Usefulness of 18F-FPP-RGD2 PET in pathophysiological evaluation of lung fibrosis using a bleomycin-induced rat model.

PURPOSE: Integrins αv are key molecules in the pathogenesis of fibrosis in multiple organs. To assess the potential utility of integrin αvß3 imaging for idiopathic pulmonary fibrosis (IPF), we evaluated an 18F-FPP-RGD2 PET probe in a rat model of bleomycin-induced lung fibrosis. METHODS: Pulmonary fibrosis was induced by single intratracheal instillation of bleomycin (3 mg/rat). Positron emission tomography (PET)/computerized tomography scans were performed 4 weeks after bleomycin administration using 18F-FPP-RGD2. Total distribution volume (VT) was estimated using one-tissue/two-compartment, two-tissue/three-compartment models, and Logan graphical analysis (Logan plot; t* = 30 min). Plasma-free fractions were estimated from images of the left ventricle. Correlation between Logan VT and lung pathology was assessed by Spearman's rank correlation. RESULTS: Histopathological evaluation demonstrated the development of fibrosis in IPF-model group. Integrin αv protein expression and lung radioactivity were higher in IPF-model group compared with control group. The lung radioactivity of 18F-FPP-RGD2 rapidly reached the peak after administration and then gradually decreased, whereas left ventricular radioactivity rapidly disappeared. Logan graphical analysis was found to be suitable for 18F-FPP-RGD2 kinetic analysis in the IPF-model lung. Logan VT values for 18F-FPP-RGD2 were significantly higher in IPF rats compared with control rats and strongly correlated with lung fibrosis, pathology, integrin αv protein expression, and oxygen partial pressure. CONCLUSION: Our findings demonstrate that the integrin αvß3 PET probe 18F-FPP-RGD2 can detect pathophysiological changes in lungs, including fibrosis accompanying upregulated integrin αv of IPF-model rats. These findings support the utility of 18F-FPP-RGD2 PET imaging for the pathophysiological evaluation of pulmonary fibrosis.

A Novel Acetyl-CoA Carboxylase 2 Selective Inhibitor Improves Whole-Body Insulin Resistance and Hyperglycemia in Diabetic Mice through Target-Dependent Pathways.

Excess intramyocellular lipid (IMCL) deposition in skeletal muscle is closely associated with insulin resistance. Pharmacological inhibition of acetyl-CoA carboxylase (ACC) 2 offers a promising approach to treat insulin resistance through stimulation of mitochondrial fatty acid oxidation (FAO) and reduction of IMCL deposition. Previously reported experimental ACC2 inhibitors exhibited plasma glucose-lowering effects in diabetic rodents. However, their antidiabetic action may be potentially biased by off-target effects on triglyceride metabolism or by neurologic side effects. In this study, we investigated a safety profile, target dependency of its action, and antidiabetic efficacy of compound 2e, a novel olefin derivative potent ACC2 selective inhibitor. Four-day administration of suprapharmacological dose of compound 2e did not exhibit any obvious side effects in Sprague-Dawley rats. In db/db mice, single administration of compound 2e led to significantly elevated FAO and reduced IMCL deposition in skeletal muscle. In ACC2 knockout mice, treatment with pharmacological doses of compound 2e did not reduce plasma triglyceride levels, whereas A-908292, a previously reported ACC2 inhibitor, caused a significant triglyceride reduction, showing that compound 2e was devoid of off-target triglyceride-lowering activity. Chronic treatment of db/db mice with compound 2e improved hyperglycemia but did not decrease plasma triglyceride levels. Additionally, compound 2e showed significant improvements of whole-body insulin resistance in the clamp study and insulin tolerance test. Collectively, compound 2e demonstrated a good safety profile and significant antidiabetic effects through inhibition of ACC2-dependent pathways. These findings provide further evidence that selective inhibition of ACC2 is an attractive strategy against insulin resistance and type 2 diabetes. SIGNIFICANCE STATEMENT: This study shows that pharmacological inhibition of acetyl-CoA carboxylase (ACC) 2 leads to significant improvements in whole-body glucose homeostasis, independently of off-target metabolic pathways and toxicity, which were observed in previously reported ACC2 inhibitors. These findings support the concept that ACC2-selective inhibitors will be a novel remedy for treatment of type 2 diabetes.

Pharmacological MRI responses of raclopride in rats: The relationship with D2 receptor occupancy and cataleptic behavior.

Pharmacological magnetic resonance imaging (phMRI) allows the visualization of brain pharmacological effects of drugs using functional MRI (fMRI). phMRI can help us facilitate central nervous system (CNS) drug development. However, there have been few studies demonstrating the dose relationship of the fMRI response induced by CNS drugs to underlying target engagement or behavioral efficacy. To clarify these relationships, we examined receptor occupancy measurements using positron emission tomography (PET) (n = 3~5), fMRI (n = 5~8) and a cataleptic behavior (n = 6) with raclopride, a dopamine D2 receptor antagonist (8, 20, and 200 µg/kg) on Wistar rats. Dopamine D2 receptor occupancy was increased dose dependently by raclopride (41.8 ± 2.7%, 8 µg/kg; 64.9 ± 2.8%, 20 µg/kg; 83.1 ± 3.0%, 200 µg/kg). phMRI study revealed significant positive responses to raclopride at 200 µg/kg specifically in the striatum and nucleus accumbens, related to dopaminergic system. Slight fMRI responses were observed at 20 µg/kg in some areas corresponding to the striatum and nucleus accumbens. There were no noticeable fMRI responses at 8 µg/kg raclopride administration. Raclopride at 200 µg/kg significantly increased the cataleptic score, although, at 8 and 20 µg/kg, raclopride had no significant effects. These findings showed that raclopride-induced fMRI responses were observed at doses inducing cataleptic behavior and high D2 receptor occupancy, suggesting that phMRI can be useful for dose selection in clinical trial as an evaluation method of brain activity, which reflects behavioral responses induced by target engagements.

(R)- and (S)-ketamine induce differential fMRI responses in conscious rats.

(R,S)-ketamine exerts robust antidepressant effects in patients with depression when given at sub-anesthetic doses. Each of the enantiomers in this racemic mixture, (R)-ketamine and (S)-ketamine, have been reported to exert antidepressant effects individually. However, the neuropharmacological effects of these enantiomers and the mechanisms underlying their antidepressive actions have not yet been fully elucidated. Therefore, we investigated the effect of (R,S)-, (R)-, and (S)-ketamine on brain activity by functional MRI (fMRI) in conscious rats and compared these with that of N-methyl-D-aspartate receptor (NMDAR) antagonist MK-801 (n = 5~7). We also assessed their pharmacokinetic profiles (n = 4) and their behavioral effects (n = 7~9). This pharmacological MRI study revealed a significant positive response to (S)-ketamine specifically in the cortex, nucleus accumbens and striatum. In contrast, negative fMRI responses were observed in various brain regions after (R)-ketamine administration. (R,S)-ketamine, evoked significant positive fMRI responses specifically in the cortex, nucleus accumbens and striatum, and this fMRI response pattern was comparable with that of (S)-ketamine. MK-801-induced similar fMRI response pattern to (S)-ketamine. The fMRI responses to (S)-ketamine and MK-801 showed differential temporal profiles, which corresponded with brain concentration profiles. (S)-ketamine and MK-801 significantly increased locomotor activity, while (R)-ketamine produced no noticeable change. (R,S)-ketamine tended to increase locomotor activity. Our novel fMRI findings show that (R)-ketamine and (S)-ketamine induce completely different fMRI response patterns on rat, and that the response produced by the latter is similar to that elicited by an NMDAR antagonist. Our findings provide insight into the antidepressant mechanism of (R,S)-ketamine.

Time Course of Diffusion Kurtosis in Cerebral Infarctions of Transient Middle Cerebral Artery Occlusion Rat Model.

OBJECTIVE: To evaluate the relationship between fiber bundle direction and changes in diffusion kurtosis, we evaluated the apparent diffusion kurtosis coefficients (AKCs) that were perpendicular to and parallel to the principal diffusion tensor eigenvector. MATERIALS AND METHODS: Adult male Wistar rats were subjected to 30 or 60 minutes of middle cerebral artery occlusion and imaged with a 7T Magnetic Resonance Imager System (Varian MRI System 7T/210: Agilent Technologies, CA). Diffusion kurtosis images were obtained before middle cerebral artery (MCA) reperfusion and 3, 6, and 24 hours after reperfusion to generate the apparent diffusion coefficient (ADC), fractional anisotropy (FA), mean apparent diffusion kurtosis coefficient (mAKC), AKC axial to the eigenvector (axAKC), and AKC radial to the eigenvector (radAKC) images. The time course of the region/normal ratio was evaluated for the above parameters in the caudoputamen and white matter. RESULTS: Relative FA and relative ADC values decreased 3 hours after MCA reperfusion and remained decreased until 24 hours. Relative mAKC, axAKC, and radAKC values were increased 3 hours after MCA reperfusion, peaked after 6 hours, and slightly decreased after 24 hours. In the white matter, axAKC showed larger changes than radAKC. CONCLUSION: The time course of the diffusion kurtosis value showed earlier pseudonormalization than the ADC value of the lesions. For white matter lesions, the increase in axAKC was larger than that in radAKC, suggesting that the tissue changes after infarction mainly produce reduced diffusivity along the fibers and lead to increased inhomogeneity of the diffusion.

Assessment of glutamine synthetase activity by [13N]ammonia uptake in living rat brain.

Glutamine synthetase (GS) plays an important role in glutamate neurotransmission or neurological disorder in the brain. [(13) N]Ammonia blood flow tracer has been reported to be metabolically trapped in the brain via the glutamate-glutamine pathway. The present study investigated the effect of an inhibitor of GS on [(13) N]ammonia uptake in order to clarify the feasibility of measuring GS activity in the living brain. l-Methionine sulfoximine (MSO), a selective GS inhibitor was microinjected into the ipsilateral striatum in rats. [(13) N]Ammonia uptake was quantified by autoradiography method as well as small animal positron emission tomography (PET) scans. The GS activity of the brain homogenate was assayed from the γ-glutamyl transferase reaction. Autoradiograms showed a decrease of [(13) N]ammonia radioactivity on the MSO-injected side compared with the saline-injected side of the striatum. This reduction could be detected with a small animal PET scanner. MSO had no effect on cerebral blood flow measured by uptake of [(15) O]H2 O. The reduction of [(13) N]ammonia uptake was closely related to the results of GS activity assay. These results indicated that [(13) N]ammonia may enable measurement of GS activity in the living brain.

Pharmacological MRI response to a selective dopamine transporter inhibitor, GBR12909, in awake and anesthetized rats.

Pharmacological magnetic resonance imaging (phMRI) is a powerful tool for imaging the effects of drugs on brain activity. In preclinical phMRI studies, general anesthesia used for minimizing head movements is thought to influence the phMRI responses to drugs. In this study we investigated the phMRI responses to a selective dopamine transporter (DAT) inhibitor, GBR12909, and a dopamine (DA) releaser, d-amphetamine (AMPH), in the isoflurane anesthetized and awake rats using a relative cerebral blood volume (rCBV) method. AMPH (1 mg/kg i.p.) caused an increase in rCBV in the dopaminergic circuitry in the both anesthetized and awake rats. The striatal rCBV change was correlated with the change of the striatal DA concentration induced by AMPH in the both anesthetized and awake rats. GBR12909 (10 mg/kg i.p.) caused a positive rCBV response and showed a similar regional pattern of rCBV response to AMPH in the awake rats, and the correlation between the change of the striatal rCBV and the striatal DA concentration was observed. However, in the anesthetized rats, GBR12909 induced a widespread negative rCBV response, whereas an increase in striatal DA concentration was observed. These findings indicate that phMRI responses to activation of DA neurotransmission by GBR12909 or AMPH are overall identical in the awake state, while the phMRI response to a DAT inhibitor, GBR12909 but not to AMPH was changed by isoflurane anesthesia. For the evaluation of neuroactive drugs using phMRI, isoflurane anesthesia might be complicated the interpretation of pharmacodynamic effects of drugs in preclinical studies.

[11C]PK11195 PET imaging of spinal glial activation after nerve injury in rats.

The role of glial activation has been implicated in the development and persistence of neuropathic pain after nerve injury by recent studies. PK11195 binding to the translocator protein 18kDa (TSPO) has been shown to be enhanced in activated microglia. This study was designed to assess PK11195 imaging in spinal microglia during activation after nerve injury. The development of neuropathic pain was induced by partial sciatic nerve ligation (PSL). PSL rats on days 7 and 14 after nerve injury were subjected to imaging with a small-animal positron emission tomography/computed tomography (PET/CT) scanner using [(11)C]PK11195 to detect spinal microglial activation by means of noninvasive in vivo imaging. Spinal [(3)H]PK11195 autoradiography was performed to confirm the results of [(11)C]PK11195 PET in PSL rats. Quantitative RT-PCR of CD11b and GFAP mRNA, and the immunohistochemistry of Iba1 and GFAP were investigated to detect activated microglia and astrocytes. Mechanical allodynia was observed in the ipsilateral paw of PSL rats from day 3 after nerve injury and stably persisted from days 7 to 14. PET/CT fusion images clearly showed large amounts of accumulation of [(11)C]PK11195 in the lumbar spinal cord on days 7 and 14 after nerve injury. [(11)C]PK11195 enhanced images were restricted to the L3-L6 area of the spinal cord. The standardized uptake value (SUV) of [(11)C]PK11195 was significantly increased in the lumbar spinal cord compared to that of the thoracic region. Increased specific binding of [(11)C]PK11195 to TSPO in the spinal cord of PSL rats was confirmed by competition studies using unlabeled (R, S)-PK11195. Increased [(3)H]PK11195 binding was also observed in the ipsilateral dorsal horn of the L3-L6 spinal cord on days 7 and 14 after nerve injury. CD11b mRNA and Iba1 immunoreactive cells increased significantly on days 7 and 14 after nerve injury by PSL. However, changes in GFAP mRNA and immunoreactivity were slight in the ipsilateral side of PSL rats. In the present study, we showed that glial activation could be quantitatively imaged in the spinal cord of neuropathic pain rats using [(11)C]PK11195 PET, suggesting that high resolution PET using TSPO-specific radioligands might be useful for imaging to assess the role of glial activation, including neuroinflammatory processes, in neuropathic pain patients.

Effect of scan-time shortening on the 11C-PHNO binding potential to dopamine D3 receptor in humans and test-retest reliability.

OBJECTIVE: 11C-PHNO is a PET radioligand most specific to dopamine D3 receptor (D3R). The long scan duration of 120 min used in quantification of 11C-PHNO binding to D3R in previous studies is challenging to subjects. The main objective of this study was to investigate the effects of shorter scan times on the binding of 11C-PHNO to D3R and test-retest reliability using the latest digital whole-body PET system. METHODS: Two 120-min 11C-PHNO brain scans were performed in 7 healthy subjects using a digital whole-body PET/CT. The binding potential relative to non-displaceable tracer in the tissue (BPND) of D3R-rich regions: the pallidum, ventral striatum (VST), substantia nigra (SN) and hypothalamus, were quantified using the simplified reference tissue model. The bias, correlation, and test-retest reliability of BPND, which includes the test-retest variability (TRV) and intraclass correlation coefficient (ICC), were evaluated and compared between scans of shorter durations (40-110 min post-injection) and the original 120-min scan acquisitions. RESULTS: Progressively, shorter scan durations were associated with underestimation of BPND, slightly decreased correlation with 120-min derived BPND, and decrease in test-retest reliability. The BPND values of the pallidum, VST and SN from the shortened 90-min scans showed excellent correlation with those derived from the 120-min scans (determination coefficients > 0.98), and the bias within 5%. The test-retest reliability of BPND in these regions derived from 90-min scan (TRV of 3% in the VST and pallidum, 7% in the SN and the ICC exceeded 0.88) was comparable to those obtained in previous 120-min studies using brain-dedicated PET scanners. In the hypothalamus, the BPND values obtained from scan-time less than 110 min showed bias larger than 5% and the TRV more than 9%. CONCLUSION: The scan-time shortening causes bias and decreasing test-retest reliability of 11C-PHNO BPND. However, in the whole-body PET system, 90-min scan duration was sufficient for estimating the 11C-PHNO BPND in the D3R-rich striatum and SN with small bias and at the test-retest reliability comparable to those derived from 120-min scans using the brain-dedicated PET systems.

Evaluation of an Integrin αvß3 Radiotracer, [18F]F-FPP-RGD2, for Monitoring Pharmacological Effects of Integrin αv siRNA in the NASH Liver.

Purpose: Integrin αv is a key regulator in the pathophysiology of hepatic fibrosis. In this study, we evaluated the potential utility of an integrin αvß3 positron emission tomography (PET) radiotracer, 18F-labeled cyclic arginine-glycine-aspartic acid penta-peptide ([18F]F-FPP-RGD2), for detecting hepatic integrin αv and function in nonalcoholic steatohepatitis (NASH) model rats using integrin αv siRNA. Methods: NASH model rats were produced by feeding a choline-deficient, low-methionine, high-fat diet for 8 weeks. PET/computerized tomography imaging and quantification of integrin αv protein, serum aspartate aminotransferase, and alanine aminotransferase were performed 1 week after single intravenous injection of integrin αv siRNA. Results: Integrin αv siRNA (0.1 and 0.5 mg/kg) dose-dependently decreased hepatic integrin αv protein concentrations in control and NASH model rats. The hepatic mean standard uptake value of [18F]F-FPP-RGD2 was decreased dose-dependently by integrin αv siRNA. The mean standard uptake value was positively correlated with integrin αv protein levels in control and NASH model rats. Serum aspartate aminotransferase and alanine aminotransferase concentrations were also decreased by siRNA injection and correlated with liver integrin αv protein expression levels in NASH model rats. Conclusion: This study suggests that [18F]F-FPP-RGD2 PET imaging is a promising radiotracer for monitoring hepatic integrin αv protein levels and hepatic function in NASH pathology.

Vibrational modes of hydrogens in the proton ordered phase XI of ice: Raman spectra above 400 cm(-1).

Polarized Raman spectra of the proton ordered phase of ice Ih, i.e., ice XI, were measured above 400 cm(-1) in the range of librational, bending, and stretching vibrations. Vibrational modes in ice XI, of which symmetry is C(2v) (12)(Cmc2(1)), were discussed from the group theoretical point of view. In the librational mode spectra below 1200 cm(-1), several new peaks and clear polarization dependencies were observed. Assignments of the librational modes agree reasonably well with the recent MD calculations by Iwano et al. (J. Phys. Soc. Jpn. 79, 063601 (2010)). In contrast, the spectra for bands above 1200 cm(-1) show no distinct polarization dependencies and the spectra resemble those in ice Ih. In ice XI, however, fine structure composed of several weak peaks appear on the broad bending and the combination band. No direct evidence of the LO-TO splitting of the ν(3) anti-symmetric stretching mode was obtained. It is contrary to the case of the translational modes Abe and Shigenari (J. Chem. Phys. 134, 104506 (2011)). Present results suggest that the influence of the proton ordering in ice XI is weaker than the effect of inter- and intra-molecular couplings in the stretching vibrations of ice Ih.

Raman spectra of proton ordered phase XI of ICE I. Translational vibrations below 350 cm(-1).

Polarized Raman spectra from single crystals of ice XI (proton ordered phase of ice Ih) were measured and assigned for the modes below 350 cm(-1) in the translational vibration region. In contrast to the proton disordered ice Ih, the spectra in ice XI show clear polarization dependence and several new peaks are observed. Most of the vibrational modes were successfully assigned by the simplified point mass model with the symmetry C(2v) (12)(Cmc2(1)) and by the depolarization effect. In particular, LO-TO splitting of the mode near 240 cm(-1) was experimentally confirmed for the first time, which indicates that the long range force effect appears distinctly in ice XI.

Dual-frequency injection-locked nanosecond pulsed laser with arbitrary combination of two oscillation frequencies.

We report a dual-frequency injection-locked nanosecond pulsed laser oscillating at an arbitrary combination of two frequencies over the broad gain range of a Ti:sapphire laser. This performance is achieved by employing two techniques. One involves introducing two different modulation frequencies to discriminate electronically the error signals, which are used for locking the two seed frequencies to the forced oscillator. The other is a cavity design that enables us to sweep the cavity length without distorting the alignment or changing the spatial mode of the cavity. The difference frequencies in a pair of single-frequency nanosecond pulses can be selected continuously from less than 1 GHz to tens of THz without modifying the laser configuration.

Remarkable selectivity of the in vivo binding of [3H]Ro15-4513 to α5 subtype of benzodiazepine receptor in the living mouse brain.

To evaluate the binding characteristics of [(3)H]Ro15-4513 with the central benzodiazepine (BZ) receptor, inhibition experiments of [(3)H]Ro15-1788 and [(3)H]Ro15-4513 were performed both in vitro and in vivo, using two BZ ligands, flunitrazepam (FNP), and ethyl-ß-carboline-3-carboxylate (ß-CCE). FNP inhibited the binding of [(3)H]Ro15-1788 and [(3)H]Ro15-4513 in a dose-dependent manner in the mouse cerebral cortex, hippocampus, and cerebellum, both in vitro and in vivo. ß-CCE also inhibited the binding of [(3)H]Ro15-1788 and [(3)H]Ro15-4513 in all the aforementioned brain regions in vitro. However, in vivo, ß-CCE inhibited the binding of [(3)H]Ro15-4513 in the cerebral cortex and cerebellum, but not in the hippocampus, even at an injected dose of up to 1mg/kg. In contrast, more than 50% of the in vivo binding of [(3)H]Ro15-1788 was inhibited by 1 mg/kg of ß-CCE in all regions. The time-activity curve of [(3)H]Ro15-4513 in the hippocampus also showed no alteration of the peak uptake between the control group and 0.3 mg/kg of ß-CCE coinjected group. These results indicated that the binding characteristics of [(3)H]Ro15-4513 with the BZ receptor differed markedly between the in vitro and in vivo condition, and the selectivity of [(3)H]Ro15-4513 binding to α5 subtype of BZ receptor in the mouse brain seemed to be remarkable under the in vivo condition.

Effects of cAMP-dependent protein kinase activator and inhibitor on in vivo rolipram binding to phosphodiesterase 4 in conscious rats.

Rolipram is a selective inhibitor of phosphodiesterase-4 (PDE4), and positron emission tomography (PET) using [(11)C]rolipram can monitor the in vivo activity of this enzyme that is part of the cAMP second messenger cascade. cAMP-dependent protein kinase (PKA) phosphorylates PDE4 and increases both enzyme activity and affinity for rolipram. In the present PET study, we examined effects of PKA modulators in conscious rats on the binding of [(11)C](R)-rolipram in comparison to the much less active enantiomer [(11)C](S)-rolipram. Unilateral injection of a PKA activator (dibutyryl-cAMP) and a PKA inhibitor (Rp-adenosine-3',5'-cyclic monophosphorothioate) into the striatum significantly increased and decreased, respectively, the binding of [(11)C](R)-rolipram. These effects were not caused by changes in blood flow or delivery of radioligand to brain, since these agents had no effect on the binding of [(11)C](S)-rolipram binding. These results support the value of measuring in vivo [(11)C](R)-rolipram binding in brain to assess responses to physiological or pharmacological challenges to the cAMP second messenger system.

Quantitative evaluation of hepatic integrin αvß3 expression by positron emission tomography imaging using 18F-FPP-RGD2 in rats with non-alcoholic steatohepatitis.

BACKGROUND: Integrin αvß3, which are expressed by activated hepatic stellate cells in non-alcoholic steatohepatitis (NASH), play an important role in the fibrosis. Recently, we reported that an RGD peptide positron emission tomography (PET) probe is useful as a predictor of hepatic fibrosis. Kinetic analysis of the RGD PET probe has been performed in tumours, but not in hepatic fibrosis. Therefore, we aimed to quantify hepatic integrin αvß3 in a model of NASH by kinetic analysis using 18F-FPP-RGD2, an integrin αvß3 PET probe. METHODS: 18F-FPP-RGD2 PET/CT scans were performed in control and NASH rats. Tissue kinetic analyses were performed using a one-tissue, two-compartment (1T2C) and a two-tissue, three-compartment (2T3C) model using an image-derived input function (IDIF) for the left ventricle. We then conducted correlation analysis between standard uptake values (SUVs) or volume of distribution (VT), evaluated using compartment kinetic analysis and integrin αv or ß3 protein expression. RESULTS: Biochemical and histological evaluation confirmed the development of NASH rats. Integrin αvß3 protein expression and hepatic SUV were higher in NASH- than normal rats. The hepatic activity of 18F-FPP-RGD2 peaked rapidly after administration and then gradually decreased, whereas left ventricular activity rapidly disappeared. The 2T3C model was found to be preferable for 18F-FPP-RGD2 kinetic analysis in the liver. The VT (IDIF) for 18F-FPP-RGD2, calculated using the 2T3C model, was significantly higher in NASH- than normal rats and correlated strongly with hepatic integrin αv and ß3 protein expression. The strengths of these correlations were similar to those between SUV60-90 min and hepatic integrin αv or ß3 protein expression. CONCLUSIONS: We have demonstrated that the VT (IDIF) of 18F-FPP-RGD2, calculated using kinetic modelling, positively correlates with integrin αv and ß3 protein in the liver of NASH rats. These findings suggest that hepatic VT (IDIF) provides a quantitative assessment of integrin αvß3 protein in liver.

PET measurement of the in vivo affinity of 11C-(R)-rolipram and the density of its target, phosphodiesterase-4, in the brains of conscious and anesthetized rats.

UNLABELLED: A variety of phosphodiesterases hydrolyze and terminate the effects of the intracellular second messenger 3',5'-cyclic adenosine monophosphate (cAMP). Phosphodiesterase subtype 4 (PDE4) is particularly abundant in the brain and has been imaged with (11)C-(R)-rolipram, a selective inhibitor of PDE4. We sought to measure in vivo both the binding site density (B(max)) and the radioligand affinity (1/K(D)) of (11)C-(R)-rolipram in the rat brain. We also studied 2 critical factors in small-animal PET scans: the influence of anesthesia and the difference in binding under in vivo and in vitro conditions. METHODS: In vivo, B(max) and K(D) were measured in PET saturation experiments by the administration of (11)C-(R)-rolipram and various doses of carrier (R)-rolipram in conscious and isoflurane-anesthetized rats. The metabolite-corrected arterial input function was measured in each scan. To image conscious rats, the head of the rat was fixed in a holder and the animals were trained to comply with this apparatus. Bound and free (R)-rolipram levels were calculated under transient equilibrium conditions (i.e., at the time of peak specific binding). RESULTS: The B(max) and K(D) of conscious rats were significantly greater than those of anesthetized rats, by 29% and 59%, respectively. In addition, the in vitro K(D) was 3-7 times greater than was the in vivo K(D), although the B(max) was similar in both conditions. CONCLUSION: The in vivo B(max) and K(D) of (R)-rolipram were successfully measured in both conscious and anesthetized rats. K(D) was affected to a greater extent than was B(max) by the 2 conditions. That is, K(D) was increased in the conscious rat, compared with in the anesthetized rat, and K(D) was increased in vitro, compared with in vivo. The current study shows that the rat, a readily available species for research, can be used to measure in vivo both affinity and density of radioligand targets, which can later be directly assessed with standard in vitro techniques.

Role of NMDA receptor upon [14C]acetate uptake into intact rat brain.

OBJECTIVE: To clarify the role of N-methyl-D: -aspartate (NMDA) receptors upon [(14)C]acetate uptake in the rodent central nervous system (CNS), ibotenic acid (IBO) was infused into the right striatum of the rat brain. METHODS: Autoradiograms of [(14)C]acetate uptake in the brain for 2 h following the infusion of IBO (10 microg/microl) were obtained in both non-treated and MK-801 (1 mg/kg, i.v.) pretreated rats. The effect of MK-801 on [(14)C]acetate uptake in the normal rat brain was also studied. RESULTS: Infusion of IBO significantly decreased [(14)C]acetate uptake in the infused side of the striatum. The expression of monocarboxylate transporter-1 was not altered, suggesting that the activity of tricarboxylic acid (TCA) cycle in glial cells might be depressed. Pretreatment with MK-801 completely blocked the decreasing effect of IBO on [(14)C]acetate uptake. MK-801 also increased [(14)C]acetate uptake in the whole brain of normal rats. CONCLUSIONS: These results indicate the important roles of NMDA receptors on [(14)C]acetate uptake in the intact rat brain.

De-coupling of blood flow and metabolism in the rat brain induced by glutamate.

OBJECTIVE: Glutamate plays an essential role in neuronal cell death in many neurological disorders. In this study, we examined both glucose metabolism and cerebral blood flow in the same rat following infusion of glutamate or ibotenic acid using the dual-tracer technique. The effects of MK-801, an NMDA receptor antagonist, and NBQX, an AMPA-kainate receptor antagonist, on the changes in the glucose metabolism and cerebral blood flow induced by glutamate were also examined. METHODS: The rats were microinjected with glutamate (1 micromol/microl, 2 microl) or ibotenic acid (10 microg/microl, 1 microl) into the right striatum, and dual-tracer autoradiograms of [(18)F]FDG and [(14)C]IMP were obtained. MK-801 and NBQX were injected intravenously about 45 and 30 min, respectively, after the infusion of glutamate. RESULTS: De-coupling of blood flow and metabolism was noted in the glutamate-infused hemisphere (as assessed by no alteration of [(18)F]FDG uptake and significant decrease of [(14)C]IMP uptake). Pretreatments with MK-801, NBQX, or combined use of MK-801 and NBQX did not affect the de-coupling of the blood flow and metabolism induced by glutamate. A histochemical study revealed that about 20% neuronal cell death had occurred in the striatum at 105 min after the infusion of glutamate. In addition, a significant increase of the [(18)F]FDG uptake and decrease of [(14)C]IMP uptake were also seen in the rat brain infused with ibotenic acid. CONCLUSION: These results indicate that glutamate and ibotenic acid caused a significant de-coupling of blood flow and glucose metabolism in the intact rat brain during the early phase of neurodegeneration. It is necessary to evaluate the relation between metabotropic glutamate receptors and de-coupling of blood flow and metabolism.

Evaluation of hepatic function using dynamic contrast-enhanced magnetic resonance imaging in melanocortin 4 receptor-deficient mice as a model of nonalcoholic steatohepatitis.

INTRODUCTION: Melanocortin 4 receptor-deficient (MC4R-KO) mice fed a high-fat diet (HFD) develop liver pathology similar to human nonalcoholic steatohepatitis (NASH). However, although liver histology and blood biochemistry have been reported, hepatic function has not been evaluated. In the present study, we evaluated hepatic function in MC4R-KO mice fed an HFD using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with gadolinium­ethoxybenzyl­diethylenetriamine pentaacetic acid (Gd-EOB-DTPA). MATERIALS AND METHODS: Wild type (WT) mice and MC4R-KO mice were fed a standard diet (SD) or an HFD for 20 weeks. The hepatic signal intensity was obtained from DCE-MRI images, and relative enhancement (RE), the time to maximum RE (Tmax), and the half-life of RE elimination (T1/2) were calculated. Histopathological analysis was then performed. RESULTS: Histological analysis with nonalcoholic fatty liver disease activity score (NAS) revealed that MC4R-KO mice fed an HFD achieved the NAS of 5. There was moderate fibrosis in MC4R-KO mice fed an HFD. DCE-MRI with Gd-EOB-DTPA showed that Tmax and T1/2 were significantly longer in MC4R-KO mice fed an HFD compared with wild type (WT) mice (Tmax, WT, 3.9 ±â€¯0.4 min; MC4R-KO, 7.4 ±â€¯1.5 min; T1/2, WT, 23.7 ±â€¯1.9 min; MC4R-KO, 62.5 ±â€¯18.5 min). Tmax and T1/2 were significantly correlated with histopathologic score (steatosis vs. Tmax, rho = 0.48, P = 0.04; steatosis vs. T1/2, rho = 0.50, P = 0.03; inflammation vs. Tmax, rho = 0.55, P = 0.02; inflammation vs. T1/2, rho = 0.61, P < 0.01; ballooning vs. T1/2, rho = 0.51, P = 0.03;fibrosis vs Tmax, rho = 0.72, P < 0.01; fibrosis vs T1/2, rho = 0.75, P < 0.01). CONCLUSIONS: MC4R-KO mice fed an HFD developed obesity and NASH. The liver kinetics of Gd-EOB-DTPA were significantly different in MC4R-KO mice fed an HFD from WT mice, and correlated with the histopathologic score. These results suggest that MC4R-KO mice fed an HFD mimic the hepatic pathology and liver function of human NASH, and therefore might be useful for the study of hepatic dysfunction during the fibrotic stage of NASH.