的主要原因3，4。在ROP中，新生儿在封闭的培养箱中暴露于高水平氧气以提高存活率，这也是阻止血管生长的重要驱动因素。治疗完成后，新生儿的视网膜经历相对缺氧期5。其他情况见于RVO中中央或分支视网膜静脉的闭塞，并且还观察到视网膜毛细血管的损伤，这是由PDR2中的微血管病引起的。缺氧通过缺氧诱导的因子-1α（HIF-1α）信号通路进一步增加血管生成因子（如血管内皮生长因子（VEGF））的表达，进而引导血管内皮细胞生长到缺氧区域并形成新血管6，7。
ROP是早产儿血管增殖性视网膜病变的一种，是儿童失明的主要原因8，9，其特征是视网膜缺氧，视网膜新生血管形成和纤维增生10，11，12。在1950年代，研究人员发现，高浓度的氧气可以显着改善早产儿的呼吸道症状13，14。因此，氧疗在当时越来越多地用于早产儿15。然而，在早产儿广泛使用氧疗的同时，ROP的发病率逐年增加。从那时起，研究人员将氧气与ROP联系起来，探索各种动物模型以了解ROP和RNV16的发病机制。
在人类中，大多数视网膜脉管系统的发育在出生前完成，而在啮齿动物中，视网膜脉管系统在出生后发育，为研究视网膜脉管系统中的血管生成提供了一个可访问的模型系统2。随着研究的不断进展，氧诱导视网膜病变（OIR）模型已成为模拟缺血引起的病理性血管生成的主要模型。OIR模型的研究中没有特定的动物物种，该模型已经在各种动物物种中开发，包括小猫17，大鼠18，小鼠19，比格犬幼犬20和斑马鱼21。所有模型都具有相同的机制，即它们在视网膜发育早期暴露于高氧，然后返回常氧环境。Smith等人观察到，将小鼠幼崽暴露于P7的高氧中5天会导致中央视网膜血管回归的极端形式，并将它们带回P12处的室内空气中，逐渐触发新生血管簇，向玻璃体生长19。这是一个标准化的OIR小鼠模型，也称为史密斯模型。Connor等人在2009年进一步优化了协议，提供了一种普遍适用的方法来量化VO（血管闭塞）和NV（新生血管形成）的面积，提高了模型22的接受度和利用率。OIR小鼠模型因其体积小、繁殖快、遗传背景清晰、重复性好、成功率高等特点，仍然是现在应用最广泛的模型。
在小鼠中，视网膜血管形成在出生后开始，血管从视神经头向视网膜内向锯齿状动脉内生长。在正常的视网膜发育过程中，第一条视网膜血管在出生时从视神经头发芽，形成一个扩张的网络（初级神经丛），在出生后第 7 天（P7）23 左右到达外围。然后血管开始长入视网膜形成深层，穿透视网膜，并在内核层（INL）周围建立层流网络，就像人类24一样。到产后第三周（P21）结束时，更深的神经丛发育几乎完成。对于OIR小鼠模型，血管闭塞总是出现在中央视网膜中，因为在高氧暴露期间中央区域的大量未成熟血管网络迅速退化。因此，病理性新生血管的生长也发生在中外周视网膜，这是非灌注区和血管区的边界。然而，人类视网膜血管几乎在出生前就已经形成。至于早产儿，当暴露于高氧25，26时，周围视网膜没有完全血管化。所以血管闭塞和新生血管形成主要出现在周边视网膜27，28。尽管存在这些差异，但小鼠OIR模型密切概括了缺血诱导的新生血管形成期间发生的病理事件。
OIR模型的诱导可分为两个阶段29：在第1阶段（高氧阶段），由于VEGF下降和内皮细胞凋亡，视网膜血管发育因血管闭塞和退化而停止或延迟24，30;在第 2 阶段（缺氧阶段），在室内空气条件下视网膜氧气供应将变得不足29，这对于神经发育和体内平衡19，31 至关重要。这种缺血情况通常会导致不受调节的异常新生血管形成。
目前，常用的建模方法是交替的高/低氧暴露：母亲和它们的幼崽在P7下暴露在75%的氧气下5天，然后在室内空气中暴露5天，直到P17显示出可比的结果22，这是OIR小鼠模型诱导的终点。（图1）。除了模拟ROP，这种缺血介导的病理性新生血管也可用于研究其他缺血性视网膜疾病。该模型的主要测量包括量化VO和NV的面积，通过免疫荧光染色或FITC-葡聚糖灌注从视网膜平面安装中分析。由于致命的操作，每只老鼠只能研究一次。目前，在血管消退和病理血管生成过程中连续观察视网膜血管系统动态变化的方法很少32.在本文中，我们提供了OIR模型诱导的详细方案，视网膜平面安装的分析以及荧光素眼底血管造影（FFA）的工作流程，这将有助于更全面地了解OIR小鼠模型两个阶段的血管动力学变化。
Protocol
所有涉及使用小鼠的程序均由中国中山大学中山眼科中心动物实验伦理委员会批准（授权编号：2020-082），并符合中山市眼科中心动物护理和使用委员会批准的指南和视觉和眼科协会（ARVO）关于动物用于眼科和视觉研究的声明。 1. 小鼠OIR模型的归纳 使用眼睛先天性畸形率较低的小鼠，例如C57BL / 6J小鼠，并以雄性/雌性= 1：2的比例交配它们。让同一天出生的幼崽在P7开?…
Representative Results
在OIR小鼠模型中，最重要和最基本的结果是VO和NV区域的量化。从P7开始在高氧环境中生活5天后，幼崽的中央视网膜显示出最大的非灌注区域。再过5 d在缺氧的刺激下，视网膜新生血管逐渐产生，荧光比周围正常血管更强烈。P17之后，病理性新生血管化的荧光信号随着视网膜的重塑而迅速消退（图5A）。通过控制幼崽的窝产仔数和出生后体重增加，OIR小鼠模型的VO和NV面积表现…
Discussion
小鼠对OIR的易感性受到许多因素的影响。不同遗传背景和品系的幼崽无法比较。在BALB/c白化小鼠中，血管迅速重新生长到VO区，新生血管簇38明显减少，这给研究带来了一些困难。在C57BL / 6小鼠中，与BALB / cJ小鼠品系39，40相比，光感受器损伤增加。不同类型的转基因小鼠41，42，<sup cl…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢我们实验室和中山眼科中心眼科动物实验室的所有成员的技术援助。我们还要感谢刘春桥教授的实验支持。这项工作得到了中国国家自然科学基金（NSFC：81670872;北京市）、广东省自然科学基金（批准号：2019A1515011347）、中山市眼科中心眼科国家重点实验室高水平医院建设项目（批准号303020103;中国广东省广州市）。
Materials
| 1 mL sterile syringe | Solarbio | YA0550 | For preparation of retinal flat mounts and intraperitoneal injection |
| 1× Phosphate buffered saline (PBS) | Transgen Biotech | FG701-01 | For preparation of retinal flat mounts |
| 2 ml Microcentrifuge Tube | Corning | MCT-200-C | For preparation of retinal flat mounts |
| 48 Well Clear TC-Treated Multiple Well Plates | Corning | 3548 | For preparation of retinal flat mounts |
| Adhesive microscope slides | Various | For preparation of retinal flat mounts | |
| Adobe Photoshop CC 2019 | Adobe Inc. | For image analysis | |
| Carbon dioxide gas | Various | For sacrifice | |
| Cover slide | Various | For preparation of retinal flat mounts | |
| Curved forceps | World Precision Instruments | 14127 | For preparation of retinal flat mounts |
| DAPI staining solution | Abcam | ab228549 | For labeling nucleus on retinal flat mounts |
| Dissecting microscope | Olmpus | SZ61 | For preparation of retinal flat mounts |
| Fluorescein sodium | Sigma-Aldrich | F6377 | For in vivo imaging |
| Fluorescent Microscope | Zeiss | AxioImager.Z2 | For acquisition of fluorescence images of retinal flat mounts |
| Fluoromount-G Mounting media | SouthernBiotech | 0100-01 | For preparation of retinal flat mounts |
| Hydroxypropyl Methylcellulose | Maya | 89161 | For in vivo imaging |
| Isolectin B4 594 antibody | Invitrogen | I21413 | For labeling retinal vasculature on retinal flat mounts |
| Mice C57/BL6J | GemPharmatech of Jiangsu Province | For OIR model induction | |
| Micro dissecting scissors-straight blade | World Precision Instruments | 503242 | For preparation of retinal flat mounts |
| No.4 straight forceps | World Precision Instruments | 501978-6 | For preparation of retinal flat mounts |
| Normal donkey serum | Abcam | ab7475 | For preparation of retinal flat mounts |
| O2 sensor | Various | For monitoring the level of O2 | |
| OxyCycler | Biospherix | A84XOV | For OIR model induction |
| Paraformaldehyde (PFA) | Sigma | P6148-1KG | For tissue fixation |
| Pentobarbital sodium | Various | For anesthesia | |
| Soda lime | Various | For absorbing excess CO2 in the oxygen chamber | |
| SPECTRALIS HRA+OCT | Heidelberg | HC00500002 | For in vivo imaging |
| SPSS Statistics 22.0 | IBM | For statistical analysis | |
| Tansference decloring shaker | Kylin-Bell | ZD-2008 | For preparation of retinal flat mounts |
| Tissue culture dish (Low attachment) | Corning | 3261-20EA | For preparation of retinal flat mounts |
| Transfer pipettes | Various | For preparation of retinal flat mounts | |
| Triton X-100 | Sigma-Aldrich | SLBW6818 | For preparation of retinal flat mounts |
| Tropicamide | Various | For in vivo imaging | |
| ZEN Imaging Software | ZEISS | For image acquisition and export |
References
- Vavvas, D. G., Miller, J. W. Chapter 26 – Basic Mechanisms of Pathological Retinal and Choroidal Angiogenesis. Retina (Fifth Edition). 1, 562-578 (2013).
- Selvam, S., Kumar, T., Fruttiger, M. Retinal vasculature development in health and disease. Progress in Retinal and Eye Research. 63, 1-19 (2018).
- Shimizu, K., Kobayashi, Y., Muraoka, K. Midperipheral fundus involvement in diabetic retinopathy. Ophthalmology. 88 (7), 601-612 (1981).
- Ashton, N. Retinal vascularization in health and disease: Proctor Award Lecture of the Association for Research in Ophthalmology. American Journal of Ophthalmology. 44 (4), 7-17 (1957).
- Hellström, A., Smith, L. E., Dammann, O. Retinopathy of prematurity. Lancet. 382 (9902), 1445-1457 (2013).
- Xu, Y., et al. Melatonin attenuated retinal neovascularization and neuroglial dysfunction by inhibition of HIF-1α-VEGF pathway in oxygen-induced retinopathy mice. Journal of Pineal Research. 64 (4), 12473 (2018).
- Cavallaro, G., et al. The pathophysiology of retinopathy of prematurity: an update of previous and recent knowledge. Acta Ophthalmologica. 92 (1), 2-20 (2014).
- Gilbert, C., Rahi, J., Eckstein, M., O’Sullivan, J., Foster, A. Retinopathy of prematurity in middle-income countries. Lancet. 350 (9070), 12-14 (1997).
- Chen, J., Smith, L. E. Retinopathy of prematurity. Angiogenesis. 10 (2), 133-140 (2007).
- Fielder, A., Blencowe, H., O’Connor, A., Gilbert, C. Impact of retinopathy of prematurity on ocular structures and visual functions. Archives of Disease in Childhood. Fetal and Neonatal Edition. 100 (2), 179-184 (2015).
- Moshfeghi, D. M. Presumed transient reactive astrocytic hyperplasia in immature retina. Retina. 26, 69-73 (2006).
- Kandasamy, Y., Hartley, L., Rudd, D., Smith, R. The association between systemic vascular endothelial growth factor and retinopathy of prematurity in premature infants: a systematic review. British Journal of Ophthalmology. 101 (1), 21-24 (2017).
- Shah, P. K., et al. Retinopathy of prematurity: Past, present and future. World Journal of Clinical Pediatrics. 5 (1), 35-46 (2016).
- Kinsey, V. E. Retrolental fibroplasia; cooperative study of retrolental fibroplasia and the use of oxygen. AMA Archives of Ophthalmology. 56 (4), 481-543 (1956).
- Tin, W., Gupta, S. Optimum oxygen therapy in preterm babies. Archives of Disease in Childhood. Fetal and Neonatal Edition. 92 (2), 143-147 (2007).
- Liu, C. H., Wang, Z., Sun, Y., Chen, J. Animal models of ocular angiogenesis: from development to pathologies. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. 31 (11), 4665-4681 (2017).
- Ashton, N., Ward, B., Serpell, G. Effect of oxygen on developing retinal vessels with particular reference to the problem of retrolental fibroplasia. The British Journal of Ophthalmology. 38 (7), 397-432 (1954).
- Penn, J. S., Tolman, B. L., Lowery, L. A. Variable oxygen exposure causes preretinal neovascularization in the newborn rat. Investigative Ophthalmology & Visual Science. 34 (3), 576-585 (1993).
- Smith, L. E., et al. Oxygen-induced retinopathy in the mouse. Investigative Ophthalmology & Visual Science. 35 (1), 101-111 (1994).
- McLeod, D. S., Brownstein, R., Lutty, G. A. Vaso-obliteration in the canine model of oxygen-induced retinopathy. Investigative Ophthalmology & Visual Science. 37 (2), 300-311 (1996).
- Cao, R., Jensen, L. D., Söll, I., Hauptmann, G., Cao, Y. Hypoxia-induced retinal angiogenesis in zebrafish as a model to study retinopathy. PLoS One. 3 (7), 2748 (2008).
- Connor, K. M., et al. Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis. Nature Protocols. 4 (11), 1565-1573 (2009).
- Fruttiger, M. Development of the mouse retinal vasculature: angiogenesis versus vasculogenesis. Investigative Ophthalmology & Visual Science. 43 (2), 522-527 (2002).
- Stahl, A., et al. The mouse retina as an angiogenesis model. Investigative Ophthalmology & Visual Science. 51 (6), 2813-2826 (2010).
- Rivera, J. C., et al. Ischemic retinopathies: oxidative stress and inflammation. Oxidative Medicine and Cellular Longevity. 2017, 3940241 (2017).
- Bashinsky, A. L. Retinopathy of prematurity. North Carolina Medical Journal. 78 (2), 124-128 (2017).
- Flynn, J. T., et al. Retinopathy of prematurity. Diagnosis, severity, and natural history. Ophthalmology. 94 (6), 620-629 (1987).
- Aguilar, E., et al. Chapter 6. Ocular models of angiogenesis. Methods in Enzymology. 444, 115-158 (2008).
- Liegl, R., Priglinger, C., Ohlmann, A. Induction and readout of oxygen-induced retinopathy. Methods in Molecular Biology. 1834, 179-191 (2019).
- Lutty, G. A., McLeod, D. S. Retinal vascular development and oxygen-induced retinopathy: a role for adenosine. Progress in Retinal and Eye Research. 22 (1), 95-111 (2003).
- Vähätupa, M., et al. Oxygen-induced retinopathy model for ischemic retinal diseases in rodents. Journal of Visualized Experiments: JoVE. (163), (2020).
- Kim, C. B., D’Amore, P. A., Connor, K. M. Revisiting the mouse model of oxygen-induced retinopathy. Eye and Brain. 8, 67-79 (2016).
- Gammons, M. V., Bates, D. O. Models of oxygen induced retinopathy in rodents. Methods in Molecular Biology. 1430, 317-332 (2016).
- Xiao, S., et al. Fully automated, deep learning segmentation of oxygen-induced retinopathy images. Journal of Clinical Investigation Insight. 2 (24), (2017).
- McLeod, D. S., D’Anna, S. A., Lutty, G. A. Clinical and histopathologic features of canine oxygen-induced proliferative retinopathy. Investigative Ophthalmology & Visual Science. 39 (10), 1918-1932 (1998).
- Penn, J. S., Johnson, B. D. Fluorescein angiography as a means of assessing retinal vascular pathology in oxygen-exposed newborn rats. Current Eye Research. 12 (6), 561-570 (1993).
- Mezu-Ndubuisi, O. J., et al. In vivo retinal vascular oxygen tension imaging and fluorescein angiography in the mouse model of oxygen-induced retinopathy. Investigative Ophthalmology & Visual Science. 54 (10), 6968-6972 (2013).
- Zeilbeck, L. F., Müller, B., Knobloch, V., Tamm, E. R., Ohlmann, A. Differential angiogenic properties of lithium chloride in vitro and in vivo. PLoS One. 9 (4), 95546 (2014).
- Walsh, N., Bravo-Nuevo, A., Geller, S., Stone, J. Resistance of photoreceptors in the C57BL/6-c2J, C57BL/6J, and BALB/cJ mouse strains to oxygen stress: evidence of an oxygen phenotype. Current Eye Research. 29 (6), 441-447 (2004).
- Zhang, Q., Zhang, Z. M. Oxygen-induced retinopathy in mice with retinal photoreceptor cell degeneration. Life Sciences. 102 (1), 28-35 (2014).
- Okamoto, N., et al. Transgenic mice with increased expression of vascular endothelial growth factor in the retina: a new model of intraretinal and subretinal neovascularization. The American Journal of Pathology. 151 (1), 281-291 (1997).
- Ohlmann, A., et al. Norrin promotes vascular regrowth after oxygen-induced retinal vessel loss and suppresses retinopathy in mice. The Journal of Neuroscience. 30 (1), 183-193 (2010).
- Fang, L., Barber, A. J., Shenberger, J. S. Regulation of fibroblast growth factor 2 expression in oxygen-induced retinopathy. Investigative Ophthalmology & Visual Science. 56 (1), 207-215 (2014).
- Chan, C. K., et al. Differential expression of pro- and antiangiogenic factors in mouse strain-dependent hypoxia-induced retinal neovascularization. Laboratory Investigation. 85 (6), 721-733 (2005).
- Stahl, A., et al. Postnatal weight gain modifies severity and functional outcome of oxygen-induced proliferative retinopathy. The American Journal of Pathology. 177 (6), 2715-2723 (2010).
- Vanhaesebrouck, S., et al. Association between retinal neovascularization and serial weight measurements in murine and human newborns. European Journal of Ophthalmology. 23 (5), 678-682 (2013).
- Gerschman, R., Nadig, P. W., Snell, A. C., Nye, S. W. Effect of high oxygen concentrations on eyes of newborn mice. The American Journal of Physiology. 179 (1), 115-118 (1954).
- Lange, C., et al. Kinetics of retinal vaso-obliteration and neovascularisation in the oxygen-induced retinopathy (OIR) mouse model. Graefe’s Archive for Clinical and Experimental Ophthalmology. 247 (9), 1205-1211 (2009).
- Huang, S., et al. Comparison of dextran perfusion and GSI-B4 isolectin staining in a mouse model of oxygen-induced retinopathy. Eye Science. 30 (2), 70-74 (2015).
- Paques, M., et al. Panretinal, high-resolution color photography of the mouse fundus. Investigative Ophthalmology & Visual Science. 48 (6), 2769-2774 (2007).
- Fletcher, E. L., et al. Animal models of retinal disease. Progress in Molecular Biology and Translational Science. 100, 211-286 (2011).
Cite This Article
Ma, Y., Li, T. Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model. J. Vis. Exp. (170), e62410, doi:10.3791/62410 (2021).