Chemical fixation with solutions containing glutaraldehyde led to a pale yellow color change in the rat brain slices within 1 h of slice immersion. Samples fixed in glutaraldehyde solutions (even after overnight washing in PBS) remained rigid and dense relative to viable slices or slices fixed only with formaldehyde. Prior to immersion fixation, the molecular layer appeared distinct compared to the remaining cortical layers of the rat cortical slices (). The lateral edges of individual slices also demonstrated increased water diffusivity and decreased T2 due to the procurement process. Note that fixation in any of the aldehyde solutions obscured these visual differences (). These regions were excluded in the ROI used for quantitative analysis of the data. Otherwise, there were limited visual MRI image differences for cortical slices imaged prior to fixation or after fixation with the various solutions and overnight washing with PBS. The subsequent MRI property differences due to fixative treatments became more readily apparent through quantitative analysis.
FIG. 1 Representative diffusion-weighted MR images of viable rat cortical slices (a), cortical slices after fixation in 4% formaldehyde (b), rat cortical slices after fixation in Karnovsky's solution (2% formaldehyde and 2% glutaraldehyde) (c) and rat cortical (more ...)
The T1 values for viable cortical slices (2.020 ± 0.050 s) were statistically different from all other treatment groups (P < 0.001) (). After immersion fixation, the T1 decreased 21% with 4% formaldehyde fixation, 26% for Karnovsky's solution fixation, and 25% for 4% glutaraldehyde fixation (all comparisons, P < 0.001). Slices immersed in 4% formaldehyde had T1 values postfixation (1.60 ± 0.04 s) that were 6% higher than slices fixed in Karnovsky's solution (P < 0.001) and 5% higher than slices fixed in 4% glutaraldehyde (P = 0.001). The T1 of slices fixed in Karnovsky's solution did not differ significantly from slices fixed in 4% glutaraldehyde. Washing slices fixed in 4% glutaraldehyde overnight with PBS increased the slice T1 by 10% to 1.66 ± 0.03 s (P < 0.001). Washing overnight did not significantly change the T1 for slices fixed in 4% formaldehyde or Karnovsky's solution (P > 0.05). After washing, the T1 of slices fixed in Karnovsky's solution (1.53 ± 0.06) were 8% lower than slices treated with both the other fixatives (P < 0.001).
FIG. 2 Bar graph demonstrating the effects of aldehyde chemical fixation on the T1 relaxation properties of rat cortical slices (mean ± SD). Fixation with 4% formaldehyde, 4% glutaraldehyde, or Karnovsky's solution (2% formaldehyde and 2% glutaraldehyde) (more ...)
The T2 values for viable cortical slices (0.098 ± 0.006 s) also were statistically different from all other treatment groups (P ≤ 0.01) except compared to samples fixed in Karnovsky's solution and washed with PBS overnight (P = 0.987) (). Immersion fixation reduced rat cortical slice T2 by 81% for slices in 4% formaldehyde, 64% for slices in Karnovsky's solution and 54% for slices in 4% glutaraldehyde (all comparisons, P < 0.001). The T2 of slices immersed in the three different fixative solutions were all statistically different from each other (P < 0.01) such that T2 decreased approximately 6.5 ms for each 1% addition of formaldehyde to the final fixative solution (range = 0–4% formaldehyde, R2 = 0.983). Hence, slices in 4% formaldehyde had the lowest T2 (0.019 ± 0.002 s). Washing immersion-fixed cortical slices overnight in PBS significantly increased T2 in all fixative treatment groups (all comparisons, P < 0.001) such that the T2 of slices fixed in Karnovsky's solution and washed overnight in PBS no longer differed significantly from unfixed, viable slices (P = 0.987). However, the T2 of washed slices originally fixed in 4% formaldehyde (0.117 ± 0.015 s) or 4% glutaraldehyde (0.107 ± 0.003 s) were 19% and 9% higher than the T2 of viable cortical slices, respectively (both comparisons, P ≤ 0.01). After washing, slices originally immersed in the three different fixative solutions were again all statistically different from each other (P < 0.01).
FIG. 3 Bar graph demonstrating the effects of aldehyde chemical fixation on the T2 relaxation properties of rat cortical slices (mean ± SD). Fixation with 4% formaldehyde, 4% glutaraldehyde, or Karnovsky's solution (2% formaldehyde and 2% glutaraldehyde) (more ...)
Aldehyde fixation also reduced the SNR measured in proton-density-weighted images. For example, SNR was reduced 37% by immersion in 4% formaldehyde (P
< 0.001), while subsequent washing of the slices fixed with 4% formaldehyde restored SNR to 86% of prefixation values (P
< 0.001). Similar SNR reductions were observed in slices immersed in glutaraldehyde solutions (e.g., 32% decrease with Karnovsky's solution, P
< 0.001). Unlike slices fixed in 4% formaldehyde, however, there was no significant SNR improvement after washing the glutaraldehyde-fixed samples overnight in PBS. This low SNR observed in the proton density images and the short T2
of slices still immersed in their respective fixation solutions was most evident in diffusion-weighted images, especially at high b
-values and long diffusion times. Hence, the SNRs of diffusion MRI data of rat brain slices immersed in the three fixative solutions were not sufficient for acceptable fits of the two-compartment diffusion model. Similar SNR limitations for diffusion analysis were reported previously in a study of a single human brain immersed in fixative (12
The two-compartment model of water diffusion fit the data well () and indicated that aldehyde fixation had several significant effects on the cortical slice microstructures sampled by water diffusion (). It was not possible to accurately determine the intracellular diffusion coefficient due the effects of restriction in cortical slices even at relatively short diffusion times of 10 ms (31
). Fixation with 4% formaldehyde increased the ADCEX
of cortical slices by 88% compared to viable, unfixed tissue (P
= 0.019). Slice fixation with Karnovsky's solution also increased ADCEX
by 47%, although this change was not statistically significant (P
= 0.377), probably due to variability in the fits of this model parameter. Slice fixation in 4% glutaraldehyde also did not significantly change ADCEX
. Fixation with all three solutions increased the apparent restriction size (a) in the rat cortical slices 30% to 40% from viable tissue values (P
≤ 0.021, all comparisons). The cortical slice transmembrane water exchange rate (kIE
) was increased by fixation in 4% formaldehyde and Karnovsky's solutions by 239% and 244%, respectively, from control tissues (P
< 0.001, both comparisons). This corresponded to a reduction in the mean intracellular residence time of water (equivalent to 1/KIE
) from 61 ms in viable slices to 18 ms in cortical slices fixed using formaldehyde-containing solutions. The exchange rate in slices fixed with 4% glutaraldehyde, however, did not differ significantly from control slices, but was statistically different from the other two fixation treatments (P
< 0.001, both comparisons). In contrast, 4% glutaraldehyde significantly reduced the intracellular magnetization fraction (MIN
) by 25% from viable, unfixed cortical slices (P
= 0.009) whereas the other two fixation solutions did not alter MIN
significantly compared to viable slices (although these too differed significantly from the 4% glutaraldehyde-treated slices).
FIG. 4 Representative semi-log diffusion-weighted signal attenuation plots for viable, perfused rat cortical slices (a), and cortical slices fixed in 4% formaldehyde (b), Karnovsky's (2% formaldehyde and 2% glutaraldehyde) (c), or 4% glutaraldehyde solutions (more ...)