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Basic Science Investigations |
1 Chemistry Department, Brookhaven National Laboratory, Upton, New York
2 Medical Department, Brookhaven National Laboratory, Upton, New York
3 Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York
4 Department of Applied Mathematics and Statistics, State University of New York at Stony Brook, Stony Brook, Stony Brook, New York
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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25%–45% resulting in an average increase of DVR of 3.5% (range, 0%–10%) after correction. Conclusion: This study shows that the 11C-raclopride microPET-derived DVR is reproducible and suitable for studying D2R availability in the rat brain. MicroPET sensitivity was sufficient to determine reproducible DVRs from 11C-raclopride injections of 9.25 MBq (250 µCi). However, the effect of tracer mass on the DVR should be considered for studies using more than 1–2 nmol/kg raclopride, and scatter correction has a measurable impact on the results.
Key Words: microPET • rat brain • dopamine receptor • raclopride
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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The feasibility of using specially designed, dedicated animal PET cameras to study dopaminergic function in the rodent brain was first investigated >5 y ago using a custom-built tomograph with a spatial resolution of 3- to 4-mm full width at half maximum (FWHM) near the center (7). This and more recent studies of the rat D2R system (8,14,15) have demonstrated that custom-built, high-resolution PET cameras can give reproducible data that are highly correlated with established ex vivo and postmortem techniques. With the appearance of a new generation of commercial animal PET cameras, the need for similar feasibility studies is apparent. Recently, our laboratory acquired the microPET R4 (Concorde Microsystems), a high-resolution tomograph designed especially for rodent imaging. Although a performance evaluation of the microPET P4—the machine identical to the model R4 except with a larger system radius—was recently published (1), little information is available on the practical application of commercial microPET to characterizing dopamine receptor function in the rat brain. The goal of this work was to evaluate the application of the microPET R4 to D2R imaging in the rat brain using 11C-raclopride. Specifically, we address the reproducibility of 11C-raclopride binding and determinations of the distribution volume ratio (DVR) used to assess D2R availability. The reproducibility was determined using a test-retest paradigm, thus extending the work of earlier studies that describe primarily the reproducibility of kinetic measurements within a group of rats (7,13,15–17). In addition, to address the impact of 2-mm resolution of modern small-animal PET cameras, we compared measures of binding potential (DVR-1) and striatum/cerebellum (ST/CB) ratios to previously published results using lower resolution machines.
Finally, it was important to validate the quantitative capabilities of the commercial microPET, especially with respect to corrections for photon attenuation and scatter that are not yet functional in the vendor software. Although attenuation correction factors for both the rat striatum and cerebellum have been determined previously (17), little information is available on the contribution of scatter to PET measurements in the rat striatum and cerebellum. Hence, for these studies, we focused on the effect of scatter correction on dynamic microPET measurements of 11C-raclopride in the rat brain.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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11C-Raclopride Preparation
11C-Raclopride was synthesized according to the method described previously (18). 11C-Raclopride doses were small fractions of routine syntheses prepared for human subject studies. Specific activity determination was made using mass measurements acquired during radiotracer purification by high-performance liquid chromatography (HPLC) (Novapak C18, 250 x 10 mm; Waters, Milford, MA) and radioactivity measurements obtained with a calibrated ion chamber (Capintec, Inc., Ramsey, NJ).
PET
Imaging was performed using the microPET R4 tomograph (Concorde Microsystems), which has a 12-cm animal port with an image field of view (FOV) of 11.5 cm. Three animals received a single injection of 11C-raclopride, whereas 4 other animals received a double injection of 11C-raclopride to assess test-retest reproducibility (see below). Each animal was positioned prone on the microPET bed, centering the brain in the FOV. The rat head was supported and secured to the bed to approximate a flat-skull orientation (19) in the camera FOV. Fully 3-dimensional listmode data were collected for 60 min using an energy window of 250–750 keV and a time window of 10 ns (default settings recommended by the manufacturer). MicroPET data acquisition was started simultaneously with 11C-raclopride injection. The range in dose of 11C-raclopride injected for all studies was 6.11–18.54 MBq (165–501 µCi). The specific activity at the time of injection varied from a minimum of 4.07 GBq/µmol (0.11 Ci/µmol) to a maximum of 48.1 GBq/µmol (1.3 Ci/µmol). This corresponded to a range of injected raclopride mass of 0.5–15 nmol/kg. For maximum sensitivity, coincidence data were binned into 3-dimensional sinograms using the full axial acceptance angle of the scanner. To preserve axial resolution, high sampling of the polar angle was used (21 segments). The binning produced 24 time frames (6 x 10 s, 3 x 20 s, 8 x 60 s, 4 x 300 s, and 3 x 600 s) and included subtraction of random coincidences collected in a delayed time window. The resulting sinograms were then rebinned using Fourier rebinning and reconstructed with 2-dimensional filtered backprojection using a ramp filter with cutoff at Nyquist via software provided by the manufacturer. Image pixel size was 0.85 mm transaxially with a 1.21-mm slice thickness.
Test-Retest Experiments
In 3 animals, injections of 11C-raclopride were paired to address the reproducibility of microPET imaging in the same animal on the same day. In a fourth animal, raclopride (1 mg/kg) was coinjected with the second injection of 11C-raclopride. The time between 11C-raclopride injections varied from 2 to 4 h. Anesthesia was maintained during this time by intraperitoneal injection of ketamine/xylazine. The depth of anesthesia was monitored using respiration rate and whisker twitch.
Scatter Correction
For the scatter-corrected results, scattered events were subtracted from randoms-corrected sinograms based on a quadratic polynomial fit of projection data from outside the head in a manner similar to previously published tail-fitting methods (20,21). For improved statistics of the fit, radial projections from all azimuthal angles in the sinogram were summed. Thus, the same curve was subtracted from each projection within a given slice, although independent fits were made for each different slice. Because of the large transaxial FOV, it was possible to use conservative, fixed radial limits for the fit that excluded all possible true coincidences. Scatter-corrected sinograms were then subjected to the reconstruction methods described above. Note that this approach has the advantages of accounting for scatter from outside the FOV as well as any other potential sources of uniform background in the data.
Image Analysis
Region-of-interest (ROI) analysis was performed using vendor software (ASI Pro 1.1; Concorde Microsystems) on reconstructed images with and without scatter correction for each study. A circular ROI with an area of 15 pixels was drawn on the left striatum and right striatum from a single microPET image plane. Typically the striatum was delineated on 4 different coronal planes. One of 2 center planes was chosen for ROI analysis to minimize axial partial-volume effects. A 29-pixel elliptic ROI was drawn on the cerebellum from a single coronal plane. The ROI for the cerebellum was typically 9 planes caudal to the striatum, corresponding to a distance of 11 mm.
The harderian glands were used as anatomic landmarks to confirm identification of the correct plane for the striatum. The ROI for the striatum was most often located on the fifth coronal plane caudal to the last plane containing the harderian glands. This internal landmark was particularly useful for confirming proper ROI placement in low-contrast (low specific activity) studies. Left and right striatum data were averaged to give a single time-activity curve for the striatum. The DVR was computed from time-activity curves obtained from ROI analysis of both scatter-corrected and uncorrected images using the graphical analysis method without blood sampling (22).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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11% from 3.36 to 3.74 after scatter correction. Study darat2 was excluded from the ratio computation because it was a blocking study with 1 mg/kg raclopride. Study darat1 was also excluded because of low specific activity (15 nmol/kg).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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3 cm in diameter). Furthermore, image attenuation corrections determined previously for the striatum and cerebellum in adult Sprague-Dawley rats using a custom-built animal PET camera (17) were found to be the same (16%). Because the data analysis methods used here (ST/CB and DVR) rely on the comparison (ratio) of a D2R-rich region (striatum) with a region used to estimate nonspecific binding (cerebellum), a constant attenuation correction factor for the striatum and cerebellum can be ignored. Scatter, on the other hand, manifests itself more as an image offset and, thus, can potentially have a much larger effect, particularly for low-activity reference regions such as the cerebellum.
No estimates of the background from photon scatter and its impact on the determination of ST/CB and DVR using the microPET R4 with 11C-raclopride were available. With a diameter (3 cm) that is small compared with the mean free path of a 511-keV photon in tissue (10 cm), the adult rat head was expected to give microPET data with relatively small scatter fractions. However, the lack of septa in the microPET and its large axial FOV increase the probability that sinogram scatter fractions might be appreciable, especially because of activity outside the FOV. Indeed, our typical measured scatter fractions of 41% in early time frames are larger than those from the Concorde P4 primate scanner (32% for a 5-cm-diameter "rat" phantom (1)), which is consistent with an axial acceptance angle almost double that of the P4. The tail-fitting scatter correction algorithm produced a small but consistent effect on the DVR, increasing the DVR an average of 3.5% with a maximum of 10%. ROI data showed a more significant effect, especially when derived from brain regions of lower accumulation of tracer radioactivity such as the cerebellum, where mean ROI pixel values from scatter-corrected images decreased as much as 20% compared with uncorrected image data.
An interesting observation is the significant increase in sinogram background from the beginning of the study to the end. If related to scatter, it is presumably caused by a redistribution of radioactivity from inside to outside the FOV. At the low activity levels near the end of a scan, other low-level sources of uniform background might also contribute, such as an imperfect randoms correction or the natural radioactivity of lutetium oxyorthosilicate (LSO). A full explanation of this effect is under investigation but, regardless of the cause, it is significant and demonstrates the need to incorporate a background subtraction technique into the recommended configuration of the microPET R4 to obtain accurate tracer kinetic information.
Because at least 1 dimension of a brain structure (e.g., striatum) studied in this work is near (2 x FWHM) the spatial resolution of the scanner, measurements of radioactivity concentration in this region will be underestimated due to the partial-volume effect (24). An appreciation of the magnitude of partial-volume errors can be gleaned from comparisons with ex vivo measurements. For example, the ST/CB of 4 determined at 60 min after 11C-raclopride injection can be compared with ex vivo data—dissected brain regions counted in a well counter—giving ratios of 9 (7). This partial-volume error in ST/CB can be attributed primarily to errors in striatal ROI values because the cerebellum, a brain region of nonspecific 11C-raclopride binding, is surrounded by tissue of similar uptake, thereby masking the effect of spillover activity from surrounding tissue. The effects of partial volume on the ST/CB can also be observed when comparing data from PET scanners of different spatial resolutions. For example, the measurement of higher ST/CB ratios in this study (4) compared with values reported by Hume et al. (7) (2.5) is consistent with an improved radioactivity recovery that can be expected with the higher spatial resolution of the microPET R4 (2 mm), given similar ROI sizes.
Contamination of radioactivity from one region to a nearby region (spillover) due to the partial-volume effect will also give rise to errors in ROI radioactivity concentration determinations. Spillover errors in rat brain imaging can be of special concern because of the existence of 2 discrete extracerebral structures just anterior to the brain. These structures, the harderian glands, often exhibit high radioactivity uptake that can result in significant spillover errors in nearby brain regions, such as the prefrontal cortex (25). With the advent of new high-resolution animal cameras such as the microPET R4, this problem has diminished (14,26–28). For example, Nikolaus et al. (14), using the specific D2R ligand 18F-N-methyl-benperidol and a high-resolution PET camera (2.1-mm FWHM), determined the spillover error in the striatum from harderian gland uptake to be 5%.
The magnitude of the spillover errors to the striatum is not just a function of the distance between structures relative to the camera resolution but also depends on the relative uptake in the 2 regions and, therefore, must be determined for each tracer. Meyers et al. (17) and Lammertsma et al. (29) have determined the spillover fraction from the harderian glands to the striatum for 11C-raclopride in a rat imaged with a clinical human camera (FWHM = 5.5 mm) to be 0%–3.5% in control binding experiments. The higher resolution of the microPET R4 reduces this fraction below 3.5% and, therefore, this effect should be negligible in our studies.
A primary goal of this work was to measure the test-retest reproducibility of 11C-raclopride binding in the rat brain as reflected in changes in the DVR. The average change in the DVR of 8% observed in this work is similar to the reproducibility obtained in humans (30) and baboons (31) and, therefore, suggests that microPET imaging can be successfully used to study the D2R system in the rat brain. However, the large variability of the test-retest change, including the observation of a -13% change in the second paired control study, suggests that determinations of the DVR in rat brain may be more sensitive to PET experimental conditions than primate imaging performed using a standard clinical camera. Although increased noise from the use of a low injected dose (6.85 MBq [185 µCi]) may be contributing to the larger test-retest error in the second paired study (Fig. 2B), the observation that all 3 retest studies gave a lower DVR value suggests that there may be a bias in the retest experiment.
Potential PET experimental conditions creating a bias in the retest data include tracer-specific activity and anesthesia. Two of the 3 retest experiments, including the study giving the largest DVR change, had a lower raclopride mass administered to the animal, so a consistently smaller retest DVR cannot be explained by specific activity differences. The other important experimental condition that is significantly different from PET studies with primates is the method of anesthesia used in this work. The average dose of ketamine (intraperitoneal) used in this study was 85 mg/kg/h. Tsukada et al. (32) have shown that ketamine can reduce raclopride binding in the primate brain in a dose-dependent manner for 3–10 mg/kg/h given intravenously. The lower retest DVR observed in this work is consistent with the fact that retest experiments are performed (2–4 h later) after a larger ketamine dose has been administered to the animal and, therefore, may show a larger decrease in 11C-raclopride binding.
The reproducibility of the DVR of 11C-raclopride measured in 6 different rats is similar to data reported by Myers et al. (17) for a similar range of specific activity. These authors determined the binding potential (DVR-1) in 15 rats to be 0.79 ± 0.09 (coefficient of variation [CV] = 11%) using a small-animal PET camera (20). In this study, the average DVR for 6 rats was 2.43 ± 0.19 (CV = 8%). This gives an average binding potential of 1.43 ± 0.19 (CV = 13%). The variability of the binding potential measured here is similar to published data even with a relatively small sample size.
The average binding potential of 1.43 determined is almost 2 times the binding potential reported by Hume et al. (7). Assuming similar ROI sizes, this is consistent with an improved radioactivity recovery in the striatum due to a decrease in the partial-volume error associated with the higher resolution of the microPET R4. The dynamic range, or specific binding window, also increased by at least a factor of 2 in this work because blocking studies gave similar binding potentials. Even if a smaller ROI size (resulting in improved radioactivity recovery) underlies the determination of a higher binding potential in this work, the fact remains that reproducible data were obtained with 30-pixel ROIs, giving a robust change in binding potential (0.2–1.4). This result confirms that the microPET R4 has both sufficient sensitivity and resolution to perform experiments of the D2R system in the rat brain using the DVR.
Much attention has been given to the issue of tracer-specific activity and the limits it puts on high-resolution PET imaging of small laboratory animals, particularly in mice (11,23,33). It has been suggested that a receptor occupancy of 1% is required to satisfy tracer kinetic modeling (23,34). Morris and London (35) have simulated studies with primates and have concluded that a dose of raclopride of 0.5 nmol/kg was required before the binding potential was compromised. This dose corresponds to a receptor occupancy of 3% using the analysis of Hume et al. (23). Although a rigorous saturation study was not performed in this work, the specific activity of 11C-raclopride used varied by an order of magnitude. Figure 4 clearly shows an effect of specific activity (raclopride mass) on the measured DVR. From these data and a simple grouping of studies using a mesh of 9% occupancy, a minimum threshold for raclopride mass can be obtained. Under the present experimental conditions, this threshold is <1.5 nmol/kg raclopride. The mean DVR (n = 4) obtained with raclopride doses <1.5 nmol/kg was significantly different (P < 0.05) than that obtained from studies in the range of 1.5–3.7 nmol/kg (n = 3). Furthermore, within the high specific activity group (<1.5 nmol/kg) there was no correlation between raclopride mass and DVR. Previous work has demonstrated a significant change in experimentally determined binding potential in a rat when raclopride mass is in the range of 1 nmol/kg (13) to 5 nmol/kg (17).
Although this threshold does not address the issue of pharmacologic response and receptor occupancy, it does establish a guideline for microPET R4 rat brain experiments given the experimental conditions of anesthesia, camera performance, and 11C-raclopride specific activity used in these investigations. Extension of these findings to possible microPET R4 determinations of the DVR in the mouse brain, however, must be made cautiously. In fact, recent results in our laboratory have shown that, although it was possible to observe differences in both DVR and ST/CB between groups of wild-type and D2 knockout mice using the microPET R4, the average D2R occupancy in the mouse brain was estimated to be 50% (11). Therefore, further investigations must be performed before the usefulness of microPET R4 imaging of D2R function in the mouse brain with 11C-raclopride can be established.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Custom scatter correction software based on fitting of data outside the object was easy to implement and improved specific measures of D2R availability, including the ST/CB ratio and DVR, although the average improvement in the DVR was small (3.5%). Overall test-retest reproducibility of the scatter-corrected data (average = -8%) is similar to results obtained in baboons and humans using clinical PET and, therefore, should be sufficient for many types of drug challenge studies. Reduced variability is expected using larger sample sizes, although more studies may be needed to assess the contribution of the possible interaction of ketamine anesthesia with dopaminergic function (32) on test-retest reproducibility.
A threshold for raclopride mass below which a change in the DVR could not be measured was determined experimentally to be 1.5 nmol/kg. This threshold provides a useful guideline for carrying out microPET R4 experiments under the specific experimental conditions of anesthesia, camera performance, and 11C-raclopride specific activity described in this work and is similar to results obtained using other small-animal PET cameras (13).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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For correspondence or reprints contact: David L. Alexoff, BSE, Brookhaven National Laboratory, Building 555, Upton, NY 11973-5000.
E-mail: alexoff{at}bnl.gov
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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| JOURNAL OF NUCLEAR MEDICINE TECHNOLOGY | THE JOURNAL OF NUCLEAR MEDICINE |
, First injection; 
and 
, Second injection. (A) First injection, 12.84 MBq (347 µCi; 3.8 nmol/kg raclopride); second injection, 11.91 MBq (322 µCi; 1.9 nmol/kg raclopride). (B) First injection, 7.22 MBq (195 µCi; 1.2 nmol/kg raclopride); second injection, 6.51 MBq (176 µCi; 1.0 nmol/kg raclopride). (C) First injection, 5.99 MBq (162 µCi; 0.5 nmol/kg raclopride); second injection, 13.88 MBq (375 µCi; 1.7 nmol/kg raclopride). (D) Blocking study: first injection, 17.58 MBq (475 µCi; 15 nmol/kg raclopride); second injection, 18.54 MBq (501 µCi; 1 mg/kg or 2,881 nmol/kg raclopride).
