Perfusion fixation methods for preclinical biodistribution studies: A comparative assessment using automated image processing.

Studying the biodistribution of novel therapeutics and biomaterialsin vivorequires 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 (20 ± 54% fewer RBCs), and that our novel approach of 'dual-targeted' perfusion (via the right and left ventricles sequentially) had even higher efficacy (30 ± 6% 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.

Ultrasound-sensitive nanodroplets achieve targeted neuromodulation.

Focused ultrasound (FUS) offers an attractive tool for non-invasive neuromodulation, addressing a clinical need to develop more minimally invasive approaches that are safer, more tolerable and versatile. In combination with a cavitation agent, the effects of ultrasound can be amplified and localized for therapy. Using c-Fos expression mapping, we show how ultrasound-sensitive nanodroplets can be used to induce either neurosuppression or neurostimulation, without disrupting the blood-brain barrier in rats. By repurposing a commercial ultrasound contrast agent, Definity, lipid-shell decafluorobutane-core nanodroplets of 212.5 ± 2.0 nm were fabricated and loaded with or without pentobarbital. FUS was delivered with an atlas-based targeting system at 1.66 MHz to the motor cortex of rats, using a feedback-controller to detect successful nanodroplet vaporization and drug release. Neuromodulation was quantified through changes in sensorimotor function and c-Fos expression. Following FUS-triggered delivery, sham nanodroplets induced a 22.6 ± 21% increase in local c-Fos expression, whereas pentobarbital-loaded nanodroplets induced a 21.7 ± 13% decrease (n = 6). Nanodroplets, combined with FUS, offer an adaptable tool for neuromodulation, through local delivery of small molecule anesthetics or targeted mechanical effects.