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Unlabelled: Human mesenchymal stem cells (hMSCs) represent a promising treatment approach for tissue repair and regeneration. However, little is known about the underlying mechanisms and the fate of the transplanted cells. The objective of the presented work was to determine the feasibility of PET imaging and in vivo monitoring after transplantation of dopamine type 2 receptor-expressing cells., Methods: An hMSC line constitutively expressing a mutant of the dopamine type 2 receptor (D2R80A) was generated by lentiviral gene transfer. D2R80A messenger RNA expression was confirmed by reverse transcriptase-polymerase chain reaction. Localization of the transmembrane protein was analyzed by confocal fluorescence microscopy. The stem cell character of transduced hMSCs was investigated by adipogenic and osteogenic differentiation. Migration capacity was assessed by scratch assays in time-lapse imaging. In vitro specific binding of ligands was tested by fluorescence-activated cell sorting analysis and by radioligand assay using (18)F-fallypride. Imaging of D2R80A overexpressing hMSC transplanted into athymic rats was performed by PET using (18)F-fallypride., Results: hMSCs showed long-term overexpression of D2R80A. As expected, the fluorescence signal suggested the primary localization of the protein in the membrane of the transduced cells. hMSC and D2R80A retained their stem cell character demonstrated by their osteogenic and adipogenic differentiation capacity and their proliferation and migration behavior. For in vitro hMSCs, at least 90% expressed the D2R80A transgene and hMSC-D2R80A showed specific binding of (18)F-fallypride. In vivo, a specific signal was detected at the transplantation site up to 7 d by PET., Conclusion: The mutant of the dopamine type 2 receptor (D2R80A) is a potent reporter to detect hMSCs by PET in vivo., (© 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Unlabelled: Data collection in preclinical small-animal PET studies has been hindered by the small number of recordings typically obtained for a single radiosynthesis. Therefore, we tested procedures for obtaining 8 simultaneous small-animal PET recordings from the brains of 8 mice using an acrylic anesthesia distributor (the Octamouse), with the dopamine D(2/3) ligand (18)F-fallypride serving as a test substance for brain receptor imaging., Methods: The effect of scatter correction on the small-animal PET recordings was first evaluated in phantom studies in which sources of different radioactivity concentration were placed within the chambers of the Octamouse. Next, potential effects of mass on the (18)F-fallypride binding potential (BP(ND)) in the striatum were tested in groups of mice receiving (18)F-fallypride at 2 different specific activities (140 and 50 GBq/μmol), with and without scatter correction. Finally, the relationship between BP(ND) and injected dose of (18)F-fallypride (3.5-17 MBq/mouse) was tested., Results: Scatter correction improved the contrast between sources and air space within the Octamouse phantom. The magnitude of (18)F-fallypride BP(ND) in mouse striatum was invariant across the tested range of specific activities, and scatter correction increased BP(ND) by a mean of 6%; covariances of the inter- and intraoperator variability of BP(ND) were 10%. There was a positive correlation between radiochemical dose and BP(ND) with (R(2) = 0.53) and without (R(2) = 0.63) scatter correction, which was driven by increasing area under the percentage injected dose curve in the striatum., Conclusion: The quantitation of emission sources placed within the Octamouse is linear over a wide range of source activities. In the striatum of living mice, the magnitude of (18)F-fallypride BP(ND) was highly reproducible between operators and was constant over a 3-fold range of specific activities, indicating a lack of significant occupancy. Scatter correction improved quantitation but did not entirely correct for the dependence of BP(ND) on injected dose, which was deemed to arise because of effects propagating from detector dead time when the total radiochemical dose in the field of view exceeded 50 MBq. Given this consideration, we were still able to quantify (18)F-fallypride BP(ND) in 16 mice from a single radiosynthesis, an economy that should be generalizable to brain studies of diverse radioligands.

Aim: To test for alpha(2) adrenergic modulation of dopamine D(2/3) receptor availability in striatum of living mice using the high-affinity ligand [(18)F]fallypride and microPET., Methods: Groups of anesthetized mice were pretreated with saline, the alpha(2)-agonist clonidine (1 mg/kg), and the alpha(2)-antagonists RX821002 (1 mg/kg) and yohimbine (1 mg/kg). Dynamic microPET recordings lasting 120 min were then initiated upon i.v. tracer injection of [(18)F]fallypride. Parametric maps of [(18)F]fallypride binding potential (BP(ND)) were calculated using the Logan method, with cerebellum serving as the reference region., Results: Mean striatal [(18)F]fallypride BP(ND) was 10.6 +/- 1.7 in the saline control animals, 8.9 +/- 1.7 (-16%; P < 0.05) in the RX821002 group, 8.3 +/- 2.6 (-22%; P < 0.05) in the yohimbine group and 10.3 +/- 2.2 (n.s.) in the clonidine group., Conclusions: These findings are consistent with a tonic inhibition of dopamine release by alpha(2) adrenergic receptors, such that alpha(2) blockade increased the competition from endogenous dopamine at D(2/3) receptors, thus reducing the [(18)F]fallypride BP(ND) by about 20%. Absent effects of clonidine suggest a ceiling effect in the tonic inhibition of dopamine release. This in vivo PET evidence for alpha(2)/dopaminergic interaction may be relevant to putative actions of atypical antipsychotic medications via adrenergic receptors.

Aim: Molecular imaging studies with benzamide radioligands can reveal competition from endogenous binding at D(2/3)-receptors in living brain. However, single photon emission computed tomography (SPECT) methods suffer from limited spatial resolution, and [(11)C]-labeled ligands are only available at positron emission tomography (PET) research sites with cyclotron-radiochemistry facilities, whereas [(18)F] can be transported, due to its longer physical half-life. Therefore, we endeavored to characterize the vulnerabilities of the benzamide antagonist [(18)F]desmethoxyfallypride (DMFP) and its high-affinity congener [(18)F]fallypride (FP) to competition from endogenous dopamine in living mouse brain., Methods: Groups of awake mice were pretreated with saline, amphetamine (10 mg/kg), or reserpine (5 mg/kg), followed by i.v. tracer injections. Mice were killed at 2.5-90 min (DMFP) or 2.5-180 min (FP) circulation times. Brains were dissected and regional radioactivity concentration measured by gamma counting. Other groups of mice were anesthetized for dynamic microPET recordings with DMFP or FP. Binding potentials (BP(ND)) were calculated using cerebellum as reference region., Results: With 90-min circulation, DMFP BP(ND) in striatum was 2.4 by dissection and 2.2 by microPET, which showed a 62% decrease in response to amphetamine-evoked dopamine release and a 33% increase after reserpine-evoked dopamine depletion. With 120-min circulation, FP BP(ND) in striatum was 24.1 by dissection and 9.2 by microPET, which showed a 31% decrease in the amphetamine group, but no effect of reserpine. Dissection showed similar sensitivities for FP binding, but only a 29% amphetamine-evoked reduction for DMFP., Conclusions: Relative to gold standard ex vivo results, microPET estimates of DMFP BP(ND) were unbiased, whereas FP BP(ND) in striatum was substantially underestimated. Both tracers proved suitable for revealing pharmacologically evoked changes in competition at D(2/3)-receptors in striatum of living mice.