Could Diffusion MRI monitor the brain glymphatic system? A proof-of-concept study using an aquaporin-4 channel inhibitor pharmacological challenge
Clement S. Debacker1, Tomokazu Tsurugizawa 1, Boucif Djemai1, Luisa Ciobanu1, and Denis Le Bihan1

1NeuroSpin, CEA, Gif-sur-Yvette, France


Dysfunction of the Glymphatic System (GS), which clears brain tissue from waste, has been proposed as a mechanism to several brain pathologies, including the Alzheimer’s disease. GS has been investigated with preclinical imaging through the invasive intracisternal injection of Gadolinium. In this study, we have show, using a pharmacological mouse brain model, that diffusion MRI, through the Sindex approach could reveal noninvasively changes in brain cortex tissue following injection of an Aquaporin 4 channel inhibitor known to interfere with the GS via the inhibition of astrocyte swelling.


Proper neuronal function necessitates a highly regulated extracellular environment. To maintain this balance, exchanges between the brain tissue and the blood are tightly regulated. Defaults of this regulatory system may lead to the accumulation of toxic compounds. Alzheimer’s disease, for example, is thought to be associated with the accumulation of the β-amyloid (Aβ) peptide1. Other toxic compounds might enter abnormally and accumulate into the brain, such as gadolinium (Gd) used as MRI contrast agent2,3. Although the Blood Brain Barrier is thought to be the premier mechanism involved in controlling these exchanges another possible critical determinant of brain-blood exchanges has more recently been identified: the glymphatic system (GS) which corresponds to the active circulation of the cerebrospinal fluid (CSF) through the brain parenchyma to clean its interstitium1. CSF crosses the astrocyte end-feet through a mechanism dependent on Aquaporin-4 (AQP-4), a water channel membrane protein4. We hypothesized that dynamic volume change of astrocytes could be monitored noninvasively through water diffusion MRI which is exquisitely sensitive to changes in tissue microstructure, especially cell swelling. This hypothesis was tested on a mouse model using an AQP-4 inhibition pharmacological challenge.

MRI experiments

The MRI experiments were conducted on a Bruker 11.7T scanner with a cryo-cooled mouse brain coil. Eleven C57BL6 mice were allocated to two groups: 7 mice received an intra-peritoneal injection of 250mg/kg of TGN-020 (an AQP-4 channel inhibitor). The control group (n=4) received an intra-peritoneal injection of saline. Mice were anesthetized by 1.5% isoflurane with air. Diffusion-weighted EPI data sets were acquired with the following parameters: 150x150x250 μm3 resolution, 16 b-values ∈ [10 – 2250] s/mm2 along 6 directions, TE/TR = 36.3/2300 ms, diffusion time=24ms, total scan time = 3.75 min. 8 continuous DWI-EPI sets were first acquired (baseline) before TGN-020 or Saline was injected. Then, after 30 minutes (to let TGN-020 act5) 8 additional DWI-EPI data sets were acquired (Fig. 1). The body temperature was maintained at 37 °C. The respiration rate was monitored and stable (60-90 /min) throughout the experiment.

Data analysis

The 8 pre and post injection DWI-EPI data sets were first averaged for each animal to increase SNR. Data were analyzed using the non-Gaussian kurtosis model6 leading to ADCo (diffusion) and K (Kurtosis) and the Sindex method7 with Lb=250 and Hb=1750s/mm² as the “key b values” using a homemade software implemented with Matlab. The Sindex (SI) is a relative distance marker designed to identify tissue types or changes in tissue microstructure7. SI was calculated from the direction-averaged, normalized signals, SV(b) in each voxel, as the algebraic distance between the vector made of these signals and those of the signature tissue signals for gray matter, SG, and white matter, SW, each key b value:


with dSV,W,G(b)=[SV,W,G(b)-SN(b)]/SN(b). SN is taken as an intermediate signal between SW and SG. SI was then further linearly scaled as Sindex=(SI+1)*25+25 which is now centered at 50, so that Sindex=75 for a typical white matter tissue and Sindex=25 for a typical gray matter tissue. The Sindex represents a quantitative continuous scale. In this study, changes in Sindex values after TGN-020 and saline were calculated taking 3D Regions of Interest encompassing the whole brain cortex. The difference between post-injection and baseline values for each parameter, ΔP=Pafter-Pbefore, (P refers to the S-index, K or ADCo) was tested for statistical significance using a one sample t-test performed at group level (p < 0.05). We have also tested the statistical significance between saline and TGN-020 group with a two-sample t-test (p<0.05).

Results and Discussion

Fig.2 shows typical cortical absolute Sindex change maps following injection of TGN-020 and saline. TGN-020 resulted in decrease in Sindex not observed with saline. The Sindex decrease and ADCo increase were significant, while changes in K were not (Table 1). The higher sensitivity to small changes in tissue features of the Sindex, which requires no fitting and encompasses in a single parameter IVIM (incoherent flow which includes CSF), Gaussian and non-Gaussian diffusion effects, over K has already been reported7. Obviously more studies are required to confirm these findings and interpret the observed changes in Sindex and ADCo. One may speculate only at this stage that they might reflect a small increase in cortical water diffusion and, hence, a decrease in the amount of astrocytes swelling resulting from AQP-4 channel inhibition.


The gold standard method to investigate the GS in preclinical settings relies on the intracisternal injection of Gd in the cisterna magna8. Our results suggest that diffusion MRI, especially through the Sindex, could be alternative to noninvasively monitor the activity of the GS without the need for contrast agent.


This research was supported by a public grant overseen by the French National Research Agency (ANR) under the project name “MrGLY” (reference: ANR-17-CE37-0010).


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Figure 1: Experimental design

Figure 2: Sindex change in the cortex before and after injection of a saline (a) or TGN-020 (b) solution.

Table 1: Mean difference between post-injection and baseline values for K, ADCo and Sindex. Data are expressed as mean ± SEM.
*: p<0.05 for one sample t-test
✝: p<0.05 vs saline

Proc. Intl. Soc. Mag. Reson. Med. 27 (2019)