Comparative effects of perfusate composition on rat brain histology following transcardial perfusion fixation

Background: Transcardial perfusion fixation is widely used to preserve rodent brains for histological and ultrastructural analysis, but protocols can vary in the use of pre-fixation washout, fixative formulation, and osmotic additives. Relatively few studies have directly compared how the chemical composition of the perfusate affects tissue preservation. Methods: Male Sprague-Dawley rats underwent transcardial perfusion using a series of aldehyde-based fixative formulations that varied in fixative composition, use of a phosphate buffered saline washout, and the addition of mannitol or polyethylene glycol 35 kDa (PEG35). Perfusion outcomes were assessed using gross brain morphology, semi-quantitative grading of vascular blood clearance, quantitative detection of residual erythrocytes in whole-slide histological images, and light microscopic measures of cellular visualization and morphology. Perfusate osmolality was measured, and selected specimens were examined by electron microscopy. Results: Substantial vascular blood clearance was frequently achieved with fixative-only perfusion, indicating that a pre-fixation washout was not required to achieve high levels of blood clearance under the conditions tested. Routine light microscopy-based measures of cellular visibility and morphology were broadly similar across treatment groups. Adding mannitol and PEG35 to the fixative solution both led to concentration-dependent gross tissue shrinkage, particularly with PEG35, without correspondingly large or consistent changes on light microscopy. A substantial degree of case-to-case variation in perfusion quality was also observed among animals undergoing nominally similar procedures. Conclusions: Substantial variation in perfusate composition produced relatively modest differences in the light microscopy outcomes examined, despite pronounced effects of osmotic additives on gross brain morphology. There were some cases where perfusion with fixative alone led to near-complete blood vessel clearance, and the use of a preceding phosphate buffered saline washout was not associated with higher vascular clearance scores. The degree of case-to-case variability observed within treatment conditions suggests that procedural factors can play an important role on perfusion quality in addition to perfusate composition.