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

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.