Merz George, Schwenk Valerie, Shah Ruchit, Salafia Carolyn, Necaise Phillip, Joyce Michael, Villani Tom, Johnson Michael, Crider Nick
The Institute for Basic Research, The New York State Office for People with Developmental Disabilities;
The Institute for Basic Research, The New York State Office for People with Developmental Disabilities.
J Vis Exp. 2018 Mar 29(133):57099. doi: 10.3791/57099.
Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.
母体与胎儿之间的营养物质和气体交换发生在母体绒毛间隙血液与构成人类胎盘大部分实质的庞大绒毛毛细血管网络的界面处。远端绒毛毛细血管网络是脐带伸出的血管经过几代分支后胎儿血液供应的终点。这个网络有一个连续的细胞鞘，即合体滋养层屏障层，它可以防止胎儿血液与它持续浸泡其中的母体血液混合。在母体糖尿病、高血压和肥胖等疾病中发生的胎盘毛细血管网络完整性受损，会对胎儿、婴儿和成人造成严重的健康风险。为了更好地确定这些损伤的结构影响，本研究制定了一个方案，该方案能够捕捉1 - 2毫米量级的毛细血管网络结构，从而可以全面研究其拓扑特征。为此，从胎盘中解剖出终末绒毛簇，并对滋养层和毛细血管内皮进行免疫标记。然后用一种新的组织透明化方法对这些样本进行透明化处理，这使得获取深度约为1毫米的共聚焦图像堆栈成为可能。然后对这些堆栈的三维渲染图进行处理和分析，以生成诸如体积、毛细血管分支数量和毛细血管分支端点等基本毛细血管网络测量值，作为验证该方法用于毛细血管网络表征的适用性。
