Ultra-high-field pharmacological functional MRI of dopamine D1 receptor-related interventions in anesthetized rats.

The dopamine D1 receptor (D1R) is associated with schizophrenia, Parkinson's disease, and attention deficit hyperactivity disorder. Although the receptor is considered a therapeutic target for these diseases, its neurophysiological function has not been fully elucidated. Pharmacological functional MRI (phfMRI) has been used to evaluate regional brain hemodynamic changes induced by neurovascular coupling resulting from pharmacological interventions, thus phfMRI studies can be used to help understand the neurophysiological function of specific receptors. Herein, the blood oxygenation level-dependent (BOLD) signal changes associated with D1R action in anesthetized rats was investigated by using a preclinical ultra-high-field 11.7-T MRI scanner. PhfMRI was performed before and after administration of the D1-like receptor agonist (SKF82958), antagonist (SCH39166), or physiological saline subcutaneously. Compared to saline, the D1-agonist induced a BOLD signal increase in the striatum, thalamus, prefrontal cortex, and cerebellum. At the same time, the D1-antagonist reduced the BOLD signal in the striatum, thalamus, and cerebellum by evaluating temporal profiles. PhfMRI detected D1R-related BOLD signal changes in the brain regions associated with high expression of D1R. We also measured the early expression of c-fos at the mRNA level to evaluate the effects of SKF82958 and isoflurane anesthesia on neuronal activity. Regardless of the presence of isoflurane anesthesia, c-fos expression level was increased in the region where positive BOLD responses were observed with administration of SKF82958. These findings demonstrated that phfMRI could be used to identify the effects of direct D1 blockade on physiological brain functions and also for neurophysiological assessment of dopamine receptor functions in living animals.

Anatomical variability, multi-modal coordinate systems, and precision targeting in the marmoset brain.

Localising accurate brain regions needs careful evaluation in each experimental species due to their individual variability. However, the function and connectivity of brain areas is commonly studied using a single-subject cranial landmark-based stereotactic atlas in animal neuroscience. Here, we address this issue in a small primate, the common marmoset, which is increasingly widely used in systems neuroscience. We developed a non-invasive multi-modal neuroimaging-based targeting pipeline, which accounts for intersubject anatomical variability in cranial and cortical landmarks in marmosets. This methodology allowed creation of multi-modal templates (MarmosetRIKEN20) including head CT and brain MR images, embedded in coordinate systems of anterior and posterior commissures (AC-PC) and CIFTI grayordinates. We found that the horizontal plane of the stereotactic coordinate was significantly rotated in pitch relative to the AC-PC coordinate system (10 degrees, frontal downwards), and had a significant bias and uncertainty due to positioning procedures. We also found that many common cranial and brain landmarks (e.g., bregma, intraparietal sulcus) vary in location across subjects and are substantial relative to average marmoset cortical area dimensions. Combining the neuroimaging-based targeting pipeline with robot-guided surgery enabled proof-of-concept targeting of deep brain structures with an accuracy of 0.2 mm. Altogether, our findings demonstrate substantial intersubject variability in marmoset brain and cranial landmarks, implying that subject-specific neuroimaging-based localization is needed for precision targeting in marmosets. The population-based templates and atlases in grayordinates, created for the first time in marmoset monkeys, should help bridging between macroscale and microscale analyses.

Towards HCP-Style macaque connectomes: 24-Channel 3T multi-array coil, MRI sequences and preprocessing.

Macaque monkeys are an important animal model where invasive investigations can lead to a better understanding of the cortical organization of primates including humans. However, the tools and methods for noninvasive image acquisition (e.g. MRI RF coils and pulse sequence protocols) and image data preprocessing have lagged behind those developed for humans. To resolve the structural and functional characteristics of the smaller macaque brain, high spatial, temporal, and angular resolutions combined with high signal-to-noise ratio are required to ensure good image quality. To address these challenges, we developed a macaque 24-channel receive coil for 3-T MRI with parallel imaging capabilities. This coil enables adaptation of the Human Connectome Project (HCP) image acquisition protocols to the in-vivo macaque brain. In addition, we adapted HCP preprocessing methods to the macaque brain, including spatial minimal preprocessing of structural, functional MRI (fMRI), and diffusion MRI (dMRI). The coil provides the necessary high signal-to-noise ratio and high efficiency in data acquisition, allowing four- and five-fold accelerations for dMRI and fMRI. Automated FreeSurfer segmentation of cortex, reconstruction of cortical surface, removal of artefacts and nuisance signals in fMRI, and distortion correction of dMRI all performed well, and the overall quality of basic neurobiological measures was comparable with those for the HCP. Analyses of functional connectivity in fMRI revealed high sensitivity as compared with those from publicly shared datasets. Tractography-based connectivity estimates correlated with tracer connectivity similarly to that achieved using ex-vivo dMRI. The resulting HCP-style in vivo macaque MRI data show considerable promise for analyzing cortical architecture and functional and structural connectivity using advanced methods that have previously only been available in studies of the human brain.

A Novel Phosphodiesterase 1 Inhibitor DSR-141562 Exhibits Efficacies in Animal Models for Positive, Negative, and Cognitive Symptoms Associated with Schizophrenia.

In our drug discovery program, we identified a novel orally available and brain-penetrant phosphodiesterase (PDE) 1 inhibitor, 3-methyl-7-(tetrahydro-2H-pyran-4-yl)-2-{[trans-4-(trifluoromethyl)cyclohexyl]-methoxy}imidazo[5,1-f][1,2,4]triazin-4(3H)-one (DSR-141562). In the present study, we characterized the preclinical profile of DSR-141562. This compound has preferential selectivity for predominantly brain-expressed PDE1B over other PDE1 family members, and high selectivity for the PDE1 family over other PDE families and 65 other tested biologic targets. Oral administration of DSR-141562 at 10 mg/kg slightly elevated the cGMP concentration, and it potently enhanced the increase of cGMP induced by a dopamine D1 receptor agonist in mouse brains. The cGMP level in monkey cerebrospinal fluid was also elevated after treatment with DSR-141562 at 30 and 100 mg/kg and could be used as a translational biomarker. Since PDE1B is believed to regulate dopaminergic and glutamatergic signal transduction, we evaluated the effects of this compound using schizophrenia-related behavioral assays. DSR-141562 at 3-30 mg/kg potently inhibited methamphetamine-induced locomotor hyperactivity in rats, while it had only minimal effects on the spontaneous locomotor activity. Furthermore, DSR-141562 at 1-100 mg/kg did not induce any signs of catalepsy in rats. DSR-141562 at 0.3-3 mg/kg reversed social interaction and novel object recognition deficits induced by repeated treatment with an N-methyl-D-aspartate receptor antagonist, phencyclidine, in mice and rats, respectively. In common marmosets, DSR-141562 at 3 and 30 mg/kg improved the performance in object retrieval with detour tasks. These results suggest that DSR-141562 is a therapeutic candidate for positive, negative, and cognitive symptoms in schizophrenia. SIGNIFICANCE STATEMENT: This is the first paper showing that a phosphodiesterase 1 inhibitor is efficacious in animal models for positive and negative symptoms associated with schizophrenia. Furthermore, we demonstrated that this compound improved cognitive function in the common marmoset, a nonhuman primate.

A novel Tungsten-based fiducial marker for multi-modal brain imaging.

BACKGROUND: Multi-modal brain image registration is a prerequisite for accurate mapping of brain structure and function in neuroscience. Image registration is commonly performed using automated software; however, its accuracy decreases when images differ in modality, contrast, uniformity, and resolution. This limitation could be overcome by using an external reference point; however, high-contrast agents in multi-modal imaging have not been previously reported. NEW METHODS: Here, we propose a novel multi-modal fiducial marker that contains Tungsten solution and provides high contrast in magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET). The basic characteristics of this multi-modal marker were investigated by assessing major sources of image contrast in the following modalities: density and T1-, T2-relaxivity in comparison with conventional contrast agents. RESULTS: Tungsten solution had lower T1- and T2-relaxivity and high solubility, and showed high contrast in T1- and T2-weighted MR and CT images at a high-density concentration (˜3.0 g/mL), whereas other conventional solutions did not show sufficient contrast in either CT or MRI. COMPARISON WITH EXISTING METHODS: The use of this Tungsten-based multi-modal marker allowed more accurate registration than a software-only method in phantom and animal experiments. Application of this method demonstrated accurate cortical surface mapping of neurotransmitter function (dopamine transporter, DAT) using PET and MRI, and provided a neurobiologically relevant cortical distribution consistent with previous literature on histology-based DAT immunoreactivity. CONCLUSIONS: The Tungsten-based multi-modal fiducial marker is non-radioactive, easy to handle, and aids precise registration across different modalities of brain imaging.

Comparison of 125I- and 111In-labeled peptide probes for in vivo detection of oxidized low-density lipoprotein in atherosclerotic plaques.

OBJECTIVE: Oxidized low-density lipoprotein (OxLDL) plays a pivotal role in atherosclerotic plaque destabilization, which suggests its potential as a nuclear medical imaging target. We previously developed radioiodinated 125I-AHP7, a peptide probe carrying a 7-residue sequence from the OxLDL-binding protein Asp-hemolysin, for specific OxLDL imaging. Although 125I-AHP7 recognized OxLDL, it had low stability. Thus, to improve stability, we designed radiolabeled 22-residue peptide probes, 125I-AHP22 and 111In-AHP22, which include the entire AHP7 sequence, and evaluated the stability, activity, and applications of these probes in vitro and in vivo. METHODS: Probes consisting of a 21-residue peptide derived from the Asp-hemolysin sequence and an N-terminal Cys or aminohexanoic acid for labeling with 125I-N-(3-iodophenyl)maleimide or 111In diethylene triamine pentaacetic acid were termed 125I-AHP22 and 111In-AHP22. An in vitro-binding inhibition assay with OxLDL was performed using 125I-AHP7 as a radiotracer. Radioactivity accumulation in the atherosclerotic aorta and plasma intact fraction was evaluated 30 min after intravenous administration of probes in myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. RESULTS: 125I-AHP22 and 111In-AHP22 were synthesized in ~ 360 and 60 min, respectively, with > 98% radiochemical purities after RP-HPLC purification. An in vitro-binding assay revealed similar or greater inhibition of OxLDL binding by both In-AHP22 and I-AHP22 compared to I-AHP7. The fraction of intact 125I-AHP22 and 111In-AHP22 in plasma was estimated to be approximately tenfold higher than that of 125I-AHP7. Both probes were rapidly cleared from the blood. 111In-AHP22 had a 2.3-fold higher accumulation in WHHLMI rabbit aortas compared to control rabbits, which was similar to 125I-AHP7. However, 125I-AHP22 accumulated to similar levels in aortas of WHHLMI and control rabbits due to high nonspecific accumulation in normal aortas that could be due to high lipophilicity. CONCLUSIONS: 111In-AHP22, easily prepared within 1 h, showed moderate affinity for OxLDL, high stability in vivo, and high accumulation in atherosclerotic aortas. 111In-AHP22 could be a potential lead compound to develop future effective OxLDL imaging probes.

Radiofluorinated probe for PET imaging of fatty acid binding protein 4 in cancer.

INTRODUCTION: Cancer-associated adipocytes metabolically interact with adjacent cancer cells to promote tumor proliferation and metastasis. Fatty acid binding protein 4 (FABP4) participates in this interaction, and is gathering attention as a therapeutic and diagnostic target. Positron emission tomography (PET) is a useful diagnostic method that enables noninvasive in vivo quantitative imaging of biofunctional molecules with probes labeled with positron-emitting radioisotopes. Here a novel (18)F labeled probe for PET FABP4 imaging developed through dedicated drug design from a radioiodinated probe we recently reported is evaluated in vitro and in vivo. METHODS: We designed the [(18)F]-labeled FTAP1 and FTAP3 probe, composed of a single or triple oxyethylene linker and a triazolopyrimidine scaffold derived from an FABP4 inhibitor. FABP4 binding affinities for chemically synthesized FTAP1 and FTAP3 were measured using FABP4 and 8-anilino-1-naphthalene sulfonic acid. Cell membrane permeability was measured using a commercially available plate assay system. After radiosynthesis, [(18)F]FTAP1 affinity and selectivity were evaluated using immobilized FABP3, FABP4, and FABP5. Cell uptake was investigated using differentiated adipocytes expressing FABP4 with inhibitor treatment. Following biodistribution studies in C6 glioblastoma-bearing mice, ex vivo autoradiography and immunohistochemistry were performed using thin sliced tumor sections. PET/CT imaging was then performed on C6 tumor bearing mice. RESULTS: FTAP1 showed high FABP4 affinity (Ki=68±8.9 nM) and adequate cell permeability. [(18)F]FTAP1 with ≥98% radiochemical purity was shown to selectively bind to FABP4 (16.3- and 9.3-fold higher than for FABP3 and FABP5, respectively). [(18)F]FTAP1 was taken up by FABP4 expressing cells, and this uptake could be blocked by an inhibitor, indicating very low non-specific cell binding. [(18)F]FTAP1 showed high tumor accumulation, which demonstrates its potential use for in vivo tumor PET imaging, and the intratumoral radioactivity distribution corresponded to the FABP4 expression profile. CONCLUSION: [(18)F]FTAP1 is a promising PET probe to target FABP4.

Development of a radioiodinated triazolopyrimidine probe for nuclear medical imaging of fatty acid binding protein 4.

Fatty acid binding protein 4 (FABP4) is the most well-characterized FABP isoform. FABP4 regulates inflammatory pathways in adipocytes and macrophages and is involved in both inflammatory diseases and tumor formation. FABP4 expression was recently reported for glioblastoma, where it may participate in disease malignancy. While FABP4 is a potential molecular imaging target, with the exception of a tritium labeled probe there are no reports of other nuclear imaging probes that target this protein. Here we designed and synthesized a nuclear imaging probe, [123I]TAP1, and evaluated its potential as a FABP4 targeting probe in in vitro and in vivo assays. We focused on the unique structure of a triazolopyrimidine scaffold that lacks a carboxylic acid to design the TAP1 probe that can undergo facilitated delivery across cell membranes. The affinity of synthesized TAP1 was measured using FABP4 and 8-anilino-1-naphthalene sulfonic acid. [125I]TAP1 was synthesized by iododestannylation of a precursor, followed by affinity and selectivity measurements using immobilized FABPs. Biodistributions in normal and C6 glioblastoma-bearing mice were evaluated, and excised tumors were subjected to autoradiography and immunohistochemistry. TAP1 and [125I]TAP1 showed high affinity for FABP4 (Ki = 44.5±9.8 nM, Kd = 69.1±12.3 nM). The FABP4 binding affinity of [125I]TAP1 was 11.5- and 35.5-fold higher than for FABP3 and FABP5, respectively. In an in vivo study [125I]TAP1 displayed high stability against deiodination and degradation, and moderate radioactivity accumulation in C6 tumors (1.37±0.24% dose/g 3 hr after injection). The radioactivity distribution profile in tumors partially corresponded to the FABP4 positive area and was also affected by perfusion. The results indicate that [125I]TAP1 could detect FABP4 in vitro and partly in vivo. As such, [125I]TAP1 is a promising lead compound for further refinement for use in in vivo FABP4 imaging.

Radioiodinated peptide probe for selective detection of oxidized low density lipoprotein in atherosclerotic plaques.

INTRODUCTION: Despite the significant effort in developing radioprobes for atherosclerosis, few have low molecular weight. Oxidized LDL (OxLDL), a highly proinflammatory and proatherogenic factor that is abundant in atherosclerotic plaques, plays a pivotal role in plaque destabilization, which makes OxLDL a relevant probe target. We developed a radioiodinated short peptide, AHP7, as a low molecular weight probe for specific OxLDL imaging and evaluated its utility using myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits (WHHLMI). METHODS: [¹²5I]AHP7 was designed and synthesized based on the sequence of Asp-hemolysin, an OxLDL binding protein extracted from Aspergillus fumigatus. In vitro binding studies with OxLDL having varying degrees of oxidation were performed. Radioactivity accumulation in the aorta was measured 30 min post-administration in rabbits. Autoradiography and histological studies were performed using serial aorta sections. A radioiodinated scrambled peptide ([¹²5I]AHP scramble) was used as a negative control. RESULTS: [¹²5I]AHP7 bound to OxLDL in proportion to the degree of oxidation (R=0.91, P<0.0001) and was inhibited by unlabeled AHP7 in a concentration-dependent manner. The aorta accumulation level and aorta/blood and aorta/muscle ratios of [¹²5I]AHP7 in WHHLMI were 2.8-, 1.3- and 1.8-fold higher, respectively, than those in control rabbits (P<0.001). Co-administration of AHP7 significantly reduced [¹²5I]AHP7 radioactivity in aorta sections (P<0.0001). Regional radioactivity levels in the aorta sections showed nonuniformity but similarity to the immunohistochemical OxLDL density. CONCLUSIONS: The potential of radioiodinated AHP7 for selectively imaging OxLDL was demonstrated both in vitro and in vivo.

Tissue factor detection for selectively discriminating unstable plaques in an atherosclerotic rabbit model.

UNLABELLED: Tissue factor (TF), a transmembrane glycoprotein that acts as an essential cofactor to factor VII/VIIa, initiates the exogenous blood coagulation cascade leading to thrombin generation and subsequent thrombus formation in vivo. TF expression is closely related to plaque vulnerability, and high TF expression is shown in macrophage-rich atheromatous lesions, making TF a potential target for detecting atheromatous lesions in vivo. Thus, we prepared (99m)Tc-labeled anti-TF-monoclonal antibody (TF-mAb) IgG as a molecular probe and evaluated its usefulness to achieve TF-specific imaging using myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. METHODS: Anti-TF-mAb was created using a standard hybridoma technique and was labeled by (99m)Tc with 6-hydrazinonicotinic acid (HYNIC) as a chelating agent to obtain (99m)Tc-TF-mAb. The immunoreactivity of HYNIC-TF-mAb was estimated by flow cytometry. WHHLMI and control rabbits were injected intravenously with (99m)Tc-TF-mAb. Twenty-four hours after the injection, the aorta was removed and radioactivity was measured. Autoradiography and histologic studies were performed using serial aorta sections. Subclass matched antibody (IgG(1)) was used as a negative control. RESULTS: HYNIC-TF-mAb showed 93% immunoreactivity of the anti-TF-mAb. The radioactivity accumulation in WHHLMI aortas was 6.1-fold higher than that of control rabbits. Autoradiograms showed a heterogeneous distribution of radioactivity in the intima of WHHLMI aortas. Regional radioactivity accumulation was positively correlated with TF expression density (R = 0.64, P < 0.0001). The highest radioactivity accumulation in percentage injected dose × body weight/mm(2) × 10(2) was found in atheromatous lesions (5.2 ± 1.9) followed by fibroatheromatous (2.1 ± 0.7), collagen-rich (1.8 ± 0.7), and neointimal lesions (1.8 ± 0.6). In contrast, (99m)Tc-IgG(1) showed low radioactivity accumulation in WHHLMI aortas that was independent of the histologic grade of lesions. CONCLUSION: The TF-detecting ability and preferential accumulation in atheromatous lesions of (99m)Tc-TF-mAb were demonstrated, indicating its potential for selective imaging of macrophage-rich atheromatous lesions in vivo.

Imaging with radiolabelled anti-membrane type 1 matrix metalloproteinase (MT1-MMP) antibody: potentials for characterizing atherosclerotic plaques.

PURPOSE: Membrane type 1 matrix metalloproteinase (MT1-MMP) activates pro-MMP-2 and pro-MMP-13 to their active forms and plays important roles in the destabilization of atherosclerotic plaques. This study sought to determine the usefulness of (99m)Tc-labelled monoclonal antibody (mAb), recognizing MT1-MMP, for imaging atherosclerosis in a rabbit model (WHHLMI rabbits). METHODS: Anti-MT1-MMP monoclonal IgG(3) and negative control IgG(3) were radiolabelled with (99m)Tc after derivatization with 6-hydrazinonicotinic acid (HYNIC) to yield (99m)Tc-MT1-MMP mAb and (99m)Tc-IgG(3), respectively. WHHLMI and control rabbits were injected with these radio-probes. The aorta was removed and radioactivity was measured at 24 h after the injection. Autoradiography and histological studies were performed. RESULTS: (99m)Tc-MT1-MMP mAb accumulation in WHHLMI rabbit aortas was 5.4-fold higher than that of control rabbits. Regional (99m)Tc-MT1-MMP mAb accumulation was positively correlated with MT1-MMP expression (r = 0.59, p < 0.0001), while (99m)Tc-IgG(3) accumulation was independent of MT1-MMP expression (r = 0.03, p = NS). The highest (99m)Tc-MT1-MMP mAb accumulation was found in atheromatous lesions (4.8 ± 1.9, %ID×BW/mm(2) × 10(2)), followed in decreasing order by fibroatheromatous (1.8 ± 1.3), collagen-rich (1.6 ± 1.0) and neointimal lesions (1.5 ± 1.5). In contrast, (99m)Tc-IgG(3) accumulation was almost independent of the histological grade of lesions. CONCLUSION: Higher (99m)Tc-MT1-MMP mAb accumulation in grade IV atheroma was shown in comparison with neointimal lesions or other more stable lesions. Nuclear imaging with (99m)Tc-MT1-MMP mAb, in combination with CT and MRI, could provide new diagnostic imaging capabilities for detecting vulnerable plaques, although further investigations to improve target to blood ratios are strongly required.