Optimal tissue preparation for ex vivo preclinical imaging
Rachel L. C. Barrett1,2, Diana Cash1, Camilla Simmons1, Tobias Wood1, Anthony C. Vernon3,4, Marco Catani1,2, and Flavio Dell'Acqua2

1Department of Neuroimaging, IOPPN, King's College London, London, United Kingdom, 2Natbrainlab, Department of Forensic and Neurodevelopmental Sciences, IOPPN, King's College London, London, United Kingdom, 3Department of Basic and Clinical Neuroscience, IOPPN, King's College London, London, United Kingdom, 4MRC Centre for Neurodevelopmental Disorders, King's College London, London, United Kingdom


Ex vivo imaging is beneficial for studying rodent brain microstructure in healthy and pathological tissue at high resolution. There are challenges however associated with changes in tissue properties resulting from fixation. We present a tissue preparation protocol optimised for diffusion MRI in the rat brain by varying fixative concentration, gadolinium concentration and rehydration time. By altering T1 and T2 relaxivity, we show how these factors can be combined to maximise SNR efficiency. Improving SNR efficiency in ex vivo diffusion MRI will allow higher spatial and angular resolution for studying tissue microstructure in the rodent brain.


Preclinical ex vivo magnetic resonance imaging (MRI) is advantageous for imaging the rodent brain at microscopic scale through combination of high field strengths (>7T) and long imaging times. Diffusion MRI in particular offers unique opportunities to study microstructure and three-dimensional organization of white matter fibers in the rodent brain at high resolution, in normal animals and disease relevant models. However due to changes in tissue properties after perfusion fixation1,2 ex vivo MRI is not without challenges. The reduced T2 in fixed tissue and long TE necessary for diffusion contrast limits the signal to noise ratio (SNR). Here, we present a new tissue fixation protocol optimized for diffusion MRI. T1/T2 can be optimized by varying fixative concentration3-5 in perfusion, rehydration time after fixation6,2,7 and amount of gadolinium contrast agent in ‘active fixation’8,9. We demonstrate how these factors can be combined for an optimal MRI tissue preparation procedure, through maximization of SNR efficiency.


Animal preparation

Adult male rats (Sprague Dawley, n=12) were euthanized with pentobarbital (60 mg/kg i.p.) and transcardially perfused with ice-cold 0.9% saline followed by either 2% or 4% paraformaldehyde (PFA, ‘Parafix’, Pioneer Research Chemicals, UK) containing Gd-DTPA (Magnevist) in a concentration range of 0-50mM. The heads were removed and stored in the fixation solution for 4 days, then transferred to phosphate buffered saline (PBS) with either 0 or 1mM Gd-DTPA, at +4°C. Rats (n=2) were scanned at intervals between 0-35 days of rehydration, with the remainder (n=10) at 35 days. Heads were moved to room temperature 4 hours before scanning.

MRI acquisition

MR imaging was performed at the KCL BRAIN Centre (https://brain-imaging.org), on a Bruker 9.4T scanner with Rapid 39mm volume coil. For T1 mapping, a rapid acquisition with relaxation enhancement (RARE) sequence was used with 6 TRs from 200-5500ms, TE=7ms. For T2 mapping, a multi-slice multi-echo sequence (MSME) was used with 30 TEs from 8-240ms, TR=2000ms. Both acquisitions had in-plane resolution 0.23x0.23mm and slice thickness 1mm.

Analysis T1 and T2 maps were obtained using Bruker Image Sequence Analysis tools. Mean and standard deviation of T1 and T2 were measured in a 10-voxel region of interest placed at the midpoint of the corpus callosum. Curves relating T1 and T2 to [Gd-DTPA] were fitted in Matlab as2


These values were then used to calculate SNR efficiency to show the best choice of [Gd-DTPA] and TR, for different values of TE, using2:

$$\text{SNR}_{\text{efficiency}}\propto \frac{1}{\sqrt{\text{TR}}}e^{\frac{-\text{TE}}{\text{T2}}}[1-e^{\frac{-\text{TR}}{\text{T1}}}(2e^{\frac{-\text{TE}}{2\text{T1}}}-1)]$$

Results and Discussion

Figure 1a shows how T1 and T2 change with time spent soaking in PBS after fixation. T1 is relatively stable between 1400-1600ms from 0-35 days post fixation, whereas T2 increases steadily from 30 to 50ms in 4% PFA-fixed samples, and 2% PFA-fixed samples from 40 to 50ms, plateauing after approximately 4 weeks. The gain in T2 resulting from the 2% PFA is also evident in Figure 1b, and was reproduced in two additional rats not shown here, prepared without gadolinium, scanned only at 35 days. Longer T2 is advantageous for diffusion MRI because the TE needs to be longer than normal structural imaging to allow adequate diffusion time and diffusion contrast without compromising signal due to T2 relaxation.

Figure 1b shows the effect of adding gadolinium contrast agent to the perfusion and rehydration solutions. T1 decreases more rapidly with respect to [Gd-DTPA] than T2, for rats prepared both with 2 and 4% PFA. Reducing T1 with gadolinium can improve signal intensity, spatial and/or angular resolution in a given acquisition time. However, gadolinium also reduces T2, to a lesser extent, so this must be taken into account when choosing the optimal concentration.

The effect of gadolinium on SNR efficiency is shown in Figure 2, demonstrating the optimal TR for each concentration of Gd-DTPA for a range of TE values. Higher concentrations of gadolinium greatly reduce optimal TR values, however after 15mM the SNR efficiency begins to drop for TE above 20ms. Given a minimum TE dictated by other factors such as matrix size or b-value. Figure 2 can be used to determine how much Gd-DTPA with which TR will give the greatest SNR efficiency. Table 1 summarizes the improvements in SNR of our proposed protocol using 2% PFA, an optimal concentration of gadolinium, and rehydration for 5 weeks, with an example TE of 26ms.


Rat brain tissue preparation can be tailored for MRI by altering fixative concentration, using an optimal concentration of gadolinium, and rehydrating the sample after fixation. An optimal tissue preparation results in improvements in SNR efficiency, allowing more possibilities to study tissue microstructure at high resolution with ex vivo MRI.


Thanks to funding from the Wellcome Trust, and assistance and support from the BRAIN Centre for Preclinical Imaging, and the members of the Natbrainlab.


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Figure 1. a) T1 and T2 relaxation times as a function of time rehydrating in PBS for rats perfused with 2% and 4% PFA, no contrast agent. b) T1 and T2 relaxation times decreasing as a function of [Gd-DTPA] in perfusate. All samples in b) were rehydrated with 1mM Gd-DTPA for 35 days. Curves show the inverse function of best fit. In both panels, data points show mean and standard deviation of T1 and T2 in a region of interest in the corpus callosum.

Figure 2. SNR efficiency curves as a function of repetition time (TR) are shown for different echo times (TE) represented by different colored curves. Each plot has a different concentration of Gd-DTPA: a) No Gd-DTPA used in either perfusion or rehydration; b-g) 0, 5, 10, 15, 25 and 50mM Gd-DTPA used in perfusion, and 1mM in rehydration. SNR efficiency factor was calculated using T1 and T2 values estimated from rats perfused with 2% PFA, and rehydrated for 35 days.

Table 1. Different preparations with corresponding T1, T2, optimal TR (ms), and SNR efficiency % gain relative to baseline. When gadolinium is used, concentration in perfusion is chosen to maximize SNR efficiency, and in rehydration is 1mM.

Proc. Intl. Soc. Mag. Reson. Med. 26 (2018)