Hana Lahrech

Object Exploring mouse brains by rapid 3D-Diffusion Tensor Imaging (3D-DTI) of high spatial resolution (HSR) is challenging in vivo. Here we use the super resolution reconstruction (SRR) postprocessing method to demonstrate its performance on Microtubule-Associated-Protein6 Knock-Out (MAP6-KO) mice. Materials and methods Two spin-echo DTI were acquired (9.4T, CryoProbe RF-coil): (i)-multislice 2D-DTI, (echo-planar integrating reversed-gradient) acquired in vivo in the three orthogonal orientations (360 μm slice-thickness, 120 × 120 μm in-plane resolution, 56 min scan duration); used in SRR software to reconstruct SRR 3D-DTI with HSR in slice-plane (120 × 120 × 120 µm) and (ii)-microscopic 3D-DTI (µ-3D-DTI), (100 × 100 × 100 µm; 8 h 6 min) on fixed-brains ex vivo, that were removed after paramagnetic contrast-agent injection to accelerate scan acquisition using short repetition-times without NMR-signal sensitivity loss. Results White-matter defects, quantified from both 3D-DTI fiber-tracking were found very similar. Indeed, as expected the fornix and cerebral-peduncle volume losses were − 39% and − 35% in vivo (SRR 3D-DTI) versus − 34% and − 32% ex vivo (µ-3D-DTI), respectively (p<0.001). This finding is robust since the µ-3D-DTI feasibility on MAP6-KO ex vivo was already validated by fluorescent-microscopy of cleared brains. Discussion First performance of the SRR to generate rapid HSR 3D-DTI of mouse brains in vivo is demonstrated. The method is suitable in neurosciences for longitudinal studies to identify molecular and genetic abnormalities in mouse models that are of growing developments.

This work shows that the longitudinal relaxation differences observed at very low magnetic fields between invasion/migration and proliferation processes on glioma mouse models in vivo are related to differences in the transmembrane water exchange basically linked to the aquaporin expression changes. Three glioma mouse models were used: Glio6 and Glio96 as invasion/migration models and U87 as cell proliferation model. In vivo proton longitudinal relaxation-rate constants (R₁) at very low fields were measured by fast field cycling NMR (FFC-NMR). The tumor contribution to the observed proton relaxation rate, R₁^tum (U87: 12.26 ± 0.64 s⁻¹; Glio6: 3.76 ± 0.88 s⁻¹; Glio96: 6.90 ± 0.64 s⁻¹ at 0.01 MHz), and the intracellular water lifetime, τ_in (U87: 826 ± 19 ms; Glio6: 516 ± 8 ms; Glio96: 596 ± 15 ms), were found to be good diagnostic hallmarks to distinguish invasion/migration from proliferation (p < 0.01 and 0.001). Overexpression of AQP4 and AQP1 were assessed in invasion/migration models, highlighting the pathophysiological role of these two aquaporins in water exchange that, in turn, determine the lower values in the observed R₁ relaxation rate constant in glioma invasion/migration. Overall, our findings demonstrate that τ_in and R₁ (measured at very low fields) are relevant biomarkers, discriminating invasion/migration from proliferation in vivo. These results highlight the use of FFC-NMR and FFC-imaging to assess the efficiency of drugs that could modulate aquaporin functions.

Our objective was to study NMR relaxometry of glioma invasion/migration at very low field (T_2w‐MRI, HE histology, Ki‐67 immunohistochemistry (IHC) and CXCR4 RT‐qPCR, and were compared with the U87 model. R₁ dispersions of glioma tissues were acquired at low field (0.1 mT‐0.8 T) ex vivo and were fitted with Lorentzian and power‐law models to extract FFC biomarkers related to the molecular dynamics of water. In order to decipher relaxation changes, three main invasion/migration pathophysiological processes were studied: hypoxia, H₂O₂ function and the water‐channel aquaporin‐4 (AQP4). Glio6 and Glio96 were characterized with invasion/migration phenotype and U87 with high cell proliferation as a solid glioma. At very low field, invasion/migration versus proliferation was characterized by a decrease in the relaxation‐rate constant (ΔR₁ ≈ −32% at 0.1 mT) and correlation time (≈−40%). These decreases corroborated the AQP4‐IHC overexpression (Glio6/Glio96: +92%/+46%), suggesting rapid transcytolemmal water exchange, which was confirmed by the intracellular water‐lifetime τ_IN decrease (Δτ_IN ≈ −30%). In functional experiments, AQP4 expression, τ_IN and the relaxation‐rate constant at very low field were all found to be sensitive to hypoxia and to H₂O₂ stimuli. At very low field the role of water exchanges in relaxation modulation was confirmed, and for the first time it was linked to the glioma invasion/migration and to its main pathophysiological processes: hypoxia, H₂O₂ redox signaling and AQP4 expression. The method appears appropriate to evaluate the effect of drugs that can target these pathophysiological mechanisms. Finally, FFC‐NMR operating at low field is demonstrated to be sensitive to invasion glioma phenotype and can be straightforwardly extended to FFC‐MRI as a new cancer invasion imaging method in the clinic.

In glioma, the acidification of the extracellular tumor microenvironment drives proliferation, angiogenesis, immunosuppression, invasion and chemoresistance. Therefore, quantification of glioma extracellular pH (pHe) is of crucial importance. This study is focused on the application of the YbHPDO3A (ytterbium 1,4,7‐triscarboxymethyl‐1,4,7,10‐tetraazacyclododecane) probe for in vivo glioma pHe quantification using chemical exchange saturation transfer (CEST)‐MRI and its correlation with tumor metabolism assessed by immunohistochemistry.The U87 glioma mouse model was used (n = 18) and MRI performed at 4.7 T. CEST‐MRI of YbHPDO3A solutions at different pH values showed two resolved CEST spectra at 71 ppm and 99 ppm, both sensitive to pH variations, allowing therefore calculation of the ratiometric curve for in vivo pH quantification. In vivo MRI sequences consisted of T_2w for tumor localization, T_2w* to assess YbHPDO3A biodistribution by exploiting its magnetic susceptibility effect and CEST for glioma pHe mapping. T_2w* images show that YbHPDO3A extravasates in tumor in regions with damaged blood–brain barrier. The pHe is calculated only in these regions.Hematoxylin/eosin histology and Ki‐67, CA‐IX (carbonic anhydrase 9) and NHE‐1 immunohistochemical staining were performed; their expression rates were compared with the in vivo pHe values.On the basis of the cell proliferation marker Ki‐67, two groups were defined: one group with a lower mitotic index (MI% < 20% = mean value) and a mean pHe value of 7.00 (low‐proliferation/high‐pH group) and the other with MI% > 20% and an acidic pHe of 6.6 (high‐proliferation/low‐pH group). CA‐IX and NHE‐1 were over‐expressed in the high‐proliferation/low‐pH group (CA‐IX, 92 ± 7% versus 30 ± 13%; NHE‐1, 84 ± 8% versus 35 ± 11%), indicating an acidic/hypoxic microenvironment. These immunohistochemical results are consistent with our pHe mapping (Pearson correlation coefficient > 0.70) and provide evidence for the feasibility of the CEST‐MRI method with the YbHPDO3A probe for glioma pHe quantification at 4.7 T. Importantly, the YbHPDO3A probe has similar chemical and biological properties to the clinically approved MRI contrast agent GdHPDO3A. This makes the method promising for a clinical translation.

In the central nervous system, microtubule-associated protein 6 (MAP6) is expressed at high levels and is crucial for cognitive abilities. The large spectrum of social and cognitive impairments observed in MAP6-KO mice are reminiscent of the symptoms observed in psychiatric diseases, such as schizophrenia, and respond positively to long-term treatment with antipsychotics. MAP6-KO mice have therefore been proposed to be a useful animal model for these diseases. Here, we explored the brain anatomy in MAP6-KO mice using high spatial resolution 3D MRI, including a volumetric T_1w method to image brain structures, and Diffusion Tensor Imaging (DTI) for white matter fiber tractography. 3D DTI imaging of neuronal tracts was validated by comparing results to optical images of cleared brains. Changes to brain architecture included reduced volume of the cerebellum and the thalamus and altered size, integrity and spatial orientation of some neuronal tracks such as the anterior commissure, the mammillary tract, the corpus callosum, the corticospinal tract, the fasciculus retroflexus and the fornix. Our results provide information on the neuroanatomical defects behind the neurological phenotype displayed in the MAP6-KO mice model and especially highlight a severe damage of the corticospinal tract with defasciculation at the location of the pontine nuclei.

Cellular MRI, which visualizes magnetically labelled cells (cells*), is an active research field for in vivo cell therapy and tracking. The simultaneous relaxation rate measurements (R₂*, R₂, R₁) are the basis of a quantitative cellular MRI method proposed here. U937 cells were labelled with Molday ION Rhodamine B, a bi‐functional superparamagnetic and fluorescent nanoparticle (U937*). U937* viability and proliferation were not affected in vitro. In vitro relaxometry was performed in a cell concentration range of [2.5 × 10⁴–10⁸] cells/mL. These measurements show the existence of complementary cell concentration intervals where these rates vary linearly. The juxtaposition of these intervals delineates a wide cell concentration range over which one of the relaxation rates in a voxel of an in vivo image can be converted into an absolute cell concentration. The linear regime was found at high concentrations for R₁ in the range of [10⁶– 2 × 10⁸] cells/mL, at intermediate concentrations for R₂ in [2.5 × 10⁵– 5 × 10⁷] cells/mL and at low concentrations for R₂* in [8 × 10⁴– 5 × 10⁶] cells/mL. In vivo relaxometry was performed in a longitudinal study, with labelled U937 cells injected into a U87 glioma mouse model. Using in vitro data, maps of in vivo U937* concentrations were obtained by converting one of the in vivo relaxation rates to cell concentration maps. MRI results were compared with the corresponding optical images of the same brains, showing the usefulness of our method to accurately follow therapeutic cell biodistribution in a longitudinal study. Results also demonstrate that the method quantifies a large range of magnetically labelled cells*. Copyright © 2016 John Wiley & Sons, Ltd.

Monitoring glioma cell infiltration in the brain is critical for diagnosis and therapy. Using a new glioma Glio6 mouse model derived from human stem cells we show how diffusion tensor imaging (DTI) may predict glioma cell migration/invasion.In vivo multiparametric MRI was performed at one, two and three months of Glio6 glioma growth (Glio6 (n = 6), sham (n = 3)). This longitudinal study reveals the existence of a time window to study glioma cell/migration/invasion selectively. Indeed, at two months only Glio6 cell invasion was detected, while tumor mass formation, edema, blood–brain barrier leakage and tumor angiogenesis were detected later, at three months.To robustly confirm the potential of DTI for detecting glioma cell migration/invasion, a microscopic 3D‐DTI (80 μm isotropic spatial resolution) technique was developed and applied to fixed mouse brains (Glio6 (n = 6), sham (n = 3)).DTI changes were predominant in the corpus callosum (CC), a known path of cell migration. Fractional anisotropy (FA) and perpendicular diffusivity (D_⊥) changes derived from ex vivo microscopic 3D‐DTI were significant at two months of tumor growth. In the caudate putamen an FA increase of +38% (p < 0.001) was observed, while in the CC a − 28% decrease in FA (p < 0.005) and a + 95% increase in D_⊥ (p < 0.005) were observed. In the CC, DTI changes and fluorescent Glio6 cell density obtained by two‐photon microscopy in the same brains were correlated (p < 0.001, r = 0.69), validating FA and D_⊥ as early quantitative biomarkers to detect glioma cell migration/invasion. The origin of DTI changes was assessed by electron microscopy of the same tract, showing axon bundle disorganization.During the first two months, Glio6 cells display a migratory phenotype without being associated with the constitution of a brain tumor mass. This offers a unique opportunity to apply microscopic 3D‐DTI and to validate DTI parameters FA and D_⊥ as biomarkers for glioma cell invasion.