Studying the biodistribution of novel therapeutics and biomaterials in vivo requires effective and consistent perfusion and fixation of major organs. Standard methods for removing red blood cells (RBCs) and fixing tissue often involve transcardial perfusion, such as brain-targeted perfusion (via the left ventricle) or lung-targeted perfusion (via the right ventricle). Using autofluorescence measurements and a bespoke ImageJ macro to quantify RBC content from histology, we compared the efficacy and consistency of three whole-body perfusion techniques. We show that lung-targeted perfusion evacuates more blood from the lung vasculature than brain-targeted perfusion (2054% fewer RBCs), and that our novel approach of ‘dual-targeted’ perfusion (via the right and left ventricles sequentially) had even higher efficacy (306% fewer RBCs). Furthermore, by combining aspects of brain- and lung-targeted methods, dual-targeted perfusion achieved the highest consistency in autofluorescence emissions from major organs (64% and 65% lower variance than brain- and lung-targeted perfusion respectively). Since RBC content and autofluorescence can be confounding factors in biodistribution studies using fluorescent probes, our findings and proposed novel approach offer insight into perfusion fixation techniques for pre-clinical studies.

研究新疗法和生物材料在体内的生物分布需要对主要器官进行有效和一致的灌注和固定。去除红细胞 (RBC) 和固定组织的标准方法通常涉及经心灌注，例如脑靶向灌注（通过左心室）或肺靶向灌注（通过右心室）。使用自体荧光测量和定制的 ImageJ 宏来量化组织学中的 RBC 含量，我们比较了三种全身灌注技术的功效和一致性。我们表明，与脑靶向灌注相比，肺靶向灌注从肺脉管系统中排出更多血液（RBC 减少 2054%），并且我们的“双靶向”灌注新方法（依次通过右心室和左心室）具有更高的功效（红细胞减少 306%）。此外，通过结合大脑和肺靶向方法的各个方面，双靶向灌注在主要器官的自发荧光发射中实现了最高的一致性（分别比脑靶向灌注和肺靶向灌注低 64% 和 65%）。由于红细胞含量和自发荧光可能是使用荧光探针进行生物分布研究的混杂因素，因此我们的研究结果和提出的新方法为临床前研究的灌注固定技术提供了见解。