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Robert R. Maronpot, Abraham Nyska, Sean P. Troth, Kathleen Gabrielson, Polina Sysa-Shah, Vyacheslav Kalchenko, Yuri Kuznetsov, Alon Harmelin, Yael Schiffenbauer, David Bonnel, Jonathan Stauber, Yuval Ramot
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Available imaging systems for use in preclinical toxicology studies increasingly show utility as important tools in the toxicologic pathologist’s armamentarium, permit longitudinal evaluation of functional and morphological changes in tissues, and provide important information such as organ and lesion volume not obtained by conventional toxicology study parameters. Representative examples of practical applications in toxicology research and preclinical studies are presented for ultrasound, PET/SPECT, optical, MRI and MALDI-MSI imaging. Some of the challenges for making imaging systems GLP-compliant for regulatory submission are presented. Use of imaging data on case-by-case basis as part of safety evaluation in regulatory submissions is encouraged.

Introduction

The use of non-invasive in vivo imaging and the option of using ex vivo imaging of fresh and fixed specimens provide a unique adjunct to conventional histopathology evaluation in preclinical animal studies. The number of available imaging methods has increased in recent years, and include ultrasound, optical imaging, positron emission tomography (PET), single photon emission computed tomography (SPECT), computed tomography (CT) and magnetic resonance imaging (MRI). Images provide detailed morphological and functional insights in addition to 2D, 3D and 4D quantitative data on progression and regression of lesions in preclinical disease models and in conventional toxicity and carcinogenicity studies. The aim of this opinion paper is to provide a brief overview of imaging modalities with examples applicable to toxicologic pathology and our recommendations for regulatory submission of imaging data. Representative imaging applications have been selected from an International Academy of Toxicologic Pathology educational course presented at the 13th European Congress of Toxicologic Pathology in Surrey, UK, September 2015.

Overview of selected imaging modalities useful in preclinical studies

Ultrasound

Ultrasound as an imaging modality utilizes high frequency sound waves that travel through tissues, organs and the body to produce images. A transducer (probe) placed against a body converts electrical signals to sound waves, sends them into the body, and the sound waves that are reflected back are turned into electrical signals for processing by a computer to generate an image. The pulses are sent into the animal at recorded time intervals. The distance and direction of the reflected return echo and its time of arrival back to the transducer permits the construction of a two-dimensional image of internal structures in the animal. Resolution increases while depth of penetration decreases at higher frequencies. Use of high frequency sound waves permits real-time monitoring of blood flow. Gas-filled microbubbles can be used to increase the signal when imaging blood flow. Attaching specific proteins to microbubbles (such as antibodies directed against endothelial cell proteins) allows ultrasound to be used to target specific intravascular biochemical processes (Kiessling et al., 2012). Advantages of ultrasound include relative low cost, portability of equipment, good temporal resolution, good safety profile and excellent sensitivity when microbubbles are used. Limitations are poor imaging of bone and airand limits on depth penetration.

PET and SPECT

Positron emission tomography (PET) and single-photon emission tomography (SPECT) utilize radionuclides to allow study of biochemical changes and levels of molecular targets in living animals (Khalil et al. 2011; Yao et al. 2012; Vaquero and Kinahan 2015). For PET to work well one must identify and produce a radiolabeled imaging agent that is specific and selective for the target of interest. A common PET radionuclide is 18F with a half-life of just under two hours. Other radionuclides include 64Cu, 76Br, and short-lived radionuclides 11C, 13N and 15O. A small quantity of the specific radiolabeled agent is given intravenously to the test subject with subsequent tracing of its distribution in the body using a PET camera. The short-lived radionuclide is generated by a cyclotron and decay of the radionuclide occurs by positron emission and subsequent collision of the positron with an electron. Each collision (annihilation) generates two photons that have significantly higher energy (511 keV) than conventional X-rays. A ring-like detector surrounding the subject detects annihilation events and, after detector normalization and other corrections, converts their electrical signal to tomographic images.

Primary use of PET in the clinic is to image cancer most often using 18F-labeled glucose. The assumption is that the metabolic demands of the cancer cells exceed that of normal cells and 18F-glucose will preferentially localize in the cancer cells. Preclinical use of PET is in basic research and in rodent models to investigate mechanism of human disease (Yao et al. 2012). Miniaturized PET scanners for preclinical use have a relatively low spatial resolution of 1 to 2 mm (Moses 2011) but a high sensitivity.

SPECT uses radionuclides that decay with emission of single gamma rays. Typical radionuclides include 99mTc, 123I and 111In and a gamma camera that rotates around the subject is used to capture data from differing positions to allow tomographic reconstruction. Technical implementation (use of a collimator) to cancel out photons that are spatially uninformative can markedly decrease the sensitivity of SPECT in contrast to PET but sensitivity can be increased using micro-pinhole apertures to maintain sensitivity with reasonable spatial resolution. Miniaturized SPECT instruments are available for preclinical studies and have been used for imaging cancer xenografts in mice, rat hearts following ischemia-reperfusion, and for imaging dopamine transporters in rat brain (Scherfler et al., 2002). New versions of SPECT instrumentation for preclinical use have been developed with spatial resolutions of less than a millimeter (van der Have et al., 2009).

The advantage of PET and SPECT is that they detect biochemical changes that typically precede anatomical changes (Bernsen et al. 2014; Franc et al. 2008). Another advantage is the possibility of continuous acquisition of scan data from a radionuclide or radionuclide-labeled experimental substance. Temporal data may be acquired in the form of a dynamic scan. For example, an uptake of a specific radiolabeled antibody-drug conjugate by the tumor can be recorded at multiple time points after the injection, including minutes after the drug delivery, followed by hours and days post-injection in the same animal – providing data which are impossible to obtain using other methods (ter Weele et al., 2015).

A major limitation of PET and SPECT is poor anatomical localization. The lack of an anatomical reference frame is easily overcome by combining PET or SPECT with CT or MRI (Chatziioannou 2005; Shao et al. 1997). The radiation exposure safety concern of using radionuclide imaging for PET and SPECT limits clinical use but is of less concern for preclinical studies provided care is taken regarding exposure of preclinical technical staff.

Optical fluorescence and bioluminescence

Optical imaging, a technique for non-invasively looking inside the body, measures light produced by optical reporters that are visualized through the tissues (typically the skin) of a live animal (Dufort et al. 2010). This imaging platform allows monitoring of function in its natural structural context. There are two broad categories of optical imaging: fluorescence and bioluminescence. Fluorescent technology uses non-ionizing visible, ultraviolet, or infrared light and the special properties of photons to obtain detailed images of organs, tissues, cells, and even molecules. Optical imaging is useful for visualizing soft tissues and takes advantages of different colors of light to simultaneously measure multiple features in an organ or tissue. Bioluminescence relies on introduction of one of several luciferase enzymes into the subject that then interact with its substrate to produce photons of visible light (Sadikot and Blackwell 2005). Both are cost-effective, non-invasive, sensitive, physiological, and high throughput with spatial and temporal resolutions up to 0.25 mm, and can be designed to target a specific protein or pathway of interest. The primary limitation of optical imaging is its shallow depth of penetration limiting its use to visualize internal organs in animals larger than rodents. Novel optical imaging techniques are constantly evolving providing capability to monitor dynamic functional processes at the cellular and molecular level in biological systems and the whole animal. Different varieties of optical, in vivo imaging modalities include optical coherence tomography (Vakoc et al., 2009, Wenzel et al., 2015), photo-acoustic microscopy (Stein et al., 2009), photo-acoustic tomography (Burton et al., 2013, Hudson et al., 2014), multi-photon microscopy (Breckwoldt et al., 2014, Harb et al., 2013), hybrid methods such as laser speckle and fluorescence (Kalchenko et al., 2011, Kalchenko et al., 2010, Towle et al., 2012, Zhongchan et al., 2013), and bioluminescence (Corson et al., 2014, Hirano, 2016) imaging.

CT or CAT- Computed Tomography (Computer-Aided Tomography or Computed Axial Tomography)

Computed tomography uses X-ray imaging of sections or slices (tomography) through the body or a specific tissue to produce a 3-D image of the subject being imaged. CT is basically an anatomic imaging process where the X-ray source is coupled to a detector array and both rotate around the animal. Tissues that strongly absorb X-rays such as bone show up as white, air shows up as black, with soft tissues showing up in shades of gray. The degree of X-ray attenuation by different tissue components is assigned Hounsfield units (HUs) based on their differences in density and composition. Software automatically assigns HUs to every voxel on the CT scan to assist in image interpretation. An iodinated contrast agent can be used during CT imaging to improve spatial resolution and soft tissue contrast. Scaled-down versions of hospital CT scanners are used in preclinical research for small animals, including invertebrates.

Computed tomography allows for rapid acquisition time and high spatial resolution. Micro-CT imaging systems used for preclinical studies offer resolutions as low as 3.25-9 µm (Lombardi et al., 2014; Rueckel et al., 2014). There is essentially limitless depth of penetration. Technical advances in X-ray detection and use of helical scanning and multi-detector systems offer increasingly lower levels of radiation to obtain suitable images. However, exposure to radiation remains a limitation in clinical applications. While soft tissue contrast can be enhanced by use of iodinated contrast agents, soft tissue detail is less than that from MRI. CT remains an important and highly efficient preclinical imaging modality and when combined with PET, SPECT, optical or MRI modalities provides an anatomical reference frame for these other imaging modalities. Numerous examples of CT imaging applications in toxicologic pathology are available in the published literature (Badea et al., 2004; Badea et al., 2005; Vasquez et al., 2013; van Deel et al., 2016; Solomon et al., 2016; Ashton et al., 2015; Brown et al., 2008; Martiniova et al., 2010; Wang et al., 2016; Cavanaugh et al., 2004; Wise et al., 2010).

MRI – Magnetic Resonance Imaging

MRI uses a powerful magnet and radio frequency (RF) energy to image atomic nuclei within the body. It is based on the constant angular momentum of protons and neutrons as they spin about their axes. In addition to their angular momentum, these nuclear particles have a small magnetic field called a magnetic moment. Both the angular momentum and the magnetic moment are vector quantities and, thus, have directionality and spin or wobble features like the w