Convenient Application of a Compact-Type Magnetic Resonance Imager with Blood Pool Contrast Agent Gadolinium-Conjugated Dextran to Assess Vascular Abnormalities in Experimental Animals

Abstract

Vascular lesions are symptomatic of lifestyle-related diseases and include blood clots, coarctations, aneurysms, and apoplexy. Furthermore, increased blood vessel permeability is usually observed in tumors. To develop therapeutic drugs treating vascular lesions and tumors, methods with which the vascular abnormalities can be readily assessed in experimental animals are necessary. In this paper, a laboratory-size magnetic resonance imaging (MRI) system with permanent magnets, a compact-type MRI, was used to assess vascular abnormalities. Blood vessels in the head of a mouse were clearly visualized with the compact-type MRI in combination with gadolinium-diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid chelate (Gd-DTPA)-linked dextran (Gd-Dex) as blood pool contrast agents. The rat middle cerebral artery was imaged, and artery occlusion was identified. The difference between normal and occluded rats became more apparent upon intravenous injection of sodium nitroprusside, a nitric oxide (NO) donor. The system also visualized poor circulation in a rat saphenous artery by femoral artery occlusion. In a tumor-bearing mouse, a compact-type MRI visualized accumulation of Gd-Dex similar to that of small molecular Gd-DTPA, in the rim of tumor. Gd-Dex accumulation was more consistent than that of Gd-DTPA. Tumor vasculature was characterized by estimating the plasma-to-tumor interstitial tissue transfer constant, K^trans, of Gd-Dex and fractional plasma volume, V_p, using image data. These results demonstrate the efficacy of a compact-type MRI in combination with Gd-Dex for vascular abnormality assessment in both mice and rats.

INTRODUCTION

Vascular lesions include blood clots, coarctations, aneurism, and apoplexy. Vascular lesion frequencies are increasing in line with the recent increase in incidences of lifestyle-related diseases such as hypertension, hyperlipidemia, and diabetes. Additionally, increased blood vessel permeability is observed in tumors. Macromolecules penetrate blood vessels and accumulate in tumors due to insufficient lymphatic vessels, a phenomenon known as the enhanced permeability and retention (EPR) effect.^1,2) To develop therapeutic drugs for vascular lesions and tumors, methods to readily assess vascular abnormalities in experimental animals are necessary. Magnetic resonance imaging (MRI) yields 2-dimensional (2D) image slices or 3-dimensional (3D) images in high resolution. Gadolinium (Gd)-based contrast agents are used to shorten spin-lattice proton relaxation times (T₁) to markedly enhance the magnetic resonance (MR) signals of nearby water. In this manner, the distribution of contrast agents in experimental animals is visualized on T₁-weighted MR images. Macromolecular Gd contrast agents, including Gd-diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid chelate (Gd-DTPA)-conjugates of dextran (Gd-Dex), albumin, and dendrimers, have been used for assessment of vascular abnormalities, because they remain within normal blood vessels.³⁾ The assessment is usually performed using MRI systems with 1.5–2 T, sometimes 3–7 T to gain high resolution, superconducting magnets.^4–6) However, MRI systems using superconducting magnets impose significant costs in installation, maintenance, and operation.

Recent developments in laboratory-sized MRI systems with permanent magnets have produced a compact-type MRI, enabling convenient imaging analyses of experimental animals such as mice and rats. This system does not require shielding and is free of complicated maintenance. A few pharmaceutical studies about assessment of controlled release of a model drug, Gd-DTPA, from gel, tablet, and capsule formulations in mouse and rat stomachs have been performed with a compact-type MRI.^7–9) To assess vascular abnormalities, however, high resolution should be required. Therefore, in this study, we examined whether or not a compact-type MRI is applicable to the assessment of vascular abnormalities in small experimental animals. Among macromolecular Gd contrast agents, Gd-Dex was employed. Dextran with an approximate molecular weight of 40 kDa has been clinically used as plasma expander for more than 70 years. Dextran has low or no immunogenicity and rarely causes inflammation. Moreover, Gd-Dex is easily synthesized from dextran. Gd-Dex receives degradation in a blood circulation; but, its half-life is sufficient to obtain MR images (t_1/2: approx. 20 min).¹⁰⁾ Using a compact-type MRI and a contrast agent Gd-Dex, we demonstrated that occlusions of middle cerebral artery and femoral artery in rats and increased vascular permeability around tumor in mouse could be analyzed.

MATERIALS AND METHODS

Reagents

Dextran (molecular weight 32000–45000), DTPA dianhydride, 4-dimethylaminopyridine (DMAP), gadolinium chloride hexahydrate, 2,3,5-triphenyl-2H-tetrazolium chloride (TTC), sodium nitroprusside (SNP), and isoflurane were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Gd-DTPA was purchased from Bayer HealthCare (Osaka, Japan) as a solution of meglumine gadopentetate (Magnevist^® iv inj.). Dulbecco’s phosphate-buffered saline without Mg²⁺ or Ca²⁺ (PBS) was purchased from Gibco (Invitrogen; Carlsbad, CA, U.S.A.). Pure water freshly prepared with a Millipore Milli-Q Labo (Bedford, MA, U.S.A.) was used unless otherwise noted. All other reagents were of the highest purity commercially available.

Gd-Dex

Dextran was esterified with DTPA dianhydride in dimethyl sulfoxide dehydrated in the presence of DMAP for 2 h at 40 °C, dialyzed, and then lyophilized as per the previous report.¹¹⁾ The residual material (DTPA-linked dextran) was dissolved in water, and gadolinium chloride hexahydrate, dissolved in a small volume of water, was added dropwise to the stirred solution. The pH value was maintained below 6 during the reaction. After stirring for 40 h, the mixture was dialyzed against double-distilled water and then lyophilized. Gd-Dex was obtained as a white flocculating residue. The number of glucose units was estimated using the colorimetric method¹²⁾ with D-glucose as a standard. The Gd content was analyzed by inductively coupled plasma atomic emission spectroscopy (SPS 1200VR, Seiko Instruments Inc., Chiba, Japan, or iCAP7400, Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.). The molar ratio of Gd to glucose unit was 0.3. The hydrodynamic diameter of the primary population of Gd-Dex in PBS was 158 ± 39 nm (mean ± standard deviation (S.D.)) as measured with an ELS-Z zeta-potential & particle size analyzer (Otsuka Electronics Co., Ltd., Osaka, Japan). The main population of Gd-Dex molecule accounted for approximately 60% based on light scattering intensity. The longitudinal relaxivity of Gd-Dex was measured by a previously described method^13,14) and was 9.6 L mmol⁻¹ s⁻¹ at 1.5 T, approximately 2-times larger than that of Gd-DTPA (3.9 L mmol⁻¹ s⁻¹ at 1.5 T).

Experimental Animals

Mice (ddY, female, 4 weeks old, 18–20 g body weight (b.w.)) and rats (Wistar, female, 7 weeks old, 170–190 g b.w.) were purchased from Japan SLC (Hamamatsu, Japan). Animals were fed a commercial diet (CLEA Japan, Inc., Tokyo, Japan) and water ad libitum in a temperature- and humidity-controlled room. Surgeries and MRI measurements were performed under anesthesia with isoflurane inhalation (1.5% in air, 0.7 L/min). All animal experiments were performed according to the guideline of the Laboratory Protocol of Animal Handling, Sojo University under the permission of the Committee of Laboratory Animal Experiments in our university (Permission Nos. 2019-P-019 and 2019-P-027).

Middle Cerebral Artery Occlusion in Rats

Middle cerebral artery occlusion (MCAO) in rats was induced by the method of Koizumi with some modifications.^15,16) Briefly, the right common carotid artery, external carotid artery, and internal carotid artery were surgically exposed. The right external carotid artery was ligated, and a nylon surgical thread (4-0, 25 mm long) coated with silicon (Genie Ultra Hydrophilic Impression Material, Sultan Healthcare, York, PA, U.S.A.) was inserted from the right internal carotid artery to block the origin of the middle cerebral artery (MCA). The wound was then stitched closed. A heating pad was used to maintain body temperature at 37 °C by monitoring rectal temperature during surgery. The thread was withdrawn after MRI measurement (approximately 1.5 h ischemia), and the occlusion was confirmed by TTC staining of slices of the brain removed on the next day.¹⁶⁾

Femoral Artery Occlusion in Rats

Occlusion of femoral artery (FA) was performed as previously reported.¹⁷⁾ The right inferior limb femoral artery was surgically exposed and isolated approx. 3 mm distal to the inguinal ligament. The artery was ligated with a nylon surgical thread (4-0), then the wound was stitched closed.

Tumor Cell Transplantation to Mice

S180 tumor sarcoma cells were transplanted following a previously reported method.¹⁸⁾ Tumor cells were maintained by weekly passage of mouse abdominal cavity ascites. Cells (2 × 10⁵ cells) were washed with PBS and subcutaneously implanted in the back of mouse. Mice whose tumor reached a diameter of about 12 mm were used for MRI measurement.

MRI Experiment

Contrast agents were dissolved in a physiological saline solution and intravenously injected into the mouse or rat via the tail vein. MRI was performed with an MR VivoLVA compact MRI system (DS Pharma Biomedical Co., Osaka, Japan), with 1.5 T permanent magnets and an 80-mm gap. Radio frequency (RF) coils for mouse head and whole body (30 mm in diameter), for rat head (38.5 mm in diameter), and for rat inferior limb (50 mm height and 80 mm width) were used. T₁-weighed 2D and 3D images were obtained using a gradient echo sequence. Parameters for the 3D images were as follows; radio frequency pulse repetition time (T_R), 50 ms; echo time (T_E), 6 ms; and a flip angle (FA), 40°. Parameters for the 2D slice images were T_R, 75 ms; T_E, 6 ms; FA, 45°; with a slice thickness of 1 mm. 3D images were analyzed with INTAGE Realia Professional software (Cybernet, Tokyo, Japan), and 2D images were analyzed with ImageJ 1.48 (ver. 5) software.

Pharmacokinetic Analysis

MR images of animal were taken together with Teflon tubes (i.d. of 3 mm, length of 17 mm) filled with an aqueous solution of 0.5 mmol/L GdCl₃ or pure water. Gd concentrations (mmol/L) of the regions of interest (ROIs) and aorta were estimated, for convenience, by comparing intensity enhancement ratio (IER) of ROI with that of 0.5 mol/L GdCl₃ solution by reference to the previous reports,¹⁴⁾ where IER was calculated as follows,

  

The plasma-to-tumor interstitial tissue transfer constant, K^trans, of contrast agent and fractional plasma volume, V_p, were estimated by two-compartment model with the following equation, assuming that backflux from the tumor interstitial tissue into the blood plasma is negligible:

  

where C_t and C_p are concentrations (mmol/L) of contrast agent in tissue and plasma of aorta, respectively.^20–22) The values of K^trans and V_p were obtained by Patlak plot.^20–22) Image data of pre- and 1, 20, and 45 min post-administration of Gd-Dex were used.

RESULTS AND DISCUSSION

Gd-Dex or Gd-DTPA was intravenously injected into a healthy mouse, then 3D MR images from the head to chest were taken. As shown in Fig. 1, Gd-Dex clearly visualizes blood vessels in maximum intensity projection (MIP) images obtained from the 3D images. This image was comparable to previous reports using MRI systems with superconducting magnets.⁴⁾ However, blood vessels were unclear for high intensity over the images obtained at 0.1 and 14 min after Gd-DTPA-injection. This may result from quick diffusion of this agent into the extracellular fluid space.³⁾ Blood vessel visualization was effective for at least 45 min after Gd-Dex injection. As expected from the longitudinal relaxivity, the limit of detection for Gd-Dex (< 0.04 mmol/L) was smaller than that for Gd-DTPA (0.06 mmol/L), as examined with in vitro 2D image (Supplementary Fig. S1). These observations demonstrate the advantage of Gd-Dex for vascular images.

Fig. 1. Ability to Visualize Blood Vessels with Compact-Type MRI and Gd Contrast Agents

Either Gd-Dex or Gd-DTPA (2 µmol Gd equivalent/mouse) was intravenously injected. Dose of Gd-DTPA was comparable to that used clinically in humans (0.1 mmol/kg. b.w.). Images obtained from T₁-weighed 3D gradient echo images are shown. The time indicated on the left-hand side is the data collection start time after contrast agent injection. One excitation was used, and resolution was 0.234 × 0.234 × 0.234 mm³.

As blood vessels were clearly visualized with Gd-Dex, the MRI method was applied to the rat MCAO model (Fig. 2). Ambilateral MCAs (filled and open arrowheads) were observed in the untreated rat. However, only the left-hand side MCA (open arrowhead) was observed in a rat with MCAO induced in the right MCA before intravenous Gd-Dex injection (Figs. 2a, b). Images of MCAs in these projections were somewhat blurred; but the difference between normal and MCAO rats became clearer upon SNP treatment, an NO donor (Figs. 2c, d). The MCA diameter is approximately 0.1–0.2 mm, which is smaller than the resolution of this MRI system (0.313 × 0.313 × 0.313 mm³). The blurred MCA images before SNP treatment may result from the insufficient resolution of this MRI system. SNP administration increases MCA diameter via the vasodilating activity of NO. This vasodilation by SNP administration improves MCA visualization. Previous literature reported that SNP treatment induced arterial vasodilation of 24% in the brain of a mouse, as measured by functional optical-resolution photoacoustic microscopy.²³⁾ It was also reported that SNP administration increased cerebral blood flow as monitored by laser-Doppler flowmetry in an MCAO-treated rat.²⁴⁾ The present results demonstrate that MRI visualized improved blood flow in the MCA by SNP treatment in addition to the MCA vascular occlusion.

Fig. 2. Rat Brain MRI Contrast Images

A photograph of the removed rat brain is shown on the left-hand side (upper panel). The brain was placed upside down. Positions of the left (open arrowhead) and right (filled arrowhead) MCAs are indicated. a–d, MIP images obtained from T₁-weighed 3D gradient echo images. The rat head direction used in the images is indicated on the left-hand side (lower panel). Gd-Dex (4 µmol Gd equivalent/rat) was intravenously injected into the normal (a) and MCAO-treated rat (b). Images were taken 1 min after injection. A saline solution of SNP (0.77 mg/rat) was intravenously injected into normal (c) and MCAO-treated rats (d). After 10 min, Gd-Dex (4 µmol Gd equivalent/rat) was intravenously injected into the rats, and images were taken 1 min after injection. One excitation was used, and the resolution was 0.313 × 0.313 × 0.313 mm³. Images were trimmed to remove vascular images interfering with MCA ascertainment.

The second angiography example is that of FA occlusion. The vessels of inferior limbs of rat were clearly imaged by the compact-type MRI after intravenous administration of Gd-Dex, although the vessels could not be recognized before the administration (Fig. 3). The FA ramifies into the popliteal artery (PA), saphenous artery (SA), and lateral circumflex femoral artery (LCFA). The right FA is ligated between the inguinal ligament and the branch point. The occluded vessel at the SA position disappeared distal to the branch point and reappeared in the periphery, and the vessel at the LCFA position became thin compared to the opposite one. The MR image spatial resolution may not be sufficient to discriminate the artery and vein, and arteries are usually located parallel to the veins. Therefore, the vascular image should include both the artery and vein. The saphenous vein and lateral circumflex femoral vein are thinner than the popliteal vein. This may be why only the image of the vessel at the SA position disappeared by femoral ligation. Moreover, the inferior gluteal artery is reported as an alternative blood supply in a murine hind-limb ischemia model.²⁵⁾ The reappearance of SA in the periphery may be due to this mechanism.

Fig. 3. Rat Inferior Limb MRI Contrast Images

T₁-weighed 3D gradient echo images were taken before (a) and immediately after Gd-Dex intravenous injection (12 µmol Gd equivalent/rat) into the femoral artery occlusion rat (b). The femoral artery was occluded at the position indicated with a short white bar. The right SA position is indicated with an open arrowhead. Two excitations were used, and the resolution was 0.391 × 0.391 × 0.391 mm³. MIP images were shown.

Finally, a tumor-bearing mouse was intravenously injected with either Gd-Dex or Gd-DTPA. The 2D slice images including the tumor were then compared (Fig. 4A). Both Gd-Dex and Gd-DTPA clearly accumulated in the tumor periphery 1 min after injection. The increased intensity in the tumor periphery likely indicates the leak of contrast agents from blood vessels. The estimated mean Gd-Dex hydrodynamic size was 158 nm. Gd-Dex accumulation in the extracellular space around the tumor may result from the EPR effect. During the initial stages, the tumor boundary enhancement of Gd-Dex was less intense than that for Gd-DTPA, but over time the Gd-Dex intensity gradually increased. The accumulated Gd-Dex around tumor was detectable for at least 1 d (data not shown). This contrasts with Gd-DTPA, which washed out of the tumor after approximately 20 min.

Fig. 4. MRI Contrast Images of Tumor-Bearing Mice

Either Gd-Dex or Gd-DTPA (3 µmol Gd equivalent/mouse) was intravenously injected into S180 tumor-bearing mice. T₁-weighed slice images were taken by 2D gradient echo sequence. Three excitations were used, and the resolution was 0.234 × 0.234 mm². A. Time–course of sagittal slice images. The top images (Pre) are MR images obtained before contrast agent injection. Images obtained before contrast agent injection were subtracted from images after to obtain difference images. The difference image intensities were multiplied by a factor of 2. The open arrowhead indicates an accumulation of contrast agents in the tumor periphery. The time indicated on the left-hand side is the starting time of data collection after the contrast agent injection. B. (a) Positions of ROIs on the transverse slice image. ROIs 1 and 4 are parts of high Gd accumulation, and ROIs 2 and 3 are parts of low Gd accumulation. (b) Patlak plot of the data from ROIs. Concentrations of Gd in the ROIs and plasma of aorta were estimated, as described in Materials and Methods, and K^trans and V_p, were obtained as slop and y-intercept of Patlak plot, respectively. The values were; K^trans: 0.0017 min⁻¹ for ROI 1, 0.0004 min⁻¹ for ROI 2, 0.0001 min⁻¹ for ROI 3, and 0.0017 min⁻¹ for ROI 4; V_p: 0.12 for ROI 1, 0.045 for ROI 2, 0.029 for ROI 3, and 0.11 for ROI 4.

To characterize tumor vasculature, ROIs were chosen within tumor in transvers image of tumor bearing mouse administered with Gd-Dex; ROIs 1 and 4 are parts of high Gd accumulation, and ROIs 2 and 3 are parts of low Gd accumulation (Fig. 4B). Using estimated concentration of Gd in ROIs and plasma, K^trans and V_p were graphically obtained. As expected, the values of K^trans for the parts of high accumulation (0.0017 min⁻¹) were larger than those for the parts of low accumulation (0.0001–0.0004 min⁻¹). Similarly, the values of V_p for the parts of high accumulation (0.11–0.12) were larger than those for the parts of low accumulation (0.029–0.045). Those values obtained here should be less accurate because relationship between signal enhancement and concentration of contrast agents should not be so simple; microstructure of tissue and structure of contrast agents affect relaxivity of the contrast agents. However, it was possible to characterize the tumor vasculature to some extent with a compact-type MRI and Gd-Dex.

The present study demonstrated the use of a compact-type MRI system and the blood pool contrast agent Gd-Dex which clearly visualized vascular abnormalities in three examples: a rat MCAO model, a rat femoral occlusion model, and a mouse tumor model. This system was fully usable to detect occluded parts of blood vessels and to characterize vascular condition of tumor, although the spatial resolution is limited. Because this system is convenient and laboratory-scale, the system is suitable for routine evaluation of drug efficiency. Therefore, this imaging system is an effective tool to be adapted in the pharmaceutical sciences.

Acknowledgments

We heartily thank Dr. Mayumi Yamato (Kyushu University) for technical instruction of MCAO, Professor Yasuhisa Tsujimoto (Nihon University School of Dentistry at Matsudo) for advice and gift of silicon material, Dr. Hideaki Nakamura (Sojo University) for technical advice in particle-size measurement, and our students, Ms. Marie Tokunaga and Ms. Sayaka Hashigaki, for their excellent technical assistance. This study was supported in part by JSPS KAKENHI (Grant Nos. 25460053 and 19K07023).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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