Developing formalin-based fixative agents for post-mortem brain MRI at 9.4T

Figure 1: The B1-field at 400MHz (9.4T) of each fixative solution was measured in an elliptic 2.5Liter container in polycarbonate designed for measuring whole post-mortem human brain samples (upper row). The B1-pattern and dielectric properties of Fix01 are close to the ones encountered in vivo at 9.4T. The measurements were repeated for each fixative solution in a 4-chamber elliptic container, that was 3D-printed using poly-lactic-acid and had polycarbonate lids (lower row). The upper left quarter of the ellipsoid was filled with the fixative alone, while the other three contained the same fixative in presence of pig brain samples which had been preserved in the fixative for 38 (Fix04), 39 (Fix03), 41 (Fix01) or 42days (Fix02). The B1 maps suggest that the containers themselves and also the presence of tissue can affect B1-field uniformity. The (mean) dielectric properties of each fixative at 400MHz and room temperature (± standard deviation from 4 repetitions) were: 0.60±0.01/1.55±0.08/1.98±0.02/1.89±0.01 S/m for the conductivity of Fix01/02/03/04, respectively and 71.2±0.24/70.3±0.16/73.8±0.05/74.0±0.23 for the permittivity. — **Figure 1:** The B₁-field at 400MHz (9.4T) of each fixative solution was measured in an elliptic 2.5Liter container in polycarbonate designed for measuring whole post-mortem human brain samples (upper row). The B₁-pattern and dielectric properties of Fix01 are close to the ones encountered in vivo at 9.4T. The measurements were repeated for each fixative solution in a 4-chamber elliptic container, that was 3D-printed using poly-lactic-acid and had polycarbonate lids (lower row). The upper left quarter of the ellipsoid was filled with the fixative alone, while the other three contained the same fixative in presence of pig brain samples which had been preserved in the fixative for 38 (Fix04), 39 (Fix03), 41 (Fix01) or 42days (Fix02). The B₁ maps suggest that the containers themselves and also the presence of tissue can affect B₁-field uniformity. The (mean) dielectric properties of each fixative at 400MHz and room temperature (± standard deviation from 4 repetitions) were: 0.60±0.01/1.55±0.08/1.98±0.02/1.89±0.01 S/m for the conductivity of Fix01/02/03/04, respectively and 71.2±0.24/70.3±0.16/73.8±0.05/74.0±0.23 for the permittivity.

**Figure 1:** The B₁-field at 400MHz (9.4T) of each fixative solution was measured in an elliptic 2.5Liter container in polycarbonate designed for measuring whole post-mortem human brain samples (upper row). The B₁-pattern and dielectric properties of Fix01 are close to the ones encountered in vivo at 9.4T. The measurements were repeated for each fixative solution in a 4-chamber elliptic container, that was 3D-printed using poly-lactic-acid and had polycarbonate lids (lower row). The upper left quarter of the ellipsoid was filled with the fixative alone, while the other three contained the same fixative in presence of pig brain samples which had been preserved in the fixative for 38 (Fix04), 39 (Fix03), 41 (Fix01) or 42days (Fix02). The B₁ maps suggest that the containers themselves and also the presence of tissue can affect B₁-field uniformity. The (mean) dielectric properties of each fixative at 400MHz and room temperature (± standard deviation from 4 repetitions) were: 0.60±0.01/1.55±0.08/1.98±0.02/1.89±0.01 S/m for the conductivity of Fix01/02/03/04, respectively and 71.2±0.24/70.3±0.16/73.8±0.05/74.0±0.23 for the permittivity.

Purpose: Ultra-high field Magnetic Resonance Imaging of whole post-mortem brains is complementary to histology and pathology studies and can be useful for tissue characterization, sectioning, 3D-reconstruction of sectioned tissue data, and for diagnosis and investigation of neurodegenerative diseases. To preserve the tissue, the cerebrospinal fluid must be replaced by a fixative agent such as formalin.
Here we show how such agents can be tailored to achieve a) dielectric properties that ensure a homogeneous B₁-field, b) a magnetic susceptibility matching the tissue to improve B₀ homogeneity, and c) a singulet ¹H-NMR spectra to prevent chemical-shift-artefacts.

Figure 2: Measured B1-values in absence and presence of brain tissue. Histograms for the fixative solutions measured in (A) a 2.5L elliptic container designed for human whole brain samples, and for (B) one of the 4-chambers of an elliptic container which contains unloaded fixative shown in Fig. 1. Fix01 has a wider histogram, while the values of the remaining fixatives more closely adhere to a Gaussian distribution. The differences in B1 between the fixatives becomes more pronounced in the tissue samples (B) but remains stable during 0.5-35days of immersion fixation both in grey matter (C) and in white matter (D). The influence of tissue depth (E,F) is prominent and exceeds the standard-deviation across 0.5-35days of immersion fixation, shown as bars. The B1-values in white matter voxels (D,F) are higher than in the grey matter (C,E), especially at earlier immersion times, at increasing tissue depths and in presence of PVP (Fix01 and Fix02). — **Figure 2:** Measured B₁-values in absence and presence of brain tissue. Histograms for the fixative solutions measured in (A) a 2.5L elliptic container designed for human whole brain samples, and for (B) one of the 4-chambers of an elliptic container which contains unloaded fixative shown in Fig. 1. Fix01 has a wider histogram, while the values of the remaining fixatives more closely adhere to a Gaussian distribution. The differences in B₁ between the fixatives becomes more pronounced in the tissue samples (B) but remains stable during 0.5-35days of immersion fixation both in grey matter (C) and in white matter (D). The influence of tissue depth (**E,F**) is prominent and exceeds the standard-deviation across 0.5-35days of immersion fixation, shown as bars. The B₁-values in white matter voxels (**D,F**) are higher than in the grey matter (**C,E**), especially at earlier immersion times, at increasing tissue depths and in presence of PVP (Fix01 and Fix02).

**Figure 2:** Measured B₁-values in absence and presence of brain tissue. Histograms for the fixative solutions measured in (A) a 2.5L elliptic container designed for human whole brain samples, and for (B) one of the 4-chambers of an elliptic container which contains unloaded fixative shown in Fig. 1. Fix01 has a wider histogram, while the values of the remaining fixatives more closely adhere to a Gaussian distribution. The differences in B₁ between the fixatives becomes more pronounced in the tissue samples (B) but remains stable during 0.5-35days of immersion fixation both in grey matter (C) and in white matter (D). The influence of tissue depth (**E,F**) is prominent and exceeds the standard-deviation across 0.5-35days of immersion fixation, shown as bars. The B₁-values in white matter voxels (**D,F**) are higher than in the grey matter (**C,E**), especially at earlier immersion times, at increasing tissue depths and in presence of PVP (Fix01 and Fix02).

Method: Dielectric properties of formalin-based agents were assessed (100MHz-4.5GHz), and four candidate fixatives with/without polyvinylpyrrolidone (PVP) and different salt concentrations were formulated. B₁-field and MR-properties (T1, R₂^*, R₂, R₂^’ and magnetic susceptibility (QSM)) were observed in white and gray matter of pig brain samples during 0.5-35days of immersion fixation. The kinetics was fitted using exponential functions. The immersion time required to reach maximum R₂^*-values at different tissue-depths was used to estimate the Medawar-coefficient for fixative penetration. The impact of replacing the fixatives with Fluoroinert and phosphate-buffered-saline (PBS) as embedding media was also evaluated.

Figure 3: Depth dependence of R2*-kinetics during immersion fixation with different fixatives. The spatial locations of different tissue depths are shown in a sagittal slice through the pig brain hemisphere (A) with corresponding curves fitted to the experimental R2*-kinetics (B). The curves from the first 1-4mm are located in the GM, while the remaining curves are dominated by WM. The day, DMAX, at which maximum R2* is reached is shown as maps obtained from pixel-wise-fits for Fix01 (C), Fix02 (D); Fix03 (E) and Fix04 (F). Median values for (DMAX*24)0.5 in GM (G) and WM (H) at different tissue depths are shown as dots. Data points fulfilling three criteria: ≥50 voxels within a certain depth-bin, dbin , going from 0.5 to 15.5mm with a bin-width of 1mm; adjusted regression-coefficient R2-square>0.8; DMAX<25 were used to fit the Medawar-coefficient of fixative diffusivity K, obtained from: dbin=Kt , where t=DMAX*24 is expressed in hours. With PVP (Fix01, Fix02) K=0.9mm/(hour)0.5 ; without PVP penetration and fixation was faster: 1.5mm/(hour)0.5 . — **Figure 3:** Depth dependence of R₂^*-kinetics during immersion fixation with different fixatives. The spatial locations of different tissue depths are shown in a sagittal slice through the pig brain hemisphere (A) with corresponding curves fitted to the experimental R₂^*-kinetics (B). The curves from the first 1-4mm are located in the GM, while the remaining curves are dominated by WM. The day, DMAX, at which maximum R₂^*is reached is shown as maps obtained from pixel-wise-fits for Fix01 (C), Fix02 (D); Fix03 (E) and Fix04 (F). Median values for *(DMAX*24*)*^0.5* in GM (G) and WM (H) at different tissue depths are shown as dots. Data points fulfilling three criteria: ≥50 voxels within a certain depth-bin, dbin , going from 0.5 to 15.5mm with a bin-width of 1mm; adjusted regression-coefficient R2-square>0.8; DMAX<25 were used to fit the Medawar-coefficient of fixative diffusivity K, obtained from: dbin=Kt , where *t=DMAX*24* is expressed in hours. With PVP (Fix01, Fix02) K=0.9mm/(hour)^0.5 ; without PVP penetration and fixation was faster: 1.5mm/*(hour)^0.5* .

**Figure 3:** Depth dependence of R₂^*-kinetics during immersion fixation with different fixatives. The spatial locations of different tissue depths are shown in a sagittal slice through the pig brain hemisphere (A) with corresponding curves fitted to the experimental R₂^*-kinetics (B). The curves from the first 1-4mm are located in the GM, while the remaining curves are dominated by WM. The day, DMAX, at which maximum R₂^*is reached is shown as maps obtained from pixel-wise-fits for Fix01 (C), Fix02 (D); Fix03 (E) and Fix04 (F). Median values for *(DMAX*24*)*^0.5* in GM (G) and WM (H) at different tissue depths are shown as dots. Data points fulfilling three criteria: ≥50 voxels within a certain depth-bin, dbin , going from 0.5 to 15.5mm with a bin-width of 1mm; adjusted regression-coefficient R2-square>0.8; DMAX<25 were used to fit the Medawar-coefficient of fixative diffusivity K, obtained from: dbin=Kt , where *t=DMAX*24* is expressed in hours. With PVP (Fix01, Fix02) K=0.9mm/(hour)^0.5 ; without PVP penetration and fixation was faster: 1.5mm/*(hour)^0.5* .

Results: The dielectric properties of formalin were non-linearly modified by increasing amounts of additives. With 5%PVP and 0.04%NaCl, the dielectric properties and B₁-field reflected in-vivo-conditions (Figure 1). The highest B₁ values were found in white matter with PVP and varied significantly with tissue depth and embedding media, but not with immersion-time (Figure 2). MR-properties depended on PVP yielding lower T₁, higher R₂* a more paramagnetic QSM values, and a lower Medawar-coefficient(0.9mm/(hour)^0.5 ; without PVP: 1.5). Regardless of fixative, switching to PBS as embedder caused a paramagnetic shift in QSM and decreased R₂^* that progressed during 1month of storage, while no differences were found with Fluorinert.

Conclusion: In vivo-like B₁-fields can be achieved in formalin-fixatives using PVP and a low salt-concentration, yielding lower T₁, higher R₂* and more paramagnetic QSM than without additives. The kinetics of R₂* allowed estimation of fixative tissue penetration (Figure 3).

Azadeh Nazemorroaya, Ali Aghaeifar, Thomas Shiozawa, Bernhard Hirt, Hildegard Schulz, Klaus Scheffler, and Gisela E. Hagberg

Developing formalin-based fixative agents for post-mortem brain MRI at 9.4T

Magnetic Resonance in Medicine. 21 Dec 2021.

DOI