Impact of brain fixation methods on ultrastructural preservation in experimental models

High-quality brain fixation is essential in experimental neuropathology, as it determines tissue preservation for ultrastructural investigations and preclinical drug evaluation. This study examined the effects of two fixation methods - immersion and transcardial perfusion - on neural preservation of adult male Wistar rats. Ultrastructural analysis was performed on the pyramidal cortex and hippocampal CA1 layer under six experimental conditions: (1) brain immersion in fixative immediately after euthanasia; (2) brain immersion in fixative 6 h post-euthanasia; (3) transcardial perfusion fixation using peristaltic pump at physiological flow rate; (4) transcardial perfusion fixation using peristaltic pump at supraphysiological flow rate; (5) transcardial perfusion fixation using gravity-fed hydrostatic system at physiological pressure; and (6) transcardial perfusion fixation using gravity-fed hydrostatic system at supraphysiological pressure. Light microscopy revealed no major differences among groups fixed through transcardial perfusion, whereas immersion fixation was associated with artifactual dark neurons and spongiform-like appearance of the neuropil. Electron microscopy demonstrated that immersion fixation caused poor morphological preservation and prominent artifacts. In contrast, perfusion fixation achieved superior preservation with physiological volumes. However, deviations from appropriate volume or pressure resulted in autolytic changes and artifacts. These findings highlight the critical need for optimal fixation protocols in experimental neuropathological research to prevent fixation-induced artifacts and ensure ultrastructural integrity, especially in pre-clinical drug evaluation in animal models for neurological diseases.