SUPPLEMENTARY FIGURE LEGENDS
Figure S1: schematic representation of the image analysis pipeline for quantification of multitemporal MRI image analysis. The different steps of the MRI image analysis pipeline (panel 1) for
quantifying the 3D T2*-weighted MRI data are illustrated with intermediate results (panel 2) from a 1 day
p.i. representative study image comparing LV-eGFP in the right striatum of C57BL/6 mice to a PBS
sham injection in the contralateral hemisphere. Following MRI image acquisition, the pre-processing
module (panel 1, panel 2 a - c) corrects for RF bias field inhomogeneity and extracts a brain mask
excluding the non-brain tissues, where (a) raw MRI image, (b) bias field removed from the raw MRI
image, (c) bias corrected, brain mask extracted MRI image are shown. The spatial alignment of the preprocessed images from different time points to a MRI reference template in the Paxinos coordinate
system facilitates atlas based segmentation of our volume-of-interest (VOI). The image registration
module (d-f), spatially aligns the study image (e) to the Paxinos atlas reference frame (d) and the result
of image registration is shown in (f), where the study image is overlaid by the MRI template (panel 2, g i). In (g) the quantification module helps to define the VOI's (e.g. PBS (red), eGFP (green) based on
stereotactic surgery), and the reference region (PAG - blue based on MRI template). While the intensity
normalization step first corrects for inter-scan intensity variation, the final quantification step then
segments the hypointense MRI vector contrast (h – k, see arrows) and the subsequent contrast volume
plots (j) are obtained. Each module in panel 1 represents at least one step in the processing, written in
Matlab (The Mathworks , Natick, MA, USA).
Figure S2: Evaluation of the suitability of T2 and T2* values for the assessment of vector
expression. T2 and T2* values were determined from a slice positioned around the injection tract of the
vector (based on the 3D T2*-weighted MR images) using C57BL/6 mice injected with PBS in the left and
LV-eGFP in the right striatum (n=5) and NOD-SCID mice injected with LV-eGFP-T2A-fLuc (left striatum)
and LV-FerrH-T2A-fLuc (right striatum) (n=3). Regions of interest were determined for the needle tract as
defined by the hypointense contrast in the 3D FLASH MRI (region of interest (ROI) 1) and a surrounding
area (excluding the needle tract) corresponding to the GFP expression in histochemically stained tissue
sections (ROI 2, panel c). For the hemispheres with PBS injections, ROI 2 was defined based on a
mirror image of ROI 2 from the contralateral hemisphere (vector injection). Panel a shows a slide from
the MSME experiment (TE=10ms) with the two ROIs indicated. Panel b shows the respective T 2 map.
Panel c shows three tissue sections that were immunohistochemically stained for eGFP, illustrating the
area of transgene expression. The images were obtained from a C57BL/6 mice injected with PBS (left
striatum) and LV-eGFP (right striatum). T2 and T2* values are graphically represented for three different
time points after stereotactic injections and for the fixated (ex vivo) brain (graphs, panel d). T2/ T2*
values for ROI 1 showed a large standard deviation and values close to the shortest TE, indicating
unreliability due to fitting problems and partial volume effects (contributions from surounding tissue). T 2/
T2* values for ROI 2 did not show significant differences (P>0.05) between the three conditions, although
T2* values were shortest for the area surrounding the LV-FerrH-T2A-fLuc injection site from time point 1
month p.i.
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Figure S3: time course of hypointense MRI contrast. Representative 3D T2*-weighted in vivo MR
images before (top panel) and after pipeline processing (middle panel), acquired at baseline (a) and at 1
day (b), 1 week (c) , 1 month (d) and 3 months (e) p.i. from a C57BL6 mouse injected with LV-eGFP in
the right striatum and a sham injection with PBS in the left striatum. After the last time point, the mouse
was sacrificed and ex vivo MR images acquired (f). The pipeline segmented hypointense contrast after
LV-eGFP injection is shown for the same data in the bottom panel.
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