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Early MR Lymphography with Gadofluorine M in Rabbits¹

¹ From Corporate Research Business Area Diagnostics and Radiopharmaceuticals, MRI and X-Ray Research, Schering, Müllerstrasse 178, D-13342 Berlin, Germany. From the 2002 RSNA scientific assembly. Received August 13, 2002; revision requested October 8; final revision received September 8, 2003; accepted October 14. Address correspondence to B.M. (e-mail: bernd.misselwitz@schering.de).

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To investigate the dose and time dependency of gadofluorine M for lymph node imaging and the detection of lymph node metastases in an animal model and to compare gadofluorine M with Gadomer (both, Schering, Berlin, Germany) for lymph node enhancement.

MATERIALS AND METHODS: Enhancement of popliteal and iliac lymph nodes was studied in VX2 tumor–bearing rabbits before injection and at 5–120 minutes and 24 hours after intravenous bolus injection of 0.025, 0.05, and 0.1 mmol gadolinium per kilogram of body weight gadofluorine M (six rabbits) or 0.5 mmol/kg Gadomer (eight rabbits). Effects of treatment and time point at enhancement were evaluated with repeated measures analysis of variance. Means were separated with all-pairs comparison with Tukey-Kramer adjustment. After 1.5-T magnetic resonance (MR) imaging, lymph nodes were removed, and prepared sections were stained with hematoxylin-eosin for microscopic examination.

RESULTS: MR images in VX2 tumor–bearing rabbits revealed rapid and strong signal intensity increase in the functional lymph node tissue by 15 minutes after intravenous injection of gadofluorine M. Maximum enhancement of 165%–309% was observed 60–90 minutes after injection (enhancement with 0.05 and 0.1 mmol/kg significantly different from that with 0.025 mmol/kg, P .05). Metastatic tissue showed only slight enhancement at early time points, resulting in high-contrast differentiation between functional and metastatic tissue. Intravenous injection of the blood-pool agent Gadomer induced only short and inhomogeneous lymph node enhancement (enhancement significantly lower [P .05] than that with gadofluorine M).

CONCLUSION: Findings in the study showed that gadofluorine M produces rapid lymph node accumulation. Diagnosis of lymph node metastases was shown with intravenous injection of gadofluorine M with a minimum effective diagnostic dose of 0.025 mmol/kg.

© RSNA, 2004

Index terms: Experimental study, 99.12943 • Gadolinium • Lymphatic system, MR, 99.12943 • Magnetic resonance (MR), contrast media, 99.12943

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Detection of tumor metastases in lymph nodes is clinically important for tumor staging and therapy planning in patients with cancer. Several imaging modalities are used to help diagnose metastatic involvement in lymph nodes: nuclear medicine imaging (mainly for intraoperative localization of sentinel lymph nodes), computed tomography (CT), and magnetic resonance (MR) imaging (1). However, differential diagnosis between malignant and benign lymph nodes is a problem with most imaging modalities because the size and shape of the lymph nodes are the only criteria. Detection of small metastases in normal-sized lymph nodes at MR imaging and CT is often inadequate, and differentiation of enlarged nodes is poor (2–5). Therefore, there is an unmet medical need for a lymphotropic contrast agent that accumulates in functional lymphatic tissue but does not accumulate in metastatic deposits.

Contrast material–enhanced MR lymphography is a potential noninvasive method for analysis of the lymphatic system after interstitial (intracutaneous or subcutaneous) or intravenous administration of contrast media. Interstitial MR lymphography results in high accumulation in regional lymph nodes and could be performed with several types of contrast media, including extracellular contrast agents (6), extracellular contrast agents encapsulated in liposomes (7), superparamagnetic iron oxide particles (8), polymeric compounds (9,10), and lipophilic compounds that form aggregates or micelles (11,12). Potential problems of the interstitial route of contrast media administration are evaluations of lymph nodes distal to a lymphatic obstruction, which are not opacified, and of lymph nodes at the contralateral side, which would necessitate multiple injections (13,14). Gadolinium-enhanced MR images exhibit higher spatial resolution, higher signal-to-noise ratio, and fewer artifacts than do MR images enhanced with T2* agents such as iron oxide particles (9,15).

Intravenous injection of a lymphotropic contrast medium is preferable to interstitial administration because the contrast medium is distributed to each individual lymph node. The first contrast medium for intravenous MR lymphography, dextran-coated ultrasmall superparamagnetic iron oxide particles, did not show maximum lymphographic effect until 24–48 hours after administration (15,16). Another intravenous lymphographic contrast medium, a macromolecule consisting of a central gadolinium diethylenetriaminepentaacetic acid (DTPA)–bearing polymer covered with dextran molecules (Gd-polylysin-DTPA-dextran) was described by Harika et al (9). The T1-type compound did not show maximum lymph node accumulation and enhancement in different animal species before 24 hours after injection (9,17).

Gadofluorine M (Schering, Berlin, Germany) is a macrocyclic gadolinium chelate with a perfluorinated side chain, which results in formation of micelles in aqueous solutions. The polymeric Gadomer (Schering) is currently in clinical development as a T1 contrast agent for blood-pool MR imaging (18,19). The purpose of this study was to investigate the dose and time dependency of gadofluorine M for lymph node imaging and the detection of lymph node metastases in an animal model and to compare gadofluorine M with Gadomer for lymph node enhancement.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
All experimental protocols were performed in accordance with state regulations governing animal experiments.

Contrast Media
Gadofluorine M is an amphiphilic gadolinium complex with a molecular weight of about 1,530 g/mol (patent application DE 10040381). The contrast medium is synthesized by adding a perfluoroctyl chain to a gadolinium-containing macrocycle. The complex also contains a sugar moiety, which leads to increased hydrophilicity. The test formulation was an aqueous solution that contained NaCl/Tris buffer and CaNa₃DTPA (pH of 7.4) and had a final concentration of 200 mmol of gadolinium per liter.

Gadomer is a synthetic polymeric paramagnetic complex with 24 gadolinium atoms bonded to a dendritic backbone (U.S. patent 5,820,849). The apparent molecular weight of the compound is approximately 30 kd. The pharmacokinetics of Gadomer are described in detail by Misselwitz et al (19). The test formulation was an aqueous solution that contained NaCl/Tris buffer and Ca-Butrol (pH of 7.4), with a final concentration of 500 mmol gadolinium per liter.

Relaxivity
The T1 and T2 relaxation times of water and bovine plasma (Kraeber, Ellerbek, Germany) containing increasing concentrations of gadofluorine M (0, 0.3, 0.6, 1.2 mmol/L) were determined at 40°C by using a nuclear MR pulse spectrometer (Minispec PC 20; Bruker, Rheinstetten, Germany) at 0.47 T (J.P.). In addition, T1 relaxation times of water and bovine plasma containing increasing concentrations of gadofluorine M (0, 0.025, 0.1, 0.25 mmol/L) were measured at 20°C with a 1.5-T head MR imaging system (Allegra; Siemens, Erlangen, Germany) with an optimized inversion-recovery turbo spin-echo sequence (echo time of 8 msec, turbo factor of 7) (J.P.). T1 and T2 relaxation times of dog (beagle) blood (Winkelmann; Borchen, Germany) (freshly collected and stored at 4°C until use) containing two concentrations of gadofluorine M (0.25 and 0.5 mmol/L) were also measured at 37°C with the same MR imaging system by using an optimized inversion-recovery turbo spin-echo sequence (echo time of 8 msec, turbo factor of 17) for determination of T1 and an optimized multiecho spin-echo sequence (repetition time of 4,000, echo spacing of 7 msec, 32 echoes) for determination of T2 (J.P.).

Tumor Model
Detection of lymphatic tumor metastases was investigated in VX2 tumor–bearing rabbits. The 26 rabbits (Hare; Charles River, Kisslegg, Germany) weighed 2–3 kg and were inoculated intramuscularly in the left (22 rabbits) or right (four rabbits) thigh with two to three 1 x 1-mm pieces of VX2 carcinoma cells to produce metastases in iliac lymph nodes (B.M.). MR imaging experiments were performed 3–6 weeks after injection of the tumor cells.

MR Imaging
The MR imaging experiments were performed with the 1.5-T head MR imaging system with a three-dimensional T1-weighted sequence (magnetization-prepared rapid gradient-echo [repetition time msec/echo time msec of 11.1/4.3, flip angle of 15°, 80 sections, section thickness of 1 mm]).

Detection of lymphatic tumor metastases was investigated in VX2 tumor–bearing rabbits (weight range, 2.6–4.4 kg) (six rabbits per dose of gadofluorine M, and eight female rabbits for Gadomer). The animals were kept in normal laboratory conditions at 22°C ± 2 (mean ± SD) with a dark-light rhythm of 12 hours. Food and water were supplied ad libitum.

The rabbits were anesthetized with subcutaneous injection of 50 mg/kg ketamine hydrochloride (Ketavet; Parke-Davis, Freiburg, Germany) and 10 mg/kg xylazine hydrochloride (Rompun; Bayer, Leverkusen, Germany). Anesthesia was maintained with continuous infusion of a mixture of ketamine hydrochloride (170 mg) and xylazine hydrochloride (6 mg) in saline (14 mL).

MR images of iliac and popliteal lymph nodes were obtained before injection and at 5–120 minutes and about 24 hours (only gadofluorine M) after one intravenous administration of 0.025, 0.05, or 0.1 mmol/kg gadofluorine M or 0.5 mmol/kg Gadomer.

Signal intensity (SI) values were determined by choosing an appropriate region of interest (about 1.0–6.2 mm² [placed in consensus by B.M. and H.J.W.]) in functional, metastatic lymph node, and muscle tissues. SIs were related to the SI of a water phantom containing 0.25 mmol/L gadopentetate dimeglumine (Magnevist; Schering, Berlin, Germany). Enhancement (E) was calculated according to the following equation: E = (SI_post – SI_pre)/SI_pre. In addition, ratios of SIs of lymph node versus muscle tissue and lymph node versus metastasis were calculated.

Histologic Examination
After MR imaging, the tumor-bearing rabbits were sacrificed by means of intravenous administration of 1 mL/kg T61 (0.2 g embutramide, 0.05 g mebezoniumiodide) (Hoechst Roussel Vet, Unterschleissheim, Germany). The iliac and popliteal lymph nodes were removed, dissected from surrounding tissue, and fixed in Lille buffered formalin. The lymph nodes were embedded in paraffin, and the prepared sections (approximately 5 µm thick) were stained with hematoxylin-eosin for microscopic examination (presence of metastases) by a pathologist.

Elimination
To study the elimination of gadofluorine M, six rats (Shoe: WIST, Tierzucht Schoenwalde, Schoenwalde, Germany) (body weight at beginning of study, 95–114 g) were placed in metabolic cages to collect urine and feces separately. A dose of 0.1 mmol/kg (1.0 MBq/kg) gadolinium 153–labeled gadofluorine M was administered with an intravenous bolus injection into the tail vein. Urine and feces were collected fractionated for quantitative analysis until day 7 after injection (B.M.).

All samples were measured by using a ¹⁵³Gd gamma-ray counter (Compugamma CS 1282; LKB Wallac, Turku, Finland) for 10 minutes. The limit of detection was set to two times the background SI.

Acute Toxicity
The orientating acute systemic tolerance (lethal dose) was investigated in three mice (NMRI; Charles River, Buch, Germany) (body weight at beginning of study, 18–22 g) by injecting 1.0, 5.0, or 7.5 mmol/kg gadofluorine M at a rate of 2 mL/min into a lateral tail vein. Mortality was observed for 7 days (B.M.).

Statistical Analysis
Computer-based software (SigmaStat 2.0, SPSS Science, Chicago, Ill; SAS for Windows, version 8.2, SAS Institute, Cary, NC) was used for statistical analysis. Time dependence and treatment dependence of the enhancement in iliac and popliteal lymph nodes were investigated by means of repeated measures analysis of variance. The means were separated at each time point with all-pairs comparisons with Tukey-Kramer adjustment for multiple comparisons only if the results of repeated measures analysis of variance indicated that the dose was a factor. Apart from the Tukey-Kramer adjustment, no other adjustments were applied because of the exploratory character of the experiment. All P values should be interpreted as descriptive rather than confirmatory. In Figures 1 and 2, a difference is indicated as significant if the corresponding Tukey-Kramer adjusted P value was less than .05.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Line graph depicts percentage SI enhancement in functional tissue of iliac lymph nodes after intravenous administration of 0.025 (), 0.05 (), and 0.1 () mmol/kg gadofluorine M (six rabbits) and 0.5 mmol/kg () Gadomer (eight rabbits). * = enhancement significantly different (P .05) from that with 0.025 mmol/kg gadofluorine M, # = enhancement significantly different (P .05) from that with 0.5 mmol/kg Gadomer. gadofluorine M induced rapid and strong SI increase in functional lymph node tissue to maximum values of 165% (0.025 mmol/kg), 274% (0.05 mmol/kg), and 309% (0.1 mmol/kg). In contrast, high-dose (0.5 mmol/kg) Gadomer induced only a short-term SI increase in functional iliac lymph node tissue, with maximum enhancement of 183% at 15 minutes after injection.

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Line graph depicts percentage SI enhancement in functional tissue of popliteal lymph nodes after intravenous administration of 0.025 (), 0.05 (), and 0.1 () mmol/kg gadofluorine M (six rabbits) and 0.5 mmol/kg () Gadomer (eight rabbits). * = enhancement significantly different (P .05) from that with 0.025 mmol/kg gadofluorine M, # = enhancement significantly different (P .05) from that with 0.5 mmol/kg Gadomer. Gadofluorine M induced rapid and strong SI increase in functional lymph node tissue to maximum values of 191% (0.025 mmol/kg), 286% (0.05 mmol/kg), and 345% (0.1 mmol/kg). In contrast, high-dose (0.5 mmol/kg) Gadomer induced only short-term SI increase in functional popliteal lymph node tissue, with maximum enhancement of 150% at 15 minutes after injection.

The Student t test was performed to test for significant differences in the enhancement values between popliteal and iliac lymph nodes after gadofluorine M injection.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Relaxivity
T1 and T2 relaxivities of gadofluorine M measured at 0.47-T MR spectroscopy were about 16 and 20 L/mmol · sec^–1 at 40°C in water and about 16 and 21 L/mmol · sec^–1 at 40°C in plasma (Table). At 1.5-T MR imaging at 20°C, the values were slightly lower. There was almost no difference in relaxivity between water and plasma. In blood (37°C), T1 relaxivity at 1.5 T was about 17 L/mmol · sec^–1; T2 relaxivity was about 27 L/mmol · sec^–1 (Table).

Enhancement with gadofluorine M in the popliteal lymph node tissue increased to 191% ± 14 (0.025 mmol/kg), 286% ± 82 (0.05 mmol/kg), and 345% ± 66 (0.1 mmol/kg) (enhancement with 0.05 and 0.1 mmol/kg significantly better than that with 0.025 mmol/kg starting at 60 minutes after injection, P 0.05) (Fig 2). After injection of high-dose Gadomer (0.5 mmol/kg), enhancement increased to 150% ± 41. Enhancement was significantly less (P .05) than that with both the higher doses (starting at 30 minutes after injection) and the low dose of gadofluorine M (starting at 60 minutes after injection) (Fig 2).

In all treatment groups, there was no statistically significant difference in SI enhancement between popliteal and iliac lymph nodes (with the exception of 0.025 mmol/kg at 60 minutes after injection).

Nodes relative to muscle.—Compared with precontrast MR images, gadofluorine M–induced SI increases in functional lymph node tissue relative to the SI of muscle tissue allowed excellent detection of the lymph. There was a high ratio of SI between lymph nodes and muscle tissue in all dose groups. For the iliac lymph nodes versus muscle, the ratio increased by 113% (0.025 mmol/kg, 15 minutes after injection) and 138% (0.05 and 0.1 mmol/kg, 15 minutes after injection). For the popliteal lymph nodes versus muscle, enhancement increased by 125% (0.025 mmol/kg, 60 minutes after injection), 150% (0.05 mmol/kg, 90 minutes after injection), and 156% (0.1 mmol/kg, 90 minutes after injection). In contrast, high-dose Gadomer (0.5 mmol/kg) augmented the ratio of SI increase between lymph node and muscle tissue only by 64% for the iliac lymph nodes (5 minutes after injection) and by 40% for the popliteal lymph nodes (5 minutes after injection).

Nodal Metastases
Twelve of the 26 rabbits developed tumor metastases in iliac lymph nodes, but none of the animals developed tumor metastases in popliteal lymph nodes. Except for one animal with two small metastases (diameter, <1 mm), all 10 metastatic iliac lymph nodes were identified at gadofluorine M–enhanced MR imaging. At Gadomer-enhanced MR imaging, only one of two metastatic iliac lymph nodes could be detected. The metastasis that was missed was about 2.5 mm in diameter. Furthermore, with Gadomer enhancement, a lymph node metastasis was depicted in one additional animal that was not confirmed at histopathologic examination (false-positive finding).

The SI of the metastatic tissue increased only gradually during the experiment and reached maximum values at 120 minutes to 24 hours after injection of gadofluorine M. The highest ratio (2.3–5.4) of SI between functional and metastatic lymph node tissues was observed at 5–30 minutes after injection of gadofluorine M.

Detection of lymph node metastases was effective with all doses of gadofluorine M. Figure 3 shows metastatic iliac lymph nodes in a VX2 tumor–bearing rabbit in a series of T1-weighted gradient-echo MR images (11.1/4.3 msec, flip angle of 15°) before and at different time points after intravenous injection of 0.025 mmol/kg gadofluorine M. At 15 minutes after injection, bright and homogeneous enhancement is demonstrated in the functional lymph node tissue, whereas the nonfunctional metastatic tissue shows only minimal SI increase. Compared with the baseline MR image, the gadofluorine M–enhanced MR images show the metastatic lesions clearly, and diagnosis was confirmed at microscopic examination of the dissected and histopathologically stained (hematoxylin-eosin) nodes. At later time points (24 hours after injection), SI in the functional lymph node tissue was already reduced, while SI in the metastatic lesion increased. The SI increase in the urinary bladder confirms the renal elimination of this compound.

View larger version (163K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Nonenhanced (baseline) and gadofluorine M-enhanced (0.025 mmol/kg) T1-weighted gradient-echo MR images (11.1/4.3, flip angle of 15°) show iliac lymph nodes in one rabbit at different time points after injection (p.i.). High contrast between enhanced functional lymph node tissue and only slightly enhanced malignant tissue enables early detection of metastases. Photomicrograph confirms lesions detected at MR imaging. Arrows = iliac lymph nodes with metastases, B = urinary bladder, M = metastases. (Hematoxylin-eosin [H/E] stain.) MR images and histopathologic specimen do not exactly align because section orientation is not completely identical.

View larger version (140K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Nonenhanced (baseline) and Gadomer-enhanced (0.5 mmol/kg) T1-weighted gradient-echo MR images (11.1/4.3, flip angle of 15°) show iliac lymph nodes in one rabbit at different time points after injection (p.i.). Note short-term and rather inhomogeneous enhancement of functional lymph node tissue. Photomicrograph reveals one metastatic lesion (arrow) that was not detected at MR imaging. B = urinary bladder, M = metastases. (Hematoxylin-eosin [H/E] stain.) MR images and histopathologic specimen do not exactly align because section orientation is not completely identical.

View larger version (108K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Gadofluorine M-enhanced T1-weighted gradient-echo MR images (11.1/4.3, flip angle of 15°) obtained at 15 minutes after injection (p.i.) of 0.025, 0.05, and 0.1 mmol/kg doses shows iliac lymph nodes in different rabbits. Arrows = iliac lymph nodes with metastases, B = urinary bladder. Note high contrast between enhanced functional lymph node tissue and only slightly enhanced malignant tissue after each dose.

On the basis of the survival pattern observed in the acute toxicity experiment, the orientating acute systemic tolerance (lethal dose) after one intravenous injection of gadofluorine M in mice may be on the order of magnitude of the medium dose of 5 mmol/kg.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Basic requirements for selection of treatment of malignant tumors include histopathologic diagnosis, tumor size, growth pattern, grade, lymph node involvement, and pTNM classification (20–24). Therefore, the main indication for lymphographic contrast media is the improved detection and characterization of metastatic lymphatic involvement. Further indications are preoperative lymphatic mapping before lymph node dissection and biopsy, detection of primary lymphatic tumors (eg, lymphomas), and diagnosis of dysfunction of the lymphatic vessels (especially lymphedema).

Although dissection and histologic examination of lymph nodes are still the reference standards for determination of metastases, the invasiveness and surgical complications of the dissection procedure can lead to chronic lymphatic insufficiency with lymphedema (17,20).

Modern lymphographic imaging techniques (CT, MR imaging, ultrasonography) are becoming more clinically routine. However, the main problem is their use of only size and shape to differentiate between malignant and benign lymph nodes (2–5,24).

Although nonenhanced MR imaging also has limited value for the staging of lymph node metastases, its contrast resolution and imaging quality are higher than those of CT or nuclear medicine techniques (25–27). Therefore, a contrast agent that accumulates either in healthy lymphatic tissue or in metastatic deposits may greatly increase sensitivity and specificity of diagnosis. Most desirable would be a lymphographic contrast agent that accumulates in all lymph nodes after intravenous injection. Ideal characteristics of the contrast agent are high contrast between functional and metastatic lymph node tissue, good systemic tolerance, rapid accumulation in the lymph nodes (to allow only one session for pre- and postcontrast MR imaging), and a T1 effect in the target tissue (positive contrast, SI increase in the lymph nodes).

The first contrast media for intravenous MR lymphography were ultrasmall superparamagnetic iron oxide particles (eg, ferumoxtran [Combidex, Advanced Magnetics, Cambridge, Mass; and Sinerem, Laboratoire Guerbet, Aulnay-sous-Bois, France]) (15,28) and monocrystalline iron oxide nanoparticles (MION-46) (29). These types of compounds accumulate in phagocytic cells of the lymph nodes. Because of the T2* effects of the iron oxide particles, the accumulating functional tissue (phagocytic macrophages) appears dark (SI decrease), while the nonphagocytic metastatic tissue remains unchanged (15,30). However, the heterogeneous distribution pattern among the different lymph node groups of the body is still critical (16,31). One practical disadvantage is the slow accumulation in the lymph nodes, which requires performance of postcontrast MR imaging at 24–48 hours after injection and therefore two imaging sessions (16,28,32).

The coating material, dextran, which is probably responsible for the lymphotropic effect of the ultrasmall superparamagnetic iron oxide particles, could also be conjugated with poly-L-lysine-DTPA. After the graft copolymer was labeled with gadolinium, a T1-type polyglucose-associated macrocomplex (Gd-DTPA-PGM) resulted (9,17). The general mechanism of lymph node accumulation after intravenous injection of both types of contrast media is phagocytosis by macrophages, which are located in functional lymph node tissue but not in metastatic tissue (9,13,33,34).

In the present study, we investigated the dose and time dependency of the SI behavior and detection of tumor metastases in popliteal and iliac lymph nodes in VX2 tumor–bearing rabbits after intravenous injection of gadofluorine M, a water-soluble paramagnetic gadolinium-based T1 contrast agent, and compared them with those with Gadomer, a dendritic gadolinium chelate with blood-pool characteristics.

With a T1-weighted gradient-echo MR imaging sequence, very high and homogeneous SIs were achieved in functional lymph node tissue in rabbits after intravenous injection of gadofluorine M. The MR images demonstrated high SI contrast between the opacified lymph node and the surrounding tissue. It is surprising that the SI increase was observed by 15 minutes and lasted for at least 120 minutes. To our knowledge, this is the first contrast agent that shows such rapid lymph node–specific enhancement following this type of administration. As a result of this very effective accumulation, pre- and postcontrast MR imaging of the lymph nodes could be performed in one imaging session.

On the other hand, intravenous injection of the dendritic Gadomer produced only a short-term SI increase in functional lymph node tissue, with maximum enhancement at 15 minutes after injection. Although the dose of Gadomer was much higher than that of gadofluorine M, the maximum enhancement was considerably lower, and the SI increase in the lymph node tissue was inhomogeneous. It is known that Gadomer distributes almost exclusively in the intravascular space without significant diffusion into the interstitial space (19). Findings in pharmacokinetic studies in rabbits showed that the half-life for the elimination from plasma (ß phase) was 32 minutes (19). Therefore, the short-term SI increase in lymphatic tissue after injection of Gadomer seems to be caused by the presence of the compound in the blood vessels in the lymph node but not by accumulation of the compound in the functional lymph node tissue. This blood-pool characteristic of Gadomer is not adequate for the detection of lymph node metastases, which led to the false-negative result and the false-positive result.

In contrast, SI behavior after injection of gadofluorine M seems to result both from blood-pool effect and accumulation in the functional lymph node tissue. In general, there are two potential pathways for lymph node uptake after intravenous injection. The first pathway is nonspecific capillary extravasation through transendothelial channels into the interstitial space and subsequent uptake into primary lymphatic vessels with transport to the lymph nodes (30). This route seems to be responsible for the delayed accumulation of the iron oxide particles, as well as of the dextran-conjugated Gd-DTPA-PGM. The second pathway is direct transcapillary passage through interendothelial junctions into medullary sinuses in the lymph nodes (30,35). This process is also possible for the dextran-coated particles, but it is probably the main mechanism for the rapid lymph node uptake of gadofluorine M.

In tumor-bearing lymph nodes, the sinusoidal macrophages are replaced by tumor cells (29). It is supposed that most of the lymphographic compounds will be phagocytosed by the macrophages in the functional lymph node tissue, which will result in high SI contrast between benign and malignant structures in the nodes. The SI increase observed in the metastatic areas, especially in the late MR images (obtained 24 hours after injection), is probably caused by additional passive diffusion through more permeable blood capillaries into the metastatic lesions and delayed uptake in the necrotic parts of the malignant tumors. Because of the high contrast between functional and metastatic lymph node tissues on early MR images (obtained 15–120 minutes after injection), however, all 10 metastatic iliac lymph nodes were identified at gadofluorine M–enhanced MR imaging with the exception of one node with two small metastases (<1-mm diameter). With optimal technical equipment (field strength, coils, sequences), detection of lymph node metastases with a diameter of less than 1 mm might be possible.

In summary, findings in the current study show that detection of lymph node metastases is possible at intravenous gadofluorine M–enhanced MR imaging because the functional lymph node tissue exhibits much higher SI than does the metastatic lymph node tissue. The minimum effective diagnostic dose of gadofluorine M for detection of lymph node metastases in this rabbit model was 0.025 mmol/kg.

Practical application: Gadofluorine M–enhanced MR lymphography is a useful technique for noninvasive assessment of metastatic involvement. The especially rapid accumulation of gadofluorine M in the functional lymph node tissue allows performance of a complete pre- and postcontrast MR imaging examination in one session. Results in the current study indicate the need for larger scale experimental studies to evaluate the potential of this lymphographic contrast medium in other animal models.

	ACKNOWLEDGMENTS

 
The authors thank Ines Heinzelmann for excellent technical assistance.

	FOOTNOTES

 
Abbreviations: DTPA = diethylenetriaminepentaacetic acid, SI = signal intensity

Author contributions: Guarantors of integrity of entire study, B.M., H.J.W.; study concepts and design, B.M.; literature research, B.M.; experimental studies, B.M.; data acquisition, B.M., J.P.; data analysis/interpretation, B.M., H.J.W., J.P.; statistical analysis, B.M.; manuscript preparation, definition of intellectual content, and editing, B.M.; manuscript revision/review, B.M., J.P.; manuscript final version approval, B.M., J.P., H.J.W.

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