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Updated: Mar 19, 2026
3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse
Published on: May 19, 2015
Variations in post-perfusion immersion fixation and storage alter MRI measurements of mouse brain morphometry
A Elizabeth de Guzman1, Michael D Wong2, Jacqueline A Gleave2
1Mouse Imaging Centre, Hospital for Sick Children, 25 Orde Street, Toronto, Ontario M5T 3H7, Canada; Department of Medical Biophysics, University of Toronto, 610 University Avenue, Rm 7-411, Toronto, Ontario M5G 2M9, Canada.
Abstract:
Ex vivo magnetic resonance imaging (MRI) requires chemical fixation to preserve tissue during storage or extended imaging sessions. Although it is commonly understood that fixation may alter tissue volume and shape, the potential confounding effects of fixation and storage on morphometric analyses have not been well characterized. With increasing use of ex vivo MRI for mouse brain phenotying and opportunities for inter-study comparisons, we sought to characterize how changes in fixation and/or storage times affected tissue volume, and how this might impact phenotyping results. Mouse brain samples that had been perfusion fixed, within the skull as per our standard protocol, were immersed in formaldehyde-based fixative for 1 to 5days before being stored in saline or water. Throughout fixation and storage, samples were repeatedly scanned using magnetic resonance imaging, and analyzed for volume expansion or shrinkage. We found that most of the brain continued to shrink post fixation, with the rate of shrinkage dependent on the solution in which the samples were submerged. Maximum changes in volume of 3.5% per day and 3% per month were detected during fixation and storage (in PBS), respectively. Most notably, changes were non-uniform, with some structures shrinking slower, or even expanding, when compared to other structures in the brain. Our results highlight that caution is necessary when interpreting results from experiments with inconsistent fixation and storage protocols, so as not to mistake these changes for phenotypic differences.
Insights
Chemical fixation and storage alter ex vivo mouse brain volume, impacting morphometric analysis. These tissue volume changes are solution-dependent and non-uniform, necessitating careful consideration in phenotyping studies.
Area of Science:
- Neuroscience
- Biomedical Imaging
- Anatomical Pathology
Background:
- Ex vivo magnetic resonance imaging (MRI) relies on chemical fixation for tissue preservation.
- Fixation is known to alter tissue volume and shape, but its impact on morphometric analysis is not fully understood.
- Standardized protocols are crucial for ex vivo MRI mouse brain phenotyping and inter-study comparisons.
Purpose of the Study:
- To characterize the effects of fixation and storage duration on ex vivo mouse brain volume.
- To assess how these volume changes influence morphometric analysis and phenotyping results.
- To provide guidance for consistent experimental protocols in ex vivo MRI studies.
Main Methods:
- Mouse brains were perfusion-fixed and immersed in formaldehyde-based fixative (1-5 days).
- Samples were stored in saline or water and repeatedly scanned using MRI.
- Volume changes were analyzed throughout fixation and storage periods.
Main Results:
- Mouse brains exhibited continuous shrinkage post-fixation, with rates varying by storage solution.
- Maximum shrinkage rates of 3.5% per day (fixation) and 3% per month (storage in PBS) were observed.
- Volume changes were non-uniform across brain structures, with some expanding while others shrank at different rates.
Conclusions:
- Inconsistent fixation and storage protocols can lead to significant, non-uniform changes in ex vivo mouse brain volume.
- These volume alterations may be misinterpreted as phenotypic differences.
- Caution is advised when interpreting ex vivo MRI morphometric data; standardized protocols are essential.
