Irene Marco-Rius1, Jeremy A Gordon1, Peder EZ Larson1, Romelyn delos Santos1, Robert A Bok1, Aras Mattis2,3, Jacquelyn Maher3,4, Daniel B Vigneron1,3, and Michael A Ohliger1,3
1Department of Radiology and Biomedical Imaging, University of California San Francisco, San Francisco, CA, United States, 2Department of Pathology, University of California San Francisco, San Francisco, CA, United States, 3Liver Center, University of California, San Francisco, San Francisco, CA, United States, 4Department of Medicine, Division of Gastroenterology, University of California San Francisco, San Francisco, CA, United States
Synopsis
Diffusion weighted MRI has been widely used to
measure the movement of water molecules and study tissue microstructure in
order to characterize both diffuse and focal liver disease. In liver fibrosis,
for instance, increased collagen formation is associated with restricted
diffusion of water. However, the majority of water within the liver is either
in the vascular or intracellular space, making the diffusion of water a
potentially poor marker for fibrosis, which is an extracellular process. Here,
we investigated
applying diffusion-weighted MRI with an exogenously injected extracellular agent,
hyperpolarized 13C-urea, as a potentially more sensitive probe of
the extracellular space in the liver in a mouse model of liver fibrosis induced
with CCl4. Target Audience
Researchers interested in liver fibrosis, diffusion
and hyperpolarized
13C MRI.
Purpose
Diffusion weighted (DW) MRI is typically used to measure the
molecular movement of water in order to study tissue microstructure. In liver
fibrosis, collagen accumulates in the extracellular space and restricts the
motion of water molecules, decreasing the apparent diffusion coefficient (ADC)
1.
However, the majority of water within the liver is either intravascular or
intracellular, potentially limiting
the sensitivity of proton ADC for liver fibrosis, which
is an
interstitial, extracellular process. We investigated the progression of fibrosis using DW MRI with an exogenously injected extracellular agent, hyperpolarized
13C-urea,
as a potentially more sensitive probe of the extracellular space in the liver.
Methods
Protocol was approved by the local institutional animal care and use committee. The experimental protocol is illustrated in Figure 1. Liver fibrosis was induced in three CD1 mice by administering 1 μl/g of 1:7 mixture of CCl4 and olive oil via IP injection once every 4-5 days. Images were acquired at baseline and every 2 weeks using a 14T MRI scanner (Varian, Inc) with dedicated coils for 13C and 1H imaging. Imaging was performed on the day before the next treatment dose in order to minimize acute inflammatory effects.
1H diffusion weighted spin echo images were acquired using the following parameters: 128 x 128 matrix, 32 mm FOV, slice 2 mm, diffusion time 13 ms, b=93, 239, 538, 957, 1496 s/mm2. Combined cardiac and respiratory gating was employed.
For hyperpolarized experiments, 90 mg of [15N,13C]urea dissolved in glycerol were polarized for ~90 min using a 3T HyperSense DNP polarizer (Oxford Instruments), rapidly warmed, dissolved in PBS (final concentration 110 mM), and injected via tail vein catheter. The acquisition of 13C DW images started 20 s after injection, using an echo planar imaging sequence2 with
a
diffusion-compensated variable flip angle sequential, slice-select gradient correction to reduce bias3 and with the following parameters: 32 x 32 matrix, 32 mm FOV, slice 8 mm, diffusion time 20 ms, b=100, 200, 350, 400, 600, 750, 1000, 1500 s/mm2. Only respiratory triggering was used. Images with obvious cardiac motion artifact were excluded. For both 1H and 13C, ADC maps were calculated and a mean value determined over a region of interest (ROI) chosen in the thicker lobe of the liver (right lobe).
Two mice were treated for 12 weeks and one mouse expired at 8 weeks. The mice treated for 12 weeks were imaged 5 days after the last CCl4 injection and then again one week later. At the end of the study, each animal was sacrificed and the liver stained for collagen using trichrome and sirius red. Slides were reviewed by an experienced pathologist.
Results and Discussion
The baseline
1H ADC of water was (0.70 ± 0.07)×10-3 mm2/s, which agreed
with previously reports4,5. Urea ADC was (0.76 ± 0.15)×10-3 mm2/s, not significantly different from that of water. This was
surprising given the larger molecular weight of urea. For comparison the
solution-state ADC of water and urea at 37ºC are 2.9×10-3 mm2/s 6 and 1.5×10-3 mm2/s 7, respectively. The larger
reduction of water ADC in tissue relative to its solution value may reflect the
fact that a large fraction of water is intracellular while urea is
extracellular.
For all
animals, the 1H ADC initially increased and then decreased over time
(Figure 3, black circles). For 2/3 animals, 13C-urea ADC also
initially increased and then decreased (Figure 3, red stars). The small number
of animals used in this study did not permit statistical significance to be
determined.
Figure
4 summarizes the ADC results for the baseline, 5 days and 12 days after last
treatment, and pathology fibrosis grade. In the last post-treatment studies,
all three animals demonstrated a reduction in urea ADC compared to baseline (29%
average drop). Interestingly, for the two animals that had repeat imaging 12 days
after the final CCl4 injection, the ADC values increased compared to
the images obtained 5 days after the final CCl4 injection (25% average
increase). This effect was also surprising and may be due to either liver recovery
between the two scans or variability in the ADC measurement. Final pathologic
images and fibrosis grade for each animal are shown in Figure 5. The animal
sacrificed after 8 weeks developed severe fibrosis while the two animals
sacrificed after 12 weeks developed moderate fibrosis.
Conclusion
We have demonstrated the feasibility of measuring
the ADC of hyperpolarized [
15N,
13C]urea in a mouse model
of liver fibrosis. This pilot study establishes physical insights and
preliminary data to determine the timing and sample size for larger studies of
hyperpolarized agents in fibrosis.
Acknowledgements
We thank Daniel Tesfasilassie
for technical help and Dr. Peng Cao for fruitful discussions. This work was supported by RSNA
Research and Education Foundation, UCSF Liver Center grant P30DK026743, and NIH
grants P41EB013598 and R01EB016741. CVM was supported by NIH K01DK099451.References
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