INTRODUCTION: For delivery of MRI reporter genes (e.g. ferritin) to the rodent brain, lentiviral (LV) and adeno-associated viral (AAV) vectors are the preferred viral vector systems when aiming for stable, long-term labelling and in vivo visualization of cells. However, their potential limitations for MRI like the generation of unspecific contrast by the vector injection and host response are often insufficiently addressed. Quantification of hypointense MR contrast using the parametric T2-maps is often constrained by their low resolution. We developed an image processing pipeline for normalization and quantification of high-resolution 3D T2*-weighted MR images. This pipeline allowed accurate and objective comparison of the potential of LV and AAV to be used as MRI reporter gene expressing vectors in the rodent brain with regard to their background contrast, both in immunodeficient and immunocompetent animals. METHODS: LV/AAV encoding MRI reporter (ferritin: LV/AAV-FerrH), different batches of control vectors (LV/AAV-eGFP) or PBS were stereotactically injected in the striatum of C57BL/6 and NOD-SCID mice (n = 5 per condition). 3D T2*-w MR images (FLASH, TE=15ms, TR=150ms, flip angle 30°, isotropic resolution 59 μm), T2 (MSME) and T2* maps were acquired at different time points (t=1, 7, 14, 30, 60, 90 days) post injection (p.i.) on a 9.4T small animal scanner (Bruker Biospin, Ettlingen, Germany). After the last time point, animals were sacrificed, followed by ex vivo MRI of the excised brain (3D T2*w, isotropic resolution 32 μm) and (immuno-) histochemical stainings. Our image analysis pipeline tackles the spatial normalization of these 3D T2*-w MR time series images, using our maximization of mutual information (MMI) algorithm. Its 12-parameter affine transformation addresses the image alignment to intra/inter animal, to anatomical template, as well as for ex vivo to in vivo registration, accounting for brain shrinkage. Also, we have implemented the information minimization method, for MR intensity inhomogeneity correction approach, without which image registration and quantification derails. Finally, the inter scan intensity variation in time-series MR images were eliminated by correcting the intensity profile of the reference region. The bias corrected and normalized (spatial & intensity) 3D T2*w images were then automatically quantified for the hypointense vector contrast by thresholding the volume-of-interest’s defined in stereotactic coordinates. RESULTS: Injection in rodent brain of LV without MRI reporter (2 different batches of LV-eGFP) resulted in persisting hypointense contrast at the injection site on T2*-weighted MRI. This contrast coincided with the presence of Fe and microglia/ macrophages and could not be explained by bleeding or damage inherent to the injection (as evaluated from a PBS injection). In immunodeficient mice, the background contrast of LV was significantly reduced. For AAV, the background contrast was reduced by at least 50% as compared to LV. AAV-mediated ferritin overexpression resulted in more pronounced hypointense contrast as compared to AAV-control vector injection in immunocompetent mice (fig. a-c). This contrast coincides with the presence of Fe(III), without obvious microglia/macrophage infiltration (fig. e-f). CONCLUSIONS: LV vector injection itself results in hypointense contrast at the site of injection on T2*w MRI, which is at least partially explained by an immune response. In contrast to LV, AAV vector injection results in very low background contrast levels and AAV-mediated MRI reporter gene overexpression results in significant contrast-to-background on MRI. Our image analysis pipeline permits the comparison of 3D T2*w MR signal intensities over time points or specimen across different experimental groups.