HiSStology: High Spectral and Spatial Resolution Magnetic Resonance Imaging Detection of Vasculature Validated by Histology and Micro–Computed Tomography

Angiogenesis has been linked to tumor aggressiveness1–4 and, by extension, tumor hypoxia.5 As early as 1965, thalidomide was used to treat cancer patients.6 Since then, many antivascular or antiangiogenic agents have been used in both clinical and preclinical studies.7 Therefore, the ability to monitor angiogenesis, especially in vivo, is of great interest for cancer therapy. Clinically, computed tomography (CT) and magnetic resonance imaging (MRI) are often used with contrast agents to assess response to antivascular therapy. Two common MRI-based methods for assessing vasculature both use gadolinium (Gd), namely magnetic resonance angiography (MRA) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). In addition to concerns with nephrotoxicity, DCE-MRI suffers from poor spatial resolution and therefore is useful only as a functional imaging tool. DCE-MRI might not be sensitive to early or subtle vascular changes. MRA without contrast agents, for example, time-of-flight MRA, suffers from lower signal to noise ratio and flow artifacts.8 Susceptibility or T2*-weighted imaging is another MRI technique used for examining vasculature.9–11 CT-based angiography may limit longitudinal studies owing to fear of radiation exposure, especially with older scanners. In general, CT does not image soft tissue as well as MRI, so soft tissue with low contrast medium uptake may not be well defined, for example, a necrotic core of a tumor. Despite the limitations of low-molecular-weight Gd-based contrast agents, they are easily detectable as they leak into the extracellular-extravascular space readily. However, low-molecular-weight Gd-based contrast agents can underestimate the effects of antiangiogenic treatments.12,13 Blood-pool agents have been shown to be more effective in detecting changes in vasculature after antiangiogenic treatment.12,14,15 However, blood-pool agents produce smaller changes in signal intensity than low-molecular-weight agents, and increased sensitivity is desirable when these agents are used. We propose that magnetic resonance spectroscopic imaging of the water resonance could increase sensitivity to the effects of blood-pool agents. Echo-planar spectroscopic imaging (EPSI) can be used to acquire water and fat spectra from each small image voxel.16,17 High spectral and spatial resolution (HiSS) data can be constructed from various features of the water lines.18–20 HiSS MRI provides an extra dimension of information (not available with conventional MRI) that can be used to identify blood vessels and contrast agent effects. Because HiSS images are derived from the complete free induction decay (FID), artifacts common to standard T2*-weighted imaging are avoided or filtered out. For example, chemical shift artifacts from fat can often degrade conventional images.21,22 The morphology of breast images has been shown with more detail using HiSS MRI.23,24 (For a detailed background on EPSI, see Mulkern and Panych and Tal and Frydman.25,26) Note that EPSI is typically performed at low spatial resolution and the acronym HiSS is preferred (to describe the protocol used here) as it emphasizes the use of both high spectral and high spatial resolutions. HiSS MRI is extremely sensitive to changes in local susceptibility, for example, changes caused by deoxyhemoglobin or superparamagnetic iron oxide particles (SPIO).27 Therefore, we proposed that images derived from HiSS data are very sensitive to tumor vasculature following injection of a blood-pool contrast agent. To test this hypothesis, we developed accurate image registration methods that allow precise correlation of MRIs, CD31-stained tissue slices, and microCT images of tumor vasculature. Note that vessel structural information can be obtained at a scale below the actual image resolution, that is, like some functional images. We also demonstrate a new approach to registration of histology and tomographic images (MRI in this case). Histology is often used as a gold standard to assess the effect of an antiangiogenic agent in mouse models. It is desirable to have a system that registers multiple histologic slices with in vivo functional and anatomic images. Furthermore, the histologic slices require some degree of warping to account for tearing, shrinking, and so on. To date, many of the published image histology registration methods are missing one of these requirements or have other limitations. For example, Humm and colleagues have reported an elegant fiducial-based histology and MRI registration method.28 However, they inserted polyethylene tubes into the sample as fiducial markers. One limitation would be the possibility of inserting the fiducial into an area of interest. It would also seem that exchanging the fiducials accurately for multiple modalities could be difficult (eg, exchanging water [MRI] fiducials for positron emission tomographic [PET] fiducials). Similarly, Breen and colleagues inked tissue samples prior to histologic preparation and then inserted pins into the ink marks to visualize them better.29,30 They demonstrated a robust warping and registration system, but, again, for a small sample, the ink or pins could disturb an interesting region of the sample. Furthermore, the method does not assist in registration with multimodality in vivo images. In many of the published methods, the serial capability is not apparent; that is, one cannot likely adjust a single histology slice and have that change propagate through all of the transformations to the functional image. In addition, the non–fiducial-based systems would have difficulty registering tissue sections with a low-resolution functional image. Also, some of the image registration/immobilization systems do not lend themselves to slicing for histology owing to geometry (whole body) or the materials used.28,31 Therefore, we developed a cast immobilization/registration system that facilitates multimodality image registration. A rat prostate xenograft model was used in athymic nude mice to validate HiSS neovascular identification with immunohistochemistry. Using a syngeneic rat prostate model, vascular casts imaged with microCT were compared with HiSS images.