Two-photon excited fluorescence imaging of placental vasculature in vivo

描述（由申请人提供）：胎盘内胎儿和母体血管床的不适当建立导致胎儿健康受损；然而，对胎盘血管发育的分子决定因素的理解还远未完成。在小鼠模型中，远端无基因缺失（Dlx） 3在E9.5-E10时是胚胎致死的，因为胎盘迷路可能无法扩张并进行适当的血管形态发生。在R21应用中提出的中心假设是Dlx3在胎盘迷宫中滋养细胞群的发育决定中起关键作用，促进胎儿血管室的形成和扩张。为了检验我们的中心假设，有必要采用新的方法来成像和量化胎盘血管组织和血流。在这里，我们建议使用体内和离体双光子激发荧光（2PEF）显微镜来研究不同Dlx3基因型胎盘血管床的拓扑结构和组织，以及血管差异对胎儿血流的功能影响。具体目标是：目标1。优化双光子激发荧光显微镜对发育中的小鼠胎盘血管室的成像，并验证Dlx3是小鼠胎盘迷宫血管生成潜能的重要决定因素的假设。目的1将优化实验方案，利用2PEF显微镜对小鼠胎儿血管床拓扑结构和胶原基质组织进行离体检查。通过反复成像和通过光学消融去除组织，我们将看到不同胎龄的整个胎盘盘的血管系统。在胎儿血管室中特异性表达Tie2-GFP转基因的Dlx3+/+、Dlx3+/-和Dlx3-/-小鼠，结合母体血管/血腔的荧光标记，可以同时对母体和胎儿血管室进行成像和定量。这些影像学研究为评估Dlx3缺失对胎盘迷路血管数量、体积/表面积以及血管连通性、扭曲度和分支形态发生的影响提供了独特的机会。目标2。验证Dlx3-/-植入部位体内血流量减少的假设。在Aim 2中，我们转向在存在和不存在Dlx3的情况下迷宫内血流动力学的体内检查。单个血管的2PEF成像可以在迷宫内一次研究一根血管的血流速度。我们将确定遗传干扰Dlx3对胎儿血管室体内血流的影响，并将这些变化与Aim 1中确定的血管拓扑差异联系起来。拟议研究的长期目标是开发令人兴奋的新成像策略，以检查代表人类疾病模型的小鼠胎盘缺陷。胎盘内胎儿和母体血管床的不适当建立导致健康受损。这些疾病的范围很广，从对母亲和婴儿的直接的，通常是危及生命的健康问题，如先兆子痫，到子宫环境对成年发病的长期影响。这些疾病情况可能对患者和婴儿造成毁灭性的影响；然而，对胎盘血管发育的分子决定因素的理解还远未完成。本应用程序提出的研究优化并实现了一种新颖而令人兴奋的体内成像策略（双光子激发荧光显微镜），以表征和定量胎盘衰竭小鼠模型中的血管床拓扑结构，血管纤维胶原基质组织和血流。

DESCRIPTION (provided by applicant): Inappropriate establishment of fetal and maternal vascular beds within the placenta results in compromised fetal health; however, an understanding of the molecular determinants that underlie the development of placental vasculature are far from complete. Genetic deletion of Distal-less (Dlx) 3 in mouse models is embryonic lethal at E9.5-E10 due to putative failure of the placental labyrinth to expand and undergo proper vascular morphogenesis. The central hypothesis addressed in this R21 application is that Dlx3 plays a critical role in the developmental determination of the trophoblast population within the placental labyrinth that facilitates formation and expansion of the fetal vascular compartment. To examine our central hypothesis, it is necessary to adopt new methodologies for imaging and quantifying placental vascular organization and blood flow. Here, we propose to use in vivo and ex vivo two-photon excited fluorescence (2PEF) microscopy to study the topology and organization of placental vascular beds and the functional consequences of any vascular differences on fetal blood flow for different Dlx3 genotypes. Specific Aims are: Aim 1. To optimize the use of two-photon excited fluorescence microscopy to image vascular compartments in the developing mouse placenta and examine the hypothesis that Dlx3 is an important determinant of angiogenic potential of the mouse placental labyrinth. Aim 1 will optimize experimental protocols for the ex vivo examination of fetal vascular bed topology and collagen matrix organization in mice using 2PEF microscopy. By iteratively imaging and removing tissue through optical ablation, we will visualize the vasculature of the entire placental disk at different gestational ages. Dlx3+/+,Dlx3+/- and Dlx3-/-mice expressing the Tie2-GFP transgene specifically in the fetal vascular compartment together with fluorescent labeling of the maternal vessels/blood spaces will allow simultaneous imaging and quantification of both maternal and fetal vascular compartments. These imaging studies provide the unique opportunity to evaluate the effect(s) of loss of Dlx3 on placental labyrinth vessel number and volume/surface area as well as vessel connectivity, tortuiosity and branching morphogenesis. Aim 2. To examine the hypothesis that in vivo blood flow is reduced in the Dlx3-/- implantation sites. In Aim 2, we shift to in vivo examination of the kinetics of blood flow within the labyrinth in the presence and absence of Dlx3. 2PEF imaging of individual blood vessels enables blood flow speed to be studied one vessel at a time within the labyrinth. We will determine the effects of genetic perturbation of Dlx3 on in vivo blood flow in the fetal vascular compartment and connect these changes to differences in vascular topology determined in Aim 1. The long term goal of the proposed studies is to develop exciting new imaging strategies to examine placental defects in mice representing models of human disease. Inappropriate establishment of fetal and maternal vascular beds within the placenta results in compromised health. Conditions can range from immediate, often life threatening health issues for the mother and baby, such as with preeclampsia, to the longer term effects of the uterine environment on adult onset of disease. These disease situations can have devastating effects on the patient and baby; however, an understanding of the molecular determinants that underlie the development of placental vasculature are far from complete. The studies proposed in this application optimize and implement a novel and exciting in vivo imaging strategy (two-photon excited fluorescence microscopy) to characterize and quantitate vascular bed topology, vascular fibrillar collagen matrix organization and blood flow in an important mouse model of placental failure.