Imaging Dopamine Receptors in the Rat Striatum with the MicroPET R4: Kinetic Analysis of [11C]Raclopride Binding Using Graphical Methods
Introduction
The promise of in vivo imaging in laboratory rodents with dedicated positron emission tomography (PET) cameras lies in bringing successful PET methodologies used in clinical and large animal studies to preclinical stages of new drug and radiopharmaceutical development.1, 2, 3 For example, rapid in vivo screening of compounds labeled with positron emitters in a single rat or mouse is possible using small animal PET imaging. PET can describe more accurately the kinetics of these labeled drugs in a single rodent than traditional ex vivo methods that require sacrificing multiple animals. As well, measurements of saturation kinetics in rodents using PET, together with kinetic modeling or graphical analyses, can be used to determine pharmacokinetic constants such as Bmax and Kdin vivo (see discussion in Hume and Meyers3). By comparison, in vitro measurements are often flawed, as specific values depend greatly on methodological details and do not include effects of bioavailability inherent to in vivo measures.1 In addition, PET studies in rodents can be used to evaluate the relationship of receptor occupancy to drug efficacy in vivo, as demonstrated by Hirani et al.,4 who characterized 5-HT1A receptor occupancy in the rat brain by the drug pindolol. Finally, high-resolution PET imaging can help bridge decades of biological research in laboratory rodents with human biology by allowing direct comparisons of animal research with clinical research.5 For example, several rodent models of human diseases, including Parkinson's disease,6 Huntington's disease,6, 7, 8 and drug abuse,9 have been studied using small animal PET.
Commercialization of small animal PET cameras by at least two manufacturers (microPET; Concorde Microsystems, Knoxville, TN, and HIDAC; Oxford Positron Systems, Oxford, UK) is expected to expand the use of PET methods in basic research and new drug development by making available cost-effective PET cameras designed specially for small animal imaging. For clinical PET research groups, acquisition of a dedicated animal PET permits simultaneous operation with clinical studies, making efficient use of expensive radiochemical syntheses. The first commercially available, dedicated animal cameras were designed for nonhuman primate scanning (for review, see Chatziioannou5). One of these early machines, the SHR-2000 (Hamamatsu, Japan), a ring-based system featuring a large diameter port (50.8 cm) permitting whole body viewing of rhesus monkeys, has also been used for rodent imaging.10 A new large-aperture animal camera (SHR-7700) has become available from Hamamastu with increased sensitivity [three-dimensional (3D) mode] and better resolution.5 However, few results from rodent studies using the SHR-7700 have been reported in the literature. A second commercially available animal camera (HIDAC; Oxford Positron Systems, UK) with a variable opening or 10–20 cm was designed specifically for small animal imaging.11 More recent HIDAC designs have increased absolute sensitivity beyond 1% while still offering intrinsic resolution reported to be <1 mm. This animal camera does not function by detecting photons with arrays of scintillation detectors (like BGO and LSO), but relies on collecting and amplifying electrons created from interactions of photons with lead sheets drilled with an array of closely spaced (∼0.5 mm) holes (0.4 mm diameter). HIDAC has been used successfully to image the GABA-benzodiazepine receptor subtype containing the α5 subunit in the rat brain.12
This Chapter presents detailed methods using a third commercially available small animal PET camera, the microPET R4 (Concorde Microsystems). The MicroPET R4 is a LSO scintillator ring-based tomograph borne from original research by Cherry and co-workers13 at UCLA. A significant advance of the commercial microPET was the increase in absolute sensitivity achieved using four rings of detector blocks, resulting in an axial field of view of ∼7.8 cm. This expanded axial field of view also allows for imaging of a whole mouse body in a single bed position. This work evaluates the quantitative capabilities of the microPET R4 by examining the effects of photon scatter, attenuation, and image reconstruction methods on determinations of dopamine receptor availability using graphical methods. Graphical analysis of kinetic data in neuroimaging in human and nonhuman primates has been a very successful, efficient, general-purpose methodology. Here we extend this method and its assumptions to the analysis of PET studies in the rat brain.
Section snippets
Animal Preparation
For experiments where dynamic measurements are required starting at the time the PET radioligand is injected, complete restraint or anesthesia is generally required to prevent motion artifact during imaging. In either case, target organ function may be affected by the specific immobilization technique employed. For example, studies of the conscious primate brain have shown that PET measures can be altered significantly by the administration of anxiety-provoking drugs, suggesting that stress
[11C]Raclopride Preparation and Administration
Dopamine receptor imaging is carried out using carbon-11 labeled substituted benzamide [11C]raclopride.17 [11C]Raclopride has been shown to bind specifically to dopamine D2 receptors (D2R) in humans17 and has been used as an in vivo probe of dopaminergic function due to its sensitivity to changes in endogenous neurotransmitter.18 [11C]Raclopride is synthesized from 11CH3I according to a method described previously.1711CH3I is prepared by a gas phase reaction of 11CH4 with I2 after conversion of
MicroPET R4 Configuration
Imaging is carried out using the microPET R4 tomograph (Concorde Microsystems), which has a 12-cm animal port with an image field of view (FOV) of ∼10 cm. Each animal is positioned prone on the microPET bed, centering the brain in the field of view. The full-width half-maximum resolution (FWHM) in the center of the FOV is approximately 1.85 mm and remains <2.5 mm FWHM across the rat brain.21 The entire rat brain is easily imaged simultaneously using the large axial (rostral to caudal) FOV of
Data Corrections and Image Reconstruction
Accurate determination of radioactivity concentration using PET requires application of several corrections to coincidence lines of response recorded by the camera. These corrections include subtraction of random coincidences, correction for dead-time losses, subtraction of scattered coincidences, correction for photon attenuation, and normalization for varying individual detector efficiencies. Finally, the PET camera must be calibrated against a known radioactivity concentration. If all
Graphical Analysis of Region of Interest Data
MicroPET images are analyzed using regions of interest (ROIs) created with software provided with the camera (ASIPro v.3.2) as described previously.27 Briefly, elliptical regions of interest are drawn manually on a single plane of striatum and a single plane of the cerebellum (30 pixels). Separate ROIs are drawn on the left and right striatum (15 pixels each). The rat striatum is clearly visible as a bilateral structure on four to five coronal planes when imaged using high specific activity
Summary of Results
Using the method described in this Chapter, the binding potential was computed for 15 [11C]raclopride studies in the rat striatum using the microPET R4. In two of these studies, a 1-mg⧸kg blocking dose of raclopride was coadministered with the tracer to measure nonspecific binding of the tracer. The remaining studies were carried out with varying doses of [11C]raclopride of varying specific activity. Data are summarized in Fig. 2 plotted as the binding potential as a function of raclopride mass
Acknowledgements
This work was carried out at Brookhaven National Laboratory under Contract DE-AC02-98CH10886 with the U.S. Department of Energy and supported by its Office of Biological and Environmental Research and the National Institutes of Health (EB002630).
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