A simple method to improve the quality of diffusion-weighted magnetic resonance imaging with rapid histologic correlation in a murine model.

Diffusion-weighted magnetic resonance imaging (DW-MRI) has been used extensively in biomedical research. However, this technique has often suffered from distortion artifacts because of the magnetic field inhomogeneity surrounding the tissues. Histology is important for validating MRI interpretations, but correlating MRIs with tissue samples is challenging. Here we propose a method to improve DW-MRI and facilitate the matching between MRIs and tissue samples. A cryostat embedding medium, optimal cutting temperature (OCT) compound, was used to cover the examined target during the MRI studies. Frozen OCT compound could aid the examined target to be sectioned in parallel with the imaging plane. Phantom experiments demonstrated that embedding in OCT compound improved the magnetic field inhomogeneity while maintaining the apparent diffusion coefficient. Animal experiments revealed significantly reduced distortions in DW images in both the axial and coronal planes. The in vivo MRIs were easily matched with histologic specimens in a slice-to-slice fashion to examine the corresponding tissue microenvironment. This simple method might improve the quality of DW-MRI and provide histologic information for MRI to serve as an image biomarker.

Quantitative analysis of binding sites for 9-fluoropropyl-(+)-dihydrotetrabenazine ([¹8F]AV-133) in a MPTP-lesioned PD mouse model.

UNLABELLED: [¹8F]AV-133 is a novel PET tracer for targeting the vesicular monoamine transporter 2 (VMAT2). The aim of this study is to characterize and quantify the loss of monoamine neurons with [¹8F]AV-133 in the MPTP-lesioned PD mouse model using animal PET imaging and ex vivo quantitative autoradiography (QARG). METHODS: Optimal imaging time window of [¹8F]AV-133 was first determined in normal C57BL/6 mice (n = 3) with a 90-min dynamic scan. The reproducibility of [¹8F]AV-133 PET imaging was evaluated by performing a test-retest study within 1 week for the normal group (n = 6). For MPTP-lesioned studies, normal, and MPTP-treated [25 mg mg/kg once (Group A) and twice (Group B), respectively, daily for 5 days, i.p., groups of four normal and MPTP-treated] mice were used. PET imaging studies at baseline and at Day 4 post-MPTP injections were performed at the optimal time window after injection of 11.1 MBq [¹8F]AV-133. Specific uptake ratio (SUr) of [¹8F]AV-133 was calculated by [(target uptake-cerebellar uptake)/cerebellar uptake] with cerebellum as the reference region. Ex vitro QARG and immunohistochemistry (IHC) studies with tyrosine hydroxylase antibody were carried out to confirm the abundance of dopaminergic neurons. RESULTS: The variability between [¹8F]AV-133 test-retest striatal SUr was 6.60 ± 3.61% with less than 5% standard deviation between animals (intervariability). The percentages of MPTP lesions were Group A 0.94 ± 0.29, -42.1% and Group B 0.65 ± 0.09, -60.4%. By QARG, specific binding of [¹8F]AV-133 was reduced relative to the control groups by 50.6% and 60.7% in striatum and by 30.6% and 46.4% in substantia nigra (Groups A and B, respectively). Relatively small [¹8F]AV-133 SUr decline was noted in the serotonin and norepinephrine-enriched regions (7.9% and 9.4% in mid-brain). Results obtained from IHC consistently confirmed the sensitivity and selectivity of dopaminergic neuron loss after MPTP treatment. CONCLUSIONS: [¹8F]AV-133 PET SUr displayed a high test-retest stability. The SUr significantly declined in the caudate putamen but not in the hypothalamus and midbrain regions after MPTP treatment in the mouse brain. The results obtained for QARG and IHC were consistent and correlated well with the PET imaging studies. On the basis of these concordant results, we find that [¹8F]AV-133 should serve as a useful and reliable PET tracer for evaluating nigrostriatal degeneration.

Sigma receptor binding of tetrabenazine series tracers targeting VMAT2 in rat pancreas.

UNLABELLED: The vesicular monoamine transporter type II (VMAT2) is highly expressed in pancreatic ß-cells and thus has been proposed to be a potential target for measuring ß-cell mass (BCM) by molecular imaging. Several tracers based on the TBZ backbone, including 9-fluoropropyl-(+)-dihydrotetrabenazine ([(18)F]AV-133), have shown some promising results as potential biomarkers for BCM despite a relatively high background signal in the pancreas. In the present study, we explore the background binding characteristics of [(18)F]AV-133 in rat pancreas. METHODS: Pancreatic exocrine cells and islet cells were isolated and purified from Sprague-Dawley rats. Membrane homogenates, prepared from both pancreatic exocrine and islet cells as well as from brain striatum regions, were used for in vitro binding studies of [(18)F]AV-133 under a selective masking condition. 1,3-Di-o-tolylguanidine (DTG), displaying high and roughly equal affinity for both sigma-1 and sigma-2 receptors, was chosen at 5 µM concentration for the masking/blocking studies. RESULTS: [(18)F]AV-133 binding to rat striatum homogenates was not significantly altered by the presence of DTG. In contrast, [(18)F]AV-133 showed significant competition with DTG for binding sites in rat pancreatic exocrine homogenates as well as in rat islet cell homogenates. Importantly, in the presence of DTG, [(18)F]AV-133 showed a single high-affinity binding site on islet cell homogenates with a K(d) value of 3.8 nM which is consistent with the affinity reported previously for VMAT2 sites in rat pancreas. CONCLUSIONS: [(18)F]AV-133, in addition to a high-affinity VMAT2 binding site, binds with low affinity (but high capacity) to sigma components that are present in the rat pancreas. Identification of the cause of background binding of [(18)F]AV-133 to rat pancreatic tissue may lead to improved methods for quantification.

Binding characteristics of 9-fluoropropyl-(+)-dihydrotetrabenzazine (AV-133) to the vesicular monoamine transporter type 2 in rats.

UNLABELLED: The vesicular monoamine transporter type 2 (VMAT2) is highly expressed in pancreatic beta-cells and thus has been proposed to be a potential target for measuring beta-cell mass (BCM) by molecular imaging. C-11- and F-18-labeled tetrabenazine derivatives targeting VMAT2 have shown some promising results as potential biomarkers for BCM. In the present study, we examined the binding characteristics of 9-fluoropropyl-(+)-dihydrotetrabenzazine ([(18)F]AV-133), a potential PET tracer for BCM imaging, in rat pancreas and rat brain. METHODS: Pancreatic exocrine cells and pancreatic islet cells were isolated and purified from Sprague-Dawley rats. Membrane homogenates, prepared from both pancreatic exocrine and islet cells as well as from brain striatum and hypothalamus regions, were used for in vitro binding studies. In vitro and ex vivo autoradiography studies with [(18)F]AV-133 were performed on rat brain and rat pancreas sections. Immunohistochemistry studies were performed to confirm the distribution of VMAT2 on islet beta-cells. RESULTS: Excellent binding affinities of [(18)F]AV-133 were observed in rat striatum and hypothalamus homogenates with K(d) values of 0.19 and 0.25 nM, respectively. In contrast to single-site binding observed in rat striatum homogenates, rat islet cell homogenates showed two saturable binding sites (site A: K(d)=6.76 nM, B(max)=60 fmol/mg protein; site B: K(d)=241 nM, B(max)=1500 fmol/mg protein). Rat exocrine pancreas homogenates showed only a single low-affinity binding site (K(d)=209 nM), which was similar to site B in islet cells. In vitro autoradiography of [(18)F]AV-133 using frozen sections of rat pancreas showed specific labeling of islets, as evidenced by co-localization with anti-insulin antibody. Ex vivo VMAT2 pancreatic autoradiography in the rat, however, was not successful, in contrast to the excellent ex vivo autoradiography of VMAT2 binding sites in the brain. In vivo/ex vivo islet labeling may be complicated by the presence of the low-affinity/high-capacity site B binding in rat pancreas. CONCLUSIONS: [(18)F]AV-133 is an excellent imaging agent for mapping VMAT2 sites in rat brain and specifically binds rat islet cells in vitro and postmortem. Additional optimization may be required to achieve ex vivo islet beta-cell labeling in rats.

Pressurized water extraction of polysaccharides as secondary metabolites from Lentinula edodes.

The suitability of pressurized water extraction (PWE) of crude polysaccharides as secondary metabolites from Lentinula edodes was investigated. A series of experiments were carried out to examine the effects of extraction times and pressures. The results indicated that the maximum recovery of polysaccharides was about 90% of the crude polysaccharides from mycelia pellets when the pressure was at 10.1 MPa for 70 min (28 degrees C). This was a drastic improvement over that of boiling water extraction (BWE) at 0.1 MPa for 40 min, which gave only 27.9% recovery. A nitroblue tetrazolium (NBT) reduction assay was used to examine the macrophage stimulating activities (MSA), and it was found that the PWE polysaccharides retained the MSA. The morphology of the macrophage cells treated by PWE polysaccharides was also examined and found to be similar to that of the positive control lipopolysaccharides treated. Finally, gel chromatographic and NMR experiments revealed that both PWE and BWE polysaccharides showed the presence of four similar molecular mass components and the alpha-(1-->4)-D-Glcp and beta-(1-->6)-D-Glcp linkage residues. The improved PWE efficiency is probably due to the possibility that under high pressure, the solid polysaccharide's hydrogen bonding is partially destroyed to increase structure elasticity and water solubility.