Comparative study of pre- and post-mortem perfusion of fixative for the quality of neuronal tissue preparation

Ante-mortem transcardiac perfusion of a fixative agent is generally recommended for quality preparations for cerebral histology, ensuring rapid and deep penetration in the tissue to preserve the most fragile brain structures. Despite being performed under anesthesia and with proper analgesia, this procedure is cumbersome for the experimenter and raises ethical questions. Recently, alternative protocols have been proposed based on prior animal euthanasia followed by an injection of a fixative agent into the circulation. These so-called post-mortem perfusion protocols should in theory ensure an equivalent quality of tissue fixation, without exposing live animals to a procedure. Before adopting this new method, it is necessary to validate that sample quality is equivalent, ensuring the validity of scientific results. Here we performed a parallel comparison of several protocols of tissue fixation by ante-mortem or post-mortem transcardiac perfusion and measured the impact on the maintenance of axonal structures, dendritic spines and mitochondrial morphology. Our results showed that histological parameters show variable sensitivity to perfusion conditions and fixatives used. For instance, axon fragmentation and altered mitochondrial morphology were observed in post-mortem transcardiac perfusion groups. We furthermore determined that the fixation condition had a variable effect on immunostaining, impacting the detected expression level or pattern. Our results serve as a guide to orient the experimenter in selecting the best condition for optimal tissue fixation, which minimizes animal suffering while guaranteeing the integrity of the biological results obtained.