Transcardiac Perfusion in Mice: Comparing Heart-Beating and Non-Beating Conditions for Brain Histological and Electrophysiological Analyses

Abstract In vivo and ex vivo animal model experiments are essential for understanding the mechanisms underlying neurological diseases. These investigations bridge the gap between organism behavior and cellular pathways, providing a holistic view of brain function. A key factor is the quality of tissue, which depends on the efficacy of the perfusion process. Rapid and uniform delivery of fixative or physiological solutions is crucial to preserve tissue integrity, yet the effectiveness of perfusion under non-beating heart conditions remains debated. Our study aimed to optimize perfusion conditions for quality brain tissue samples, complying with ethical standards. We tested both beating heart conditions with xylazine/ketamine and non-beating heart conditions with xylazine/pentobarbital, with perfusion following cardiorespiratory arrest, occurring within 5 minutes in our conditions. Electroencephalography and electrocardiography measurements following pentobarbital injection demonstrated a rapid cessation of brain activity coinciding with the onset of irregular, non-sinusoidal cardiac rhythms. Death was confirmed at the time of thoracotomy, performed after cardiorespiratory arrest, which consistently occurred within a 5 minutes window post-injection. Semi-quantitative histology and neurotransmitter immunohistochemistry showed no significant differences between the 2 conditions. Electron microscopy confirmed good tissue quality in both, with similar results in functional studies using electrophysiological approaches. This study demonstrates that post-mortem transcardiac perfusion in mice, performed under conditions where perfusion is carried out very quickly, reliably yields high-quality brain samples for histology, cytology, and electrophysiology. Our findings help address controversies regarding perfusion efficacy and highlight the need to reconsider euthanasia practices to ensure sample quality while minimizing impact on animals and researchers.