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Distortion in formalin-fixed brains: using geometric morphometrics to quantify the worst-case scenario in mice

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Abstract

Although morphometric studies of fixed mammalian brains are an integral part of neuroscience, the nature of fixation-related morphometric artifacts is not well understood beyond assessments of size changes over fixation time. This study is the first to quantitatively co-evaluate the effects of the most common brain tissue fixative—formalin—on brain shape, size, and weight, using two-dimensional landmark analysis of mouse brains fixed in unbuffered, non-saline formalin from fresh specimens up to 213 days of preservation. The brains show a typical swelling reaction with subsequent decline in size and weight. Weight initially under- and later over-estimates size, so that the practice of using weight to estimate volume can be problematic. Time to recovery of original size resembled that of much larger brained mammals, suggesting that the slow reaction of formalin with tissue components mainly determines recovery times. Non-size related (anisotropic) distortion of different brain areas accounted for around a quarter of overall change suggesting that the use of “all-brain” fixation correction factors can introduce considerable error. Distortion occurs mostly after the first day of fixation, and extended fixation times impact mostly on size, not shape. Fixation effects relatively wider and stouter brain dimensions, except the cerebellum whose shape changes less. Evidence from the literature suggests that this pattern may be common to mammals due to structural commonalities.

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Acknowledgments

I thank Julie Gautrey from the Department of Physiology, Cambridge University, for providing me with the mouse specimens, N. Butterfield from Earth Sciences, Cambridge University, for the use of his lab facilities, and A. Goswami (UCL) for discussion and advice. I thank two anonymous reviewers for their valuable comments on this manuscript. This work was supported by a Volkswagen Foundation Evolution Initiative Postdoctoral grant No. 1/83 505.

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  1. School of Biological Science, University of Queensland, Brisbane, 4072, Australia

    Vera Weisbecker

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  1. Vera Weisbecker
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Correspondence to Vera Weisbecker.

Electronic supplementary material

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429_2011_366_MOESM1_ESM.doc

Online resource 1 Tables of significance of paired and conventional Holm-adjusted comparisons between specimens at different preservation times, including comparisons of weight and centroid sizes (1.1) as well as actual measurement comparisons (1.2) (DOC 116 kb)

429_2011_366_MOESM2_ESM.eps

Online resource 2 Plot of discriminant function (df) 1 against df 2 of the discriminant function analysis of size-scaled ventral landmarks; colours are as in Fig. 3. Fresh specimens score significantly lower on df1 than all preserved specimens. Specimens fixed for 213 days overlap with shorter preservation times on df 2 but score higher. Exaggerated distortion plots showing the displacement of landmarks at extreme (+10) df1 scores (fresh specimens) and high df2 values (specimens preserved for 213 days) are also shown. Warm colours (red and yellow) present relative expansion, cold colours (blue and green) represent relative contraction). For details, see results section (EPS 1,828 kb)

429_2011_366_MOESM3_ESM.eps

Online Resource 3 Boxplots of actual measurements at all preservation times on the ventral aspect of the brain affected most and least by deformation from fresh to preserved state. Cerebrum (a) and ventral midbrain (c) widen considerably after 24 h of fixation and subsequently decrease in width. Midline lengths (b) initially increase but are the only measurement to shrink to below original size after 213 days (EPS 619 kb)

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Weisbecker, V. Distortion in formalin-fixed brains: using geometric morphometrics to quantify the worst-case scenario in mice. Brain Struct Funct 217, 677–685 (2012). https://doi.org/10.1007/s00429-011-0366-1

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