Characterization of tumor angiogenesis in rat brain using iron-based vessel size index MRI in combination with gadolinium-based dynamic contrast-enhanced MRI

Abstract

This study aimed at combining an iron-based, steady-state, vessel size index magnetic resonance imaging (VSI MRI) approach, and a gadolinium (Gd)-based, dynamic contrast-enhanced MRI approach (DCE MRI) to characterize tumoral microvasculature. Rats bearing an orthotopic glioma (C6, n=14 and RG2, n=6) underwent DCE MRI and combined VSI and DCE MRI 4 h later, at 2.35 T. Gd-DOTA (200 mumol of Gd per kg) and ultrasmall superparamagnetic iron oxide (USPIO) (200 micromol of iron per kg) were used for DCE and VSI MRI, respectively. C6 and RG2 gliomas were equally permeable to Gd-DOTA but presented different blood volume fractions and VSI, in good agreement with histologic data. The presence of USPIO yielded reduced K(trans) values. The K(trans) values obtained with Gd-DOTA in the absence and in the presence of USPIO were well correlated for the C6 glioma but not for the RG2 glioma. It was also observed that, within the time frame of DCE MRI, USPIO remained intravascular in the C6 glioma whereas it extravasated in the RG2 glioma. In conclusion, VSI and DCE MRI can be combined provided that USPIO does not extravasate with the time frame of the DCE MRI experiment. The mechanisms at the origin of USPIO extravasation remain to be elucidated.

Figure 1. Protocol combining a BVf/VSI experiment with a DCE MRI experiment and images obtained at each stage of the protocol

The chronogram of MRI protocol for Experiment A is presented at the top of the figure. The names of the sequences, their respective TR, TE and durations, and the CA injections are indicated. Note that the MRI protocol of Session 1 is the same as the MRI protocol of Session 2 except that the two MGESE sequences and the injection of USPIO are missing. (A–T) examples of images obtained on one animal per tumor model using the MRI protocol of Experiment A, Sessions 1 (A–D for the C6 model and K–N for the RG2 model) and 2 (E–J for the C6 model and O–T for the RG2 model). Images acquired after CA injections are annotated with names of the CA injected and times post-injection in minutes.

Figure 2. Temporal evolution of [Gd]

Examples of temporal evolutions of [Gd] encountered during the DCE MRI analysis (A and C) in the USPIO− condition and (B and D) in the USPIO+ condition: tumor and muscle from (A–B) one animal bearing a C6 tumor and (C–D) one animal bearing a RG2 tumor. For each set of data points, the result of the fitting procedure is plotted (solid lines). Plots correspond to the time courses of a single pixel issued from the ROI.

Figure 3. BVf, VSI and DCE MRI pharmacokinetic parameter estimates in the C6 and RG2 models

Parameters obtained in Experiment A (mean±SEM) for each tumor model (C6 in black and RG2 in gray) and different tissue ROIs. BVf and VSI were obtained during Session 2. v_p, v_e, K^trans and k_ep are the results of Session 1 (USPIO−).

Figure 4. DCE MRI pharmacokinetic parameter estimates obtained in presence or in absence of USPIO

The pharmacokinetic parameters obtained in Session 1 (USPIO−, black) and in Session 2 (USPIO+, gray) are represented as mean±SEM. Data obtained in the muscle have been pooled across tumor models (n=20) while data from the C6 tumor and the RG2 tumor were kept separated (n=14 and n=6 respectively).

Figure 5. Does the USPIO extravasate in the tumor?

Temporal evolutions of the longitudinal relaxation rate after USPIO injection in Experiment B. Each graph corresponds to one ROI, and, in each graph, each time course was obtained from a single animal (four time courses per graph: 2 C6 (open symbols) and 2 RG2 (filled symbols)). Dashed lines and double arrows indicate the time at which MR acquisitions and Gd injection were performed during Experiment A, Session 2, prior and after USPIO injection (time= 0 min was defined as the beginning of the USPIO injection).

Figure 6. Histological images

Examples of histological images obtained for the C6 model (two left columns) and the RG2 model (two right columns). For each model, the tumor centre (right) and the contralateral region are represented (left). (A–D) Collagen IV staining, (E–H) BBB staining, (I–L) macrophage staining and (M–P) nucleus staining. Collagen IV and BBB stainings were performed on the same slice as well as macrophage and nucleus stainings.