Antiangiogenic Effect of Bevacizumab: Application of Arterial Spin-Labeling Perfusion MR Imaging in a Rat Glioblastoma Model

Tumor Cell Line

Human glioma cells U87 (ATCC, Rockville, Maryland) were cultured at 37°C in a humidified CO₂ incubator in RPMI 1640 Medium (Sigma Aldrich, St. Louis, Missouri), supplemented with 10% fetal bovine serum.

Animal Model

This study was approved by the Institutional Animal Care and Use Committee in Seoul National University Hospital (#12–0238-C1A0) and was performed in accordance with institutional guidelines. Nude athymic rats (200–250 g; Koatec, Gyeonggi-do, Korea) were anesthetized by intraperitoneal injection of a mixture of zolazepam and xylazine, and they were placed in a stereotaxic device. A burr-hole (2-mm-wide) was made on the right side 3 mm lateral to the midline and 2 mm proximal to the bregma by using a dental drill. Nude rats were inoculated with U87 glioma cells (3 × 10⁶ cells per 3 μL of serum-free RPMI Medium) in the right caudate-putamen region. Cells were injected into the brain by using a Hamilton syringe (Sigma Aldrich) fitted with a 28-gauge needle positioned with a syringe attachment fitted to the stereotaxic device. With stereotaxic guidance, 0, 1.4, and 3.0 mm were used in areas posterior, lateral, and dorsal to the bregma in the right caudate-putamen, respectively.

Tumor growth was verified by MR imaging at 2 weeks after implantation. Animals were randomly assigned to a control group (n = 4), 3-day treatment group (n = 6), and 10-day treatment group (n = 4). Control animals were sacrificed for brain harvest right after the first MR imaging. For the 3- and 10-day treatment groups, bevacizumab was administered intraperitoneally at 20 mg/kg in saline right after the first MR imaging.¹⁷ After 3 days, animals underwent the second MR imaging. Animals in the 3-day treatment group were sacrificed right after the second MR imaging. At 7 days after the second MR imaging, animals in the 10-day treatment group underwent the third MR imaging. They were sacrificed right after the third MR imaging. In addition, to exclude the possible effect of the tumor-volume change on the perfusion parameters, DSC perfusion MR imaging was performed at 0, 3, and 10 days in 4 rats in whom bevacizumab was not injected (10-day-without-treatment group).

The in vivo experimental design of the present study is shown in Fig 1.

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Fig 1.

Experimental design showing the timeline of each group for U87 glioma cell inoculation, bevacizumab therapy, MR imaging, and sacrifice for brain harvest.

MR Imaging Acquisition

All MR image acquisitions were performed by 2 authors (H.K. with >10 years' experience in neuroimaging and T.J.Y. with >10 years' experience in neuroradiology) by using a 9.4T MR imaging scanner (Agilent 9.4T/160AS; Agilent Technologies, Santa Clara, California). A volume coil for radiofrequency transmission and a phased array 4-channel surface coil for signal reception (Agilent Technologies) were used for the control, 3-day treatment, and 10-day treatment groups; a single-channel surface coil was used for radiofrequency transmission; and a signal reception (loop coil) (Agilent Technologies) was used for the 10-day-without-treatment group.

Before MR imaging, animals were anesthetized with 1.5% isoflurane in room air and placed inside a magnet. Animals were physiologically monitored throughout the MR imaging experiments. To avoid potential changes in cerebral blood perfusion during data collection, we carefully maintained their respiration at ∼40 beats per minute.

Following the acquisition of routine scout images in all 3 orthogonal directions, an automatic shimming procedure was performed. Unenhanced anatomic T2-weighted images were collected by using a fast spin-echo sequence with the following parameters: TR/TE_eff, 2000/45 ms; FOV, 33 × 35 mm²; matrix size, 256 × 256; echo-train length, 4; 15 sections without gap; section thickness, 1 mm; 1 signal average; 2 dummy scans; and receiver bandwidth, 100 kHz.

For perfusion data acquisitions by using the ASL technique, an amplitude-modulated pseudocontinuous arterial spin-labeling sequence was used with a single-shot spin-echo echo-planar imaging readout (mCASL; Agilent Technologies).¹⁸ Vascular and fat-suppression modules were also used with a labeling pulse duration of 3 seconds. Postlabeling delay and the gap between the labeling plane and the central imaging plane were 300 and 20 mm, respectively. Other sequence parameters were the following: TR/TE, 4000/28 ms; FOV, 33 × 35 mm²; matrix size, 64 × 64; 3 sections; section thickness, 2 mm; bandwidth, 250 kHz; 60 repetitions for labeled and control data. For quantitative analysis of cerebral blood perfusion, T1 mapping was performed by using a fat-suppressed, single-shot, inversion-recovery spin-echo echo-planar imaging sequence with TR, 8000 ms and TIs of 15, 35, 80, 200, 450, 1000, 2300, or 5400 ms. The rest of the sequence parameters were identical to those used for pseudocontinuous ASL data acquisition.¹⁸ The typical total scan time of ASL was ∼10 minutes.

Finally, DSC images were acquired from the same imaging planes as used for the pseudocontinuous ASL experiments by using a gradient-echo pulse sequence. The sequence parameters were the following: TR/TE, 25/5 ms; flip angle, 10°; matrix size, 128 × 96; 4 signal averages; bandwidth, 100 kHz; 70 repetitions. After an initial 30-second baseline acquisition, a bolus of gadoterate meglumine (0.1 mmol per kilogram of body weight; Dotarem; Guerbet, Aulnay-sous-Bois, France) was administered to the animals via a tail vein catheter by using a syringe pump (1 mL/min; Harvard Apparatus, Holliston, Massachusetts), which was immediately followed by a 1-mL saline flush. The typical total scan time of DSC was ∼11 minutes.

MR Imaging Data Analysis

All pseudocontinuous ASL images were analyzed by using Matlab (MathWorks, Natick, Massachusetts). CBF maps were derived according to previous reports.^18,19 A 3-parameter fit was used for estimating T1 maps. Once T1 maps and control and labeled images were obtained, tissue blood flow images were formed according to the following formula: CBF = (λ / T1) × (S_control − S_label) / (2α × S_control), where λ = 0.9 was the tissue/blood partition coefficient for water,¹⁹ S_control was the control image signal intensity, S_label was the labeled image signal intensity, T1 was the T1 map, and α was 0.63 as the degree of labeling efficiency.¹⁸

For DSC perfusion MR imaging, additional preprocessing was performed by using commercial software (nordicICE; NordicNeuroLab, Bergen, Norway), in which T2WIs were used for structural imaging. rCBV maps were generated by using established tracer kinetic models applied to first-pass data.^20,21 To reduce the effect of recirculation, we fitted ΔR2* (1/T2*) curves to the γ-variate function, which was an approximation of the first-pass response as it would appear in the absence of recirculation or leakage. The dynamic curves were mathematically corrected to reduce contrast agent leakage effects.²²

T2WI, rCBV, and rCBF maps based on DSC, and CBF maps based on ASL were analyzed by using a commercial PACS console. One investigator (T.J.Y. with >10 years' experience in neuroradiology) who was blinded to the experimental data drew ROIs containing the entire tumor in the plane in which the tumor area was the largest. Tumor boundaries were defined with reference to high-signal-intensity areas thought to represent tumor tissue on the T2WI. ROIs were copied and placed on coregistered rCBV and rCBF maps based on the DSC and CBF maps from ASL. The mean rCBV and mean rCBF of each tumor were measured on rCBV and rCBF maps. The mean rCBV and mean rCBF of the contralateral hemispheres were also measured as reference values for perfusion parameters. Normalized CBV (nCBV) and normalized CBF (nCBF) were derived by using the following formulas: nCBV = rCBV_tumor / rCBV_reference and nCBF = rCBF_tumor / rCBF_reference (On-line Fig 1).

Histologic Analysis

All rats were euthanized in a CO₂ chamber soon after MR imaging. Coronal sections sampled across the center of the tumors were fixed in 10% buffered formaldehyde solution and paraffin-embedded. Coronal paraffin sections were used for histologic hematoxylin-eosin staining and immunohistochemical analysis. Histologic evaluation was performed by a pathologist (J.-K.W. with >10 years' experience in neuropathology) by using a standard light microscope. Microvessel area (MVA) was determined on the basis of CD34 immunostains. Briefly, areas with the highest microvascular attenuation on CD34-stained sections were identified around the center of the tumor at scanning power. After that, MVAs consisting of endothelial area and vessel lumen were quantified with a higher power (×200 field) in the selected area. Results were expressed as the ratio of the area of microvessel to the total area of analysis within any single ×200 microscopic field.

Statistical Analysis

Paired and unpaired t tests were used to compare parameters on perfusion MR imaging and MVA on histology within subjects and between groups. For tumors for which histology was available, 1-way analysis of variance with a Scheffe post hoc test was used to analyze differences. Pearson correlation analysis was performed to determine the correlation among perfusion parameters of perfusion MR imaging and MVA. Statistical analysis was performed with the commercially available software: SPSS (Version 12.0 for Windows; IBM, Armonk, New York) and MedCalc for Windows (Version 9.3.0.0; MedCalc Software, Mariakerke, Belgium). Statistical significance was P < .05.