MAGMA (2004) 17: 133–139
DOI 10.1007/s10334-004-0049-5 RESEARCH ARTICLE
A. Vonarbourg A. Sapin L. Lemaire F. Franconi P. Menei P. Jallet J. J. Le Jeune
Characterization and detection of experimental rat gliomas using magnetic resonance imaging
Received: 20 May 2004 Accepted: 2 July 2004
Published online: 20 October 2004
© ESMRMB 2004
A. Vonarbourg·A. Sapin·L. Lemaire (✉) P. Jallet·J. J. Le Jeune
Inserm U646, “Ing´enierie de la Vectorisation Particulaire”
bˆatiment IBT, 10, rue Andr´e Boquel 49100 Angers, France
E-mail: laurent.lemaire@univ-angers.fr Tel.: +33-241-735006
Fax: +33-241-735007 F. Franconi
Service Commun d’analyses spectroscopiques
Universit´e d’Angers Angers, France P. Menei
Neurochirurgie, C.H.U. d’Angers 4, rue Larrey
49100 Angers, France
Abstract Two different experimental rat brain tumours (F98 glioma and 9L glioma) were characterized using T1 and T2, apparent diffusion coefficient (ADC) and magnetization transfer ratio (MTR). Even though both tumours appeared homogenous at the early stage of growth,
significant differences were measured for all parametric images between tumours and normal brain tissue.
Irrespective of the sequence used, tumour lesion/normal parenchyma contrast for the non-infiltrative 9L was twice that of the infiltrative F98 glioma. The use of spin preparation via an inversion pulse in a fast spin echo sequence increases contrast by a factor of 20–30.
Keywords Apparent diffusion coefficient·Magnetization transfer·T1·T2·Rat brain tumours
Introduction
In 1971, Damadian [1] demonstrated the ability of proton relaxation times to differentiate normal and tumour tis- sues but these are usually not sufficient to discriminate and delineate brain tumour tissue in vivo, particularly when surrounding edema are not present. However, the presence of edema equally jeopardizes the delineation of tumour by appearing hyperintense on T2-weighted images as do tumour [2–5]. Contrast agents such as gadolinium chelates are often used to track tumour tissues but their extravas- cular distribution equally leads to an overestimation of the tumour’s size [6–8].
Diffusion-weighted imaging (DWI) is a sensitive technique producing quantitative and non-invasive images of water motion, exchange and compartmentali- zation for characterizing tumour growth [3, 4] or response to therapy [9–12]. However, this imaging sequence can be time consuming and specific technical requirements are needed especially in terms of motion control that can jeopardize the quality of the image particularly when sub-millimetric resolution is addressed as in small animal imaging.
Magnetization transfer (MT) imaging is a unique quantitative method of magnetization resonance (MR) characterization. It is based on the coupling between
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Fig. 1 T2-weighted (TE = 60 ms) follow-up (14, 19 and 23 days after inoculation) of F98 glioma and the corresponding histology section (hematein staining) at the endpoint. The tumour appeared hyperin- tense and is pointed by anarrowon the day 14 frame. After day 19, tumour was heterogeneous as confirmed by histology
the macromolecular protons and the mobile or ‘liquid’
protons and the possibility to saturate the macromolecu- lar spins. This saturation can be transferred to the liquid spins, depending on the rate of exchange between the two spin populations, and hence can induce a decrease of the signal of the latter, thus creating a contrast. The magneti- zation transfer ratio (MTR) calculated from images per- formed with and without saturation pulses allowed very clear distinction of the tumour region characterized by a lower MTR than healthy tissues [4, 13, 14]. However, as the offset frequency and the amplitude of the radio frequency (RF) irradiating pulse highly influence the MTR, direct comparison from one study to another is difficult [14].
The aim of this study is to characterize in terms of apparent diffusion coefficient (ADC), MTR, relaxation time T1 and T2, an infiltrative (F98) and a non-infiltrative (9L) glioma at 7 T in order to define the suitable sequence for an early tumour lesion detection.
Materials and methods
Animal model
Animal care was administrated in strict accordance to French Ministry of Agriculture regulation.
Fig. 2a–d Visualization of F98 glioma 23 days after inoculation, on T2-weighted (a), T1 map (b), MT map (c) and ADC map (d). Tumour appeared heterogeneous because of edema and necrotic cores
Brain tumours were induced in 12-week-old female Fischer rats (F98 = 18, and 9L = 15) (Elevage IFFA CREDO, l’Arbre- sle, France) by stereotaxic inoculation of exponentially growing F98 or 9L-glioma cells. Rats were anesthetized with a mixture of Rompun (Xylasine, Bayer AG, Leverkusen, Germamy) and Clorketam (K´etamine, V´etoquinol, Lure, France) before being fixed in a stereotaxic holder. Through a small hole drilled in the skull (anterior−1 mm, lateral 2-mm, depth−6 mm according the bregma), 10µl of a suspension of 1,000 F98 or 9L-glioma cells were injected over a 10-min time period into the caudate putamen of the right hemisphere. Beginning at day 4, animals were scanned twice a week under Isoflurane anesthesia (1.5–3%, O23 l/min).
Histological analysis was performed on the rat brains at the end of the MR studies. Hematein staining was performed for tumour localization and description.
Imaging
Experiments were performed with a Bruker Avance DRX 300 equipped with a vertical superwide-bore magnet and shielded gradient insert. The resonant circuit of the nuclear magnetic resonance (NMR) probe was a 64-mm diameter birdcage.
Quantitative T1 and T2 MR images were obtained using spin echo sequences. For T1 estimation, three acquisitions were performed using TR = 500, 1,000 or 2,500 ms and TE = 15 ms.
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Fig. 3a–d Assessment of T2 (a), T1 (b), ADC (c) and MTR (d) evolution for F98 glioma (open square), 9L glioma (filled square) and contralateral hemisphere (filled circle) as function of time. Data are presented as mean±SEM
For T2 measurements, TR was fixed to 2,500 ms whereas eight TE ranging from 15 to 120 ms were explored. In both cases, the geometrical parameters were FOV = 3×3 cm; matrix 128×128;
nine contiguous slices of 1 mm and two acquisitions.
Rapid qualitative T2-weighted images were also obtained using a rapid acquisition with relaxation enhancement (RARE) sequence (TR = 2000 ms; mean echo time (TEm) = 31.7 ms;
RARE factor = 8; FOV = 3×3 cm; matrix 128×128; nine con- tiguous slices of 1 mm, eight acquisitions). T1 weighting was introduced in these images by using an inversion pulse prior to the RARE pattern. The duration of the inversion delay was fixed at 600 ms, enough time to allow the annulation of the normal parenchyma.
Magnetization transfer and ADC images were performed on three contiguous slices of respectively 1 and 1.5 mm thick, located at the tumour center. In order to reach an accept- able signal-to-noise ratio without dramatically increasing the acquisition time, a 96×96 matrix for FOV = 3×3 cm was used, leading to an in-plane resolution of 312µm. MT images were acquired using a spin echo sequence (TR/TE = 2500/8.7 ms, with or without saturation (ten gaussian pulses of 5 ms, interpulse delay = 300µs, offset 1.5 kHz)). Under these conditions, the spe- cific absorption rate (SAR) was under 1 W/kg.
Diffusion images were obtained using a Stejskal-Tanner- type [15] pulsed gradient stimulated echo sequence with three diffusion weighting factor values (b): [b= (γ Gδ)2(−δ/3)], with γ= gyromagnetic ratio, G= gradient, δ= gradient dura- tion,= duration between the leading edges andb= 128, 602, 1,351 s/mm2. The diffusion sensitizing gradient was along the direction of the slice selection gradient.δ= 5 ms and= 100 ms were used. The TR, TE and mixing time (TM) were fixed to TR/TE/TM = 1500/22.7/88.1 ms and two acquisitions were per- formed.
Using a T2-weighted sequence as background display, two regions of interest (ROI) were defined: one over the entire tumour lesion and one over the contralateral caudate putamen.
Data analysis and statistics
Results are expressed as mean±SEM (standard error of the mean).
Magnetization transfer ratio were calculated from the sig- nal intensity of each pixel on the MT-on image and the MT-off image and were defined as 100×[1−(MTon/MToff)]. ADC, T1 and T2 were obtained by a monoexponential fitting of the exper- imental points using the Bruker Paravision 2.0 software.
To compare the efficiency of these parameters for the detec- tion of brain tumour lesion with normal tissue, contrast (C) was defined as:C= 100×[(t−c)/c] wheretrepresents the tumour lesion MR parameter value, andcthe contralateral MR param- eter value.
Statistical analysis was performed using a bi-factorial anal- ysis of variance (ANOVA) with a multiple-least-square analysis.
Results
In the first thirteen days after F98 cell inoculation, brain tissue appeared homogenous and no signs of bleeding or scarring of tissue were observed. At this stage, tumour lesion appeared slightly hyperintense on T2-weighted images, with a mean volume of 8±4µl. F98 glioma growth was rapid and a mean volume of 300±70µl was reached by day 27 (Fig. 1). At this late stage, tumour lesion was diffuse and heterogeneous with edemas and/or necrotic cores as confirmed by histology. Paramet- ric follow-up of tumour lesion and contralateral tissue was performed (Fig. 2). As no significant changes were observed for all parameters during the entire experimental time, averaged values of contralateral T2 (56.3±0.7 ms) (p> 0.9), MTR (29.6±0.5%) (p> 0.6), T1 (728±29 ms)
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Table 1 T1, T2, ADC and MTR values measured in 9L and F98 tumor bearing rats. (means±SEM)
MR parameters 9L gliomas F98 gliomas
Controlateral Tumor lesion Controlateral Tumor lesion
T1 (ms) 800±71 1450±71 728±29 1031±29
T2 (ms) 57.8±1.6 90.7±1.6 56.3±0.7 65.8±1.2 (d14)
75.7±1.7 (d23)
ADC (103mm2/s) 0.59±0.02 0.85±0.02 0.57±0.03 0.79±0.03
MTR (%) 30.6±0.8 18.0±0.8 29.6±0.5 22.9±0.5
(p> 0.2) and ADC (0.57±0.03×103mm2/s) (p> 0.05) were calculated. MTR (22.9±0.5%) (p> 0.05), ADC (0.79±0.03×103mm2/s) (p> 0.1) and T1 (1,031±29 ms) (p> 0.8) were also unaffected in the tumour lesion whereas T2 increased significantly (p< 0.05) from 65.8±1.2 ms on day 14 to T2 = 75.7±1.7 ms on day 23 (Table 1 and Fig. 3).
Irrespective of the time considered, tumour lesion parameters were significantly increased compared to the contralateral hemisphere.
9L glioma was also observed on T2-weighted images from day 14 onward (Fig. 4). Before that day, brain tissue appeared homogenous and no signs of bleeding or scarring
Fig. 4 T2-weighted (TE = 60 ms) follow-up (14, 21 and 25 days after inoculation) of 9L glioma and the corresponding histology section (hematein staining) at the endpoint where tumour appeared hetero- geneous. The tumour appeared hyperintense and is indicated by an arrowon the day 14 frame
of tissue were noticed. At this stage, tumour lesion volume equalled 17±10µl. 9L glioma grew rapidly to reach a mean tumour lesion volume of 167±19µl on day 24.
Parametric images (Fig. 5) did not show any significant changes in the contralateral hemisphere or tumour lesion with time. The averaged values were calculated for T2 at 57.8±1.6 ms (p> 0.7), MTR = 30.6±0.8% (p> 0.1), T1 = 800±71 ms (p> 0.9) and ADC = 0.59±0.02×103mm2/s (p> 0.4) in the con- tralateral hemisphere and T2 = 90.7±1.6 ms (p> 0.4), MTR = 18.0±0.8% (p> 0.4), T1 = 1450±71 ms (p> 0.55) and ADC = 0.85±0.02×103mm2/s (p> 0.22) in the tu- mour lesion (Table 1 and Fig. 3).
Fig. 5a–d Visualization of 9L glioma 25 days after inoculation, on relaxation time T2-weighted (a), T1 map (b), MT map (c) and ADC map (d). Tumour appeared heterogeneous because of edema and necrotic cores
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Fig. 6a–c Visualization of 9L glioma with rapid acquisition with RARE sequence (a), RARE enhancement inversion-recovery sequence (b) and hematein staining histology (c)
Figure 6 shows the benefit of spin preparation using an inversion pulse prior to rapid T2-weighted sequence acquisition. Additionally, according to parenchyma and tumour lesion T1, an inversion delay of 600 ms led to the annulling of the parenchyma, which improved the tumour lesion/parenchyma contrast (Fig. 7).
Discussion
In this study, we aimed to characterize the evolution of MTR, ADC, longitudinal and transverse relaxation times in growing F98 and 9L tumours at 7 T and therefore defined the suitable sequence for an early detection of the lesion.
Measurement of MTR, ADC, T1 and T2 relaxa- tion times in the contralateral hemisphere of the rat brain
Fig. 7a–c Evolution of contrast between tumours lesions (F98 glioma (a), 9L glioma (b and c)) and contralateral hemisphere as a function of time and sequence (T2 (open diamond), T1 (filled square), ADC (open triangle), MTR (filled circle) and T2 + inversion delay (-)). Plot C corresponds to an enlargement of plot B
showed that none of these parameters were signifi- cantly modified after F98 or 9L-glioma cell inoculation regardless of the time frame between cell injection and MRI analysis (Fig. 3). Such a phenomenon has been previously reported for T2 after 9L-glioma [16] or C6- glioma cell inoculation [4] and for MTC and ADC after RG2 [13] or C6-glioma cell inoculation [4].
At the early stage of tumour growth, both tumour lesions appeared homogenous (Figs. 1 and 4). Significant differences can be measured for all parameters between tumour lesions and normal brain tissue (Fig. 3 and Table 1). Indeed, T1, T2, ADC and MTR were respectively equal to 1,500±100 ms, 86.3±3.4 ms, 0.80±0.03×103mm2/s and 19.4±1.7% for 9L tumour lesion; 1,068±54 ms, 65.8±1.2 ms, 0.72±0.05× 103mm2/s and 23.6±1.0% for F98 tumour lesion and 800±100, 56.9±0.3 ms, 0.57±0.03×103mm2/s and 30.0±1.3% for normal parenchyma (average of contralateral brain form 9L and F98 rats). As shown in Fig. 1 and 4, as tumour cells invaded the brain, the tumour lesion mass became heterogeneous. Due to this
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heterogeneity, measurements of MR parameters over the entire tumour lesion reflect an average of the contribution of tumour, edema, and necrosis. This averaging is espe- cially noticeable for T2 of F98, which increased by about 15% with respect to the initial value. For 9L glioma, de- spite the obviousness of necrosis as confirmed on histolog- ical slices (Fig. 4), no significant T2 increase was observed (Fig. 3 and Table 1). In fact, the sampling of the T2 curve appeared to be imperfect for an accurate measurement of 9L T2 since the longest TE explored was 120 ms. However, necrosis can be brought to the fore by ADC measurements, as previously reported [10, 17].
A pending issue of this systematic measurement of T1, T2, ADC or MTR is the calculation of tumour lesion/parenchyma contrast (Fig. 7) especially in the early stage of tumour cell proliferation growth when therapies can still challenge.
Whatever the sequence used, F98 tumour lesion/paren- chyma contrast was lower than 9L tumour lesion/paren- chyma contrast. Indeed, F98 tumour lesion/parenchyma contrast ranged from 15–50% whereas 9L tumour lesion/parenchyma contrast ranged from 40–80% (Fig. 7).
Calculating T1s using the saturation technique and three data points is clearly sub-optimal [18] and thus
we cannot conclude that best contrast is achievable with this parameter. However, this saturation technique al- lows rapid T1 estimation, and therefore inversion delay calculation to introduce a T1 weighting in a T2-weighted fast spin echo sequence. As shown in Fig. 6, an evident contrast enhancement between parenchyma and tumour lesion was observed and a quantitative analysis of the im- age showed that the contrast increased from 25% in the conventional fast spin echo sequence to 600% in the spin- prepared fast spin echo sequence.
In conclusion, similar contrast was observed whatever the imaging sequence used, except for T1 which seemed to provide a better contrast at least for 9L glioma. However, any of the parametric images calculated achieved a con- trast comparable to that obtained using a spin preparation prior to a fast spin echo-sequence with an inversion delay chosen to null the normal parenchyma.
AcknowledgementsWe would like to thank Dr. Anne Clavreul and Dr. Manuel Delhaye for supplying the F98 glioma cells and also recognize the employees of “animalerie hospitalo-universitaire d’Angers” for their assistance in the animal experimentation. This work is part of the INSERM-CNRS ‘Imagerie du Petit Animal’
project—grant number 8BG02H.
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