Quantitative MRI is believed to be a promising technique to investigate the tissue microstructure of the brain. Particularly, it was proposed that myelin and iron could be quantified using $$$T_1$$$ and $$$T_2^*$$$ maps [1]. However, other tissue components may also influence the MR signal, including the ExtraCellular Matrix (ECM). Previously, it was shown that ECM impacts quantitative MRI both in synthetic samples [2] and connective tissues [3,4].

The ECM of brain tissues is composed primarily of hyaluronan polymer chains anchored to the membrane of the cells and provides a scaffold for proteoglycans and other specialized structures that have a strong negative charge and are believed to determine its physiological function [5,6].

Recently it was proposed that the digestion of the ECM may lead to measurable changes in $$$T_1$$$ and $$$T_2^*$$$ mapping [7]. To investigate the origin of this effect, we performed experiments where two human brain samples were imaged simultaneously: one was processed for ECM removal while the other one was used as reference.

The hyaluronan-based ECM of the CNS, and also the specialized neuronal ECM of PNs, can be effectively removed by enzymatic digestion, which is believed to disassemble the macromolecular ECM scaffold, as shown in Fig. 1. Two post-mortem sample, fixed with $$$4\%$$$ ParaFormAldehyde (PFA), of approx. $$$40×20×5\;\mathrm{mm^3}$$$ from the left and right thalamus region (Fig. 2) of a deceased male donor (TBC age: $$$82\;\mathrm{yr}$$$, COD: colon carcinona, with no neuropathological abnormalities) were measured before and after treatment.

The samples were stored in a $$$0.1\;\mathrm{M}$$$, pH $$$7.4$$$ PBS solution at $$$4\;\mathrm{°C}$$$. Digestion of one sample was performed with hyaluronidase from bovine testis (Sigma H3884), with up to $$$\approx{}4500\;\mathrm{units/ml}$$$, at $$$37\;\mathrm{°C}$$$ for $$$14\;\mathrm{days}$$$. The other sample was kept under the same environmental conditions, except for the addition of the enzyme. For each MRI scanning session, the samples were embedded in $$$0.6\%$$$ Agar gel (with addition of $$$0.4\%$$$ NaCl), after placement in a spherical container. Images were acquired using a MAGNETOM 7T (Siemens, Erlangen, Germany) and a circularly-polarized Tx / 32-channel Rx Nova coil. During each session, quantitative $$$T_1$$$ and $$$T_2^*$$$ 3D maps (0.33mm isotropic nominal resolution) were recorded using MP2RAGE and FLASH, respectively.

Each set of experiments lasted approx. $$$2\;\mathrm{days}$$$, and the time between pre- and post- sessions was approx. $$$4\;\mathrm{weeks}$$$. Results were assessed by visual inspection of the maps, the voxel-by-voxel correlation histograms and by calculating the average and the standard deviation of the difference images.

Additionally, to verify the effectiveness of the digestion, the two samples were sliced and stained for hyaluronan (with bHABP).

$$$T_1$$$ and $$$T_2^*$$$ maps for pre- and post-digestion experiments for both samples are shown alongside with their difference as well as voxel correlation histograms and histograms for repeated-scans reproducibility under identical conditions in Figs. 3 and 4, respectively. Visual inspection of both $$$T_1$$$ and $$$T_2^*$$$ maps show contrast changes across distinct regions. Additionally, the 2D histograms present significant deviation from the identity line after the treatment. Similar deviations were not observed in the test-retest correlation histograms of repeated scans. However, the results for the two samples (i.e. digested vs. reference) are similar. Note the the shape of the histograms of the two samples behaves differently in the two cases. The hyaluronan staining (Fig.5) indicates that the digestion was effective but the initial quality of the sample may be suboptimal. Results from previously reported experiments [7] have led to the hypothesis that significant contrast changes may be related to the ECM removal. The current experiments were designed to disentangle potential contributions related to enzymatic activity from other subtle processes related to the prolonged storage of the sample in PBS. While we were able to confirm previous results, i.e. a contrast change that may be related to the hyaluronan content, our data from the reference sample also indicate that a substantial percentage of such changes may be driven by concomitant processes rather than by the digestion itself. On the other hand, there seems to be a small, but appreciable (especially in the histograms), effect that is likely to be related to the ECM digestion. More information on the spatial distribution of the ECM is still required, in order to perform an additional analysis to reliably quantify the effects associated with the ECM removal. Further experiments are required for the quantification of the impact of the ECM in quantitative MRI and for investigating the underlying biophysical mechanisms. However, we were able to show that processes concomitant to the digestion treatment, along with the hyaluronan digestion itself, substantially contribute to observable contrast changes.