Division of Cardiothoracic Surgery, Washington University in St. Louis.
Division of Thoracic Surgery, University of Maryland.
J Vis Exp. 2024 Apr 19(206). doi: 10.3791/66566.
Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in naïve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.
肺移植后的并发症主要与宿主免疫系统对移植物的反应有关。这种免疫反应受供体和受体细胞之间的串扰调节。更好地理解这些过程依赖于使用临床前动物模型，并得益于实时研究移植物内免疫细胞迁移的能力。活体双光子显微镜可用于对组织和器官进行成像，其深度可达数百微米，且光损伤最小，这比单光子共聚焦显微镜具有很大的优势。选择性地使用具有启动子特异性荧光蛋白表达的转基因小鼠，以及在活体双光子显微镜检查期间进行荧光染料标记细胞的过继转移，允许在其生理环境中对单个细胞进行动态研究。我们小组开发了一种稳定小鼠肺的技术，这使我们能够对未成熟肺和原位移植肺移植物中的细胞动力学进行成像。该技术允许对血管内和间质中的细胞行为进行详细评估，以及检查各种细胞群之间的相互作用。该程序易于学习和适应，可用于研究调节肺移植后炎症和耐受反应的免疫机制。它也可以扩展到其他致病性肺部疾病的研究。
