Effects of formalin fixation and temperature on MR relaxation times in the human brain

Abstract

Post-mortem MRI of the brain is increasingly applied in neuroscience for a better understanding of the contrast mechanisms of disease induced tissue changes. However, the influence of chemical processes caused by formalin fixation and differences in temperature may hamper the comparability with results from in vivo MRI. In this study we investigated how formalin fixation and temperature affect T1, T2 and T2* relaxation times of brain tissue. Fixation effects were examined with respect to changes in water content and crosslinking. Relaxometry was performed in brain slices from five deceased subjects at different temperatures. All measurements were repeated after 190 days of formaldehyde immersion. The water content of unfixed and fixed tissue was determined using the wet-to-dry ratio following drying. Protein weight was determined with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Fixation caused a strong decrease of all relaxation times, the strongest effect being seen on T1, with a reduction of up to 76%. The temperature coefficient of T1 was lower in the fixed than unfixed tissue, which was in contrast to T2, where an increase of the temperature coefficient was observed following fixation. The reduction of the water content after fixation was in the range of 1-6% and thus not sufficient to explain the changes in relaxation time. Results from SDS-PAGE indicated a strong increase of the protein size above 260 kDa in all brain structures examined. Our results suggest that crosslinking induced changes of the macromolecular matrix are responsible for T1 shortening and a decreased temperature dependency. The relaxation times provided in this work should allow optimization of post-mortem MRI protocols for the brain.

Neurite dispersion: a new marker of multiple sclerosis spinal cord pathology?

“…Fixation can alter tissue microstructure47, 48; therefore, NODDI metrics as shown here may not be fully representative of their in vivo counterparts. Nevertheless, our results agree well with preliminary findings in vivo , 39, 49 providing confidence that the conclusions of this study will most likely hold true in clinical scenarios.…”

The separate effects of lipids and proteins on brain MRI contrast revealed through tissue clearing

“…Firstly, we must consider the effects of protein-crosslinking induced by both tissue fixation and hydrogel embedding. PFA forms methylene bridges between active –NH groups of the proteins (Birkl et al, 2016) and hydrogel embedding leads to cross-links between proteins and the surrounding acrylamide network in the presence of PFA (Tomer et al, 2014). This reduction of active –NH groups and decreased protein mobility was previously studied within protein solutions and shown to have varied affects on T1 and T2 depending on the buffer solution, pH and whether or not the proteins had been denatured (Mäkelä et al, 2001).…”

“…This reduction of active –NH groups and decreased protein mobility was previously studied within protein solutions and shown to have varied affects on T1 and T2 depending on the buffer solution, pH and whether or not the proteins had been denatured (Mäkelä et al, 2001). Within human tissue, fixation has been shown to decrease in T1 and T2 relaxation times (Birkl et al, 2016). Specifically, at 3T, T1 in fixation was shown to decrease T1 by 67–76% in GM and 67% in WM and T2 by 5–11% in GM and 26% in WM.…”

Effects of concentration and vendor specific composition of formalin on postmortem MRI of the human brain

“…Formalin fixation of biological tissue is a procedure to stop the autolytic processes and to enable long‐term tissue storage . The major biochemical process during formalin fixation consists of building crosslinks between tissue proteins, which, not unexpected, affects the biophysical properties of the tissue and also its relaxation behavior . Fixation induced shortening of T 1 and T 2 relaxation time constants of formalin fixed tissue have been reported , and the time dependency of the fixation process has been modeled .…”

“…The major biochemical process during formalin fixation consists of building crosslinks between tissue proteins, which, not unexpected, affects the biophysical properties of the tissue and also its relaxation behavior . Fixation induced shortening of T 1 and T 2 relaxation time constants of formalin fixed tissue have been reported , and the time dependency of the fixation process has been modeled . After extensive fixation, T 1 relaxation time can be reduced by approximately 70% and T 2 by approximately 25% compared with the unfixed condition .…”