Paul D. Acton

Small-Animal SPECT and SPECT/CT: Important Tools for Preclinical Investigation

The need to study dynamic biologic processes in intact small-animal models of disease has stimulated the development of high-resolution nuclear imaging methods. These methods are capable of clarifying molecular interactions important in the onset and progression of disease, assessing the biologic relevance of drug candidates and potential imaging agents, and monitoring therapeutic effectiveness of pharmaceuticals serially within a single-model system. Single-photon–emitting radionuclides have many advantages in these applications, and SPECT can provide 3-dimensional spatial distributions of γ- (and x-) ray–emitting radionuclide imaging agents or therapeutics. Furthermore, combining SPECT with CT in a SPECT/CT system can assist in defining the anatomic context of biochemical processes and improve the quantitative accuracy of the SPECT data. Over the past decade, dedicated small-animal SPECT and SPECT/CT systems have been developed in academia and industry. Although significant progress in this arena has been realized through system development and biologic application, further innovation continues to address challenges in camera sensitivity, spatial resolution, and image reconstruction and quantification. The innumerable applications of small-animal SPECT and SPECT/CT in drug development, cardiology, neurology, and oncology are stimulating further investment in education, research, and development of these dedicated small-animal imaging modalities.

Limbic selectivity of clozapine

6motile sperm cells per mL, the inclusion criterion for the use of ICSI in the Netherlands. Both oligozoospermic (n=69) and azoospermic (n=11) men were included. All patients were karyotyped, and a consecutive subgroup (n=58) was also tested for Y-chromosomal deletions and CFTR mutations. We found an abnormal karyotype in peripheral blood lymphocytes in seven (8·8%) of 80 patients, ten-fold more than the overall population incidence of 0·85% 2 (p<0·001, 2 test; table). We analysed Y-chromosomal deletions with a multiplex PCR system with markers for the AZF-a, AZF-b, and AZF-c regions. 3 With markers sY254 and sY255, three (5%) of 58 patients were found to have an AZF-c deletion associated with the deleted-in-azoospermia (DAZ) gene. No AZF deletions were detected with this assay in a study of 100 fertile male controls. 3

2-((2-((dimethylamino)methyl)phenyl)thio)-5-iodophenylamine (ADAM): an improved serotonin transporter ligand

Serotonin transporters (SERT) are target-sites for commonly used antidepressants, such as fluoxetine, paroxetine, sertraline, and so on. Imaging of these sites in the living human brain may provide an important tool to evaluate the mechanisms of action as well as to monitor the treatment of depressed patients. Synthesis and characterization of an improved SERT imaging agent, ADAM (2-((2-((dimethylamino)methyl)phenyl)thio)-5-iodophenylamine)(7) was achieved. The new compound, ADAM(7), displayed an extremely potent binding affinity toward SERT ( K(i)=0.013 nM, in membrane preparations of LLC-PK(1)-cloned cell lines expressing the specific monoamine transporter). ADAM(7) also showed more than 1,000-fold selectivity for SERT over norepinephrine transporter (NET) and dopamine transporter (DAT) ( K(i)=699 and 840 nM, for NET and DAT, respectively). The radiolabeled compound [(125)I]ADAM(7) showed an excellent brain uptake in rats (1.41% dose at 2 min post intravenous [IV] injection), and consistently displayed the highest uptake (between 60-240 min post IV injection) in hypothalamus, a region with the highest density of SERT. The specific uptake of [(125)I]ADAM(7) in the hypothalamus exhibited the highest target-to-nontarget ratio ([hypothalamus - cerebellum]/cerebellum was 3.97 at 120 min post IV injection). The preliminary imaging study of [(123)I]ADAM in the brain of a baboon by single photon emission computed tomography (SPECT) at 180-240 min post IV injection indicated a specific uptake in midbrain region rich in SERT. These data suggest that the new ligand [(123)I]ADAM(7) may be useful for SPECT imaging of SERT binding sites in the human brain.

Pinhole SPECT of mice using the LumaGEM gamma camera

LumaGEM is a newly developed gamma camera for dedicated, small field of view, high spatial resolution imaging. The system consists of an array of 2/spl times/2/spl times/6 mm/sup 3/ NaI(Tl) pixels coupled to an array of position-sensitive photomultiplier tubes. It has a 125/spl times/125 mm/sup 2/ field of view. A pinhole collimator was used on LumaGEM to acquire SPECT images of mice that had transgenic modifications so as to model various diseases. Pinhole apertures of 1, 2 and 3 mm are interchangeable on the collimator and were used to acquire images. An iterative MLEM algorithm for pinhole SPECT was used to reconstruct the 128 projection images that covered 360/spl deg/ rotation. The reconstruction algorithm is based on a projector and backprojector pair implemented using a ray-tracing algorithm. The crucial reconstruction input parameters are the radius of rotation, center of rotation, and pinhole focal length. Ideal pinhole geometry is assumed, and no correction for attenuation has been made. The preliminary images presented here show detailed uptake in the mice subjects and are a convincing sign that animal SPECT can reach submillimeter spatial resolution and be a valuable tool in the study of diseases and the development of pharmaceuticals in animal models.

Small animal imaging with high resolution single photon emission tomography.

Molecular imaging of small animals in vivo is vital in the study of mouse and rat models of human diseases, and will provide important clues to the pathogenesis, progression and treatment of many disorders. Functional imaging of small animals using ultra-high resolution single photon emission tomography (SPECT) should be a valuable tool in the molecular imaging armamentarium. SPECT has been used to study cerebral binding sites, to image the expression of reporter genes, and in applications in cardiology and oncology. In this review, we summarize the most recent developments in SPECT imaging of small animals, with particular reference to the types of systems available, their application, and some of the potential limitations.

Optimal number of pinholes in multi-pinhole SPECT for mouse brain imaging--a simulation study.

This study simulates a multi-pinhole single-photon emission computed tomography (SPECT) system using the Monte Carlo method, and investigates different multi-pinhole designs for quantitative mouse brain imaging. Prior approaches investigating multi-pinhole SPECT were not often optimal, as the number and geometrical arrangement of pinholes were usually chosen empirically. The present study seeks to optimize the number of pinholes for a given pinhole arrangement, and also for the specific application of quantitative neuroreceptor binding in the mouse brain. An analytical Monte Carlo simulation based method was used to generate the projection data for various count levels. A three-dimensional ordered-subsets expectation-maximization algorithm was developed and used to reconstruct the images, incorporating a realistic pinhole model for resolution recovery and noise reduction. Although artefacts arising from overlapping projections could be a major problem in multi-pinhole reconstruction, the cold-rod phantom study showed minimal loss of spatial resolution in multi-pinhole systems, compared to a single-pinhole system with the same pinhole diameter. A quantitative study of neuroreceptor binding sites using a mouse brain phantom and low activity (37 MBq) showed that the multi-pinhole system outperformed the single-pinhole system by maintaining the mean and lowering the variance in the measured uptake ratio. Multi-pinhole collimation can be used to reduce the injected dose and thereby reduce the radiation exposure to the animal. Results also suggest that the nine-pinhole configuration shown in this paper is a good choice for mouse brain imaging.

Selective in vitro and in vivo binding of [125I]ADAM to serotonin transporters in rat brain

An improved iodinated tracer, ADAM (2‐((2‐((dimethylamino)methyl)‐ phenyl)thio)‐5‐iodophenylamine) for imaging serotonin transporters (SERT) with single photon emission computerized tomography (SPECT), was prepared and characterized. Scatchard analysis of saturation binding of [125I]ADAM to rat frontal cortical membrane homogenates gave a Kd value of 0.15 ± 0.03 nM and a Bmax value of 194 ± 65 fmol/mg protein. Biodistribution of [125I]ADAM in rat brain after an iv injection showed a high specific binding in the regions of hypothalamus, cortex, striatum, and hippocampus, where SERT are concentrated and the specific binding peaked at 120–240 min postinjection [(hypothalamus‐cerebellum)/cerebellum = 4.3 at 120 min post‐iv injection]. Moreover, the specific hypothalamic uptake was blocked by pretreatment with SERT selective competing drugs, such as paroxetine and (+)McN5652, while other noncompeting drugs, such as ketanserin, raclopride, and methylphenidate, showed no effect. The radioactive material recovered from rat brain homogenates at 120 min after [125I]ADAM injection showed primarily the original compound (>90%), a good indication of in vivo stability in the brain tissues. Both male and female rats showed similar and comparable organ distribution pattern and regional brain uptakes. Ex vivo autoradiograms of rat brain sections (120 min after iv injection of [125I]ADAM) showed intense labeling in several regions (olfactory tubercle, lateral septal nucleus, hypothalamic and thalamic nuclei, globus pallidus, central gray, superior colliculus, substantia nigra, interpeduncular nucleus, dorsal and median raphes, and locus coerulus), which parallel known SERT density. These results strongly suggest that the novel tracer ADAM is superior to the congers (i.e., IDAM) reported previously. When labeled with I‐123, ADAM will be an improved and useful SPECT imaging agent for SERT in the brain. Synapse 38:403–412, 2000.

Characterization of [123I]IDAM as a novel single-photon emission tomography tracer for serotonin transporters

Abstract. Development of selective serotonin transporter (SERT) tracers for single-photon emission tomography (SPET) is important for studying the underlying pharmacology and interaction of specific serotonin reuptake site inhibitors, commonly used antidepressants, at the SERT sites in the human brain. In search of a new tracer for imaging SERT, IDAM (5-iodo-2-[[2-2-[(dimethylamino)methyl]phenyl]thio]benzyl alcohol) was developed. In vitro characterization of IDAM was carried out with binding studies in cell lines and rat tissue homogenates. In vivo binding of [125I]IDAM was evaluated in rats by comparing the uptakes in different brain regions through tissue dissections and ex vivo autoradiography. In vitro binding studyshowed that IDAM displayed an excellent affinity to SERT sites (Ki=0.097 nM, using membrane preparations of LLC-PK1 cells expressing the specific transporter) and showed more than 1000-fold of selectivity for SERT over norepinehrine and dopamine (expressed in the same LLC-PK1 cells). Scatchard analysis of [125I]IDAM binding to frontal cortical membrane homogenates prepared from control or p-chloroamphetamine (PCA)-treated rats was evaluated. As expected, the control membranes showed a Kd value of 0.25 nM±0.05 nM and a Bmax value of 272±30 fmol/ mg protein, while the PCA-lesioned membranes displayed a similar Kd, but with a reduced Bmax (20±7 fmol/ mg protein). Biodistribution of[125I]IDAM (partition coefficient =473; 1-octanol/buffer) in the rat brainshowed a high initial uptake (2.44%dose at 2 min after i.v. injection) with the specific binding peaked at 60 min postinjection (hypothalamus-cerebellum/cerebellum =1.75). Ex vivo autoradiographs of rat brain sections (60 min after i.v. injection of [125I]IDAM) showed intense labeling in several regions (olfactory tubercle, lateral septal nucleus, hypothalamic and thalamic nuclei, globus pallidus, central gray, superior colliculus, substantia nigra, interpeduncular nucleus, dorsal and median raphes and locus coeruleus), which parallel known SERT density. This novel tracer has excellent characteristics for in vivo SPET imaging of SERT in the brain.

Automatic segmentation of dynamic neuroreceptor single-photon emission tomography images using fuzzy clustering

Abstract. The segmentation of medical images is one of the most important steps in the analysis and quantification of imaging data. However, partial volume artefacts make accurate tissue boundary definition difficult, particularly for images with lower resolution commonly used in nuclear medicine. In single-photon emission tomography (SPET) neuroreceptor studies, areas of specific binding are usually delineated by manually drawing regions of interest (ROIs), a time-consuming and subjective process. This paper applies the technique of fuzzy c-means clustering (FCM) to automatically segment dynamic neuroreceptor SPET images. Fuzzy clustering was tested using a realistic, computer-generated, dynamic SPET phantom derived from segmenting an MR image of an anthropomorphic brain phantom. Also, the utility of applying FCM to real clinical data was assessed by comparison against conventional ROI analysis of iodine-123 iodobenzamide (IBZM) binding to dopamine D2/D3 receptors in the brains of humans. In addition, a further test of the methodology was assessed by applying FCM segmentation to [123I]IDAM images (5-iodo-2-[[2-2-[(dimethylamino)methyl]phenyl]thio] benzyl alcohol) of serotonin transporters in non-human primates. In the simulated dynamic SPET phantom, over a wide range of counts and ratios of specific binding to background, FCM correlated very strongly with the true counts (correlation coefficient r2>0.99, P<0.0001). Similarly, FCM gave segmentation of the [123I]IBZM data comparable with manual ROI analysis, with the binding ratios derived from both methods significantly correlated (r2=0.83, P<0.0001). Fuzzy clustering is a powerful tool for the automatic, unsupervised segmentation of dynamic neuroreceptor SPET images. Where other automated techniques fail completely, and manual ROI definition would be highly subjective, FCM is capable of segmenting noisy images in a robust and repeatable manner.

Simplified quantification of small animal [18F]FDG PET studies using a standard arterial input function

PurposeArterial input function (AIF) measurement for quantification of small animal PET studies is technically challenging and limited by the small blood volume of small laboratory animals. The present study investigated the use of a standard arterial input function (SAIF) to simplify the experimental procedure.MethodsTwelve [18F]fluorodeoxyglucose ([18F]FDG) PET studies accompanied by serial arterial blood sampling were acquired in seven male Sprague-Dawley rats under isoflurane anaesthesia without (every rat) and with additional (five rats) vibrissae stimulation. A leave-one-out procedure was employed to validate the use of a SAIF with individual scaling by one (1S) or two (2S) arterial blood samples.ResultsAutomatic slow bolus infusion of [18F]FDG resulted in highly similar AIF in all rats. The average differences of the area under the curve of the measured AIF and the individually scaled SAIF were 0.11±4.26% and 0.04±2.61% for the 1S (6-min sample) and the 2S (4-min/43-min samples) approach, respectively. The average differences between the cerebral metabolic rates of glucose (CMRglc) calculated using the measured AIF and the scaled SAIF were 1.31±5.45% and 1.30±3.84% for the 1S and the 2S approach, respectively.ConclusionThe use of a SAIF scaled by one or (preferably) two arterial blood samples can serve as a valid substitute for individual AIF measurements to quantify [18F]FDG PET studies in rats. The SAIF approach minimises the loss of blood and should be ideally suited for longitudinal quantitative small animal [18F]FDG PET studies.