Differences in in vitro microglial accumulation of the energy metabolism tracers [18F]FDG and [18F]BCPP-EF during LPS- and IL4 stimulation.

The positron emission tomography probes 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and 2-tert-butyl-4-chloro-5-{6-[2-(2-[18F]fluoroethoxy)-ethoxy]-pyridin-3-ylmethoxy}-2H-pyridazin-3-one ([18F]BCPP-EF) are designed to evaluate glycolysis and oxidative phosphorylation, respectively, and are both used to estimate neuronal activity. However, previous studies have shown a discrepancy in these probes' accumulation in the compromised region, possibly due to the presence of activated microglia acting like deleterious or neuroprotective phenotypes. Hence, we evaluated lipopolysaccharide (LPS)- and interleukin 4 (IL4)-stimulated microglial uptake of [14C]2DG and [18F]BCPP-EF to give a new insight into the hypothesis that different uptake of [18F]FDG and [18F]BCPP-EF can be ascribed to the different metabolic pathways activated during microglial activation. LPS or IL4 stimulation increased the proinflammatory or anti-inflammatory marker gene expression in microglial cells. In LPS-stimulated cells, [14C]2DG uptake and glycolysis related gene expression were elevated, and [18F]BCPP-EF uptake was reduced. In IL4-stimulated cells, [18F]BCPP-EF uptake was increased, and [14C]2DG uptake was decreased. The expression of genes involved in glycolysis and mitochondrial complex I subunits was not changed by IL4 stimulation. The uptake of [14C]2DG and [18F]BCPP-EF differs in LPS- and IL4-stimulated polarized microglial cells. The present results suggest that the in vivo accumulation of metabolic tracers [18F]FDG and [18F]BCPP-EF can be influenced by the different aspects of neuroinflammation.

Conscious rat PET imaging with soft immobilization for quantitation of brain functions: comprehensive assessment of anesthesia effects on cerebral blood flow and metabolism.

BACKGROUND: Animal brain functions evaluated by in vivo imaging under anesthesia can be affected by anesthetic agents, resulting in incorrect assessment of physiological brain function. We therefore performed dynamic positron emission tomography (PET) imaging of conscious rats using recently reported soft immobilization to validate the efficacy of the immobilization for brain function assessments. We also determined the effects of six anesthetic agents-a mixed anesthetic agent (MMB), ketamine + xylazine (KX), chloral hydrate (Chloral), pentobarbital (PTB), propofol (PF), and isoflurane (IFL)-on brain function by comparison with conscious rats. RESULTS: The immobilization enabled 45-min dynamic [18F]FDG-PET acquisition with arterial blood sampling using conscious rats without the use of special techniques or invasive surgery. The spatial resolution and quantitativity of [18F]FDG-PET were not significantly lower for conscious rats than for anesthetized rats. While MMB, Chloral, PTB, and PF showed ubiquitous reduction in the cerebral metabolic rates of glucose (CMRglu) in brain regions, KX and IFL showed higher reductions in cerebellum and interbrain, and cerebellum, respectively. Cerebral blood flow (CBF) was reduced by MMB, KX, PTB, and PF; increased by IFL; and unaltered by Chloral. The magnitude of decrease in CMRglu and CBF for MMB were not larger than for other five anesthetic agents, although blood glucose levels and body temperature can be easily affected by MMB. CONCLUSION: The six anesthetic agents induced various effects on CMRglu and CBF. The immobilization technique presented here is a promising tool for noninvasive brain functional imaging using conscious rats to avoid the effects of anesthetic agents.

Macrophage-targeted, enzyme-triggered fluorescence switch-on system for detection of embolism-vulnerable atherosclerotic plaques.

The development of atherosclerotic plaques is a critical step that can result in an arterial embolism. Therefore, detection of these vulnerable plaques is of clinical significance for the diagnosis of atherosclerosis. However, there are few imaging systems able to detect such plaques easily. In this study, we designed a new platform for near-infrared fluorescence (NIRF) imaging of macrophages in atherosclerotic plaques, one using both a liposomal DDS and an activatable fluorescent probe, and evaluated the utility of this imaging for the diagnosis of atherosclerosis. We first synthesized a fluorescent switch-on probe, Peptide-ICG2, which is optically silent under normal conditions but activated in the presence of the lysosomal enzyme, cathepsin B. To achieve macrophage-specific fluorescence activation, we encapsulated Peptide-ICG2 into phosphatidylserine-containing liposome (P-ICG2-PS-Lip), since the accumulation of phosphatidylserine receptor-bearing macrophages is characteristic of embolism-vulnerable plaques. The experiments using macrophage-like RAW264 cells in culture showed that P-ICG2-PS-Lip was selectively taken up into the cells and that significant fluorescence of the probe was observed. For NIRF imaging of the atherosclerotic plaques, P-ICG2-PS-Lip was intravenously injected into ApoE-knockout atherosclerotic model mice or WHHL rabbits, and the fluorescence at the aortae was imaged. The results indicated that ICG fluorescence could be successfully observed at the plaques on the artery walls. The results of the present study thus suggest that NIRF imaging using P-ICG2-PS-Lip would be useful for detecting embolism-vulnerable atherosclerotic plaques.

Noninvasive quantitation of rat cerebral blood flow using 99mTc-HMPAO-assessment of input function with dynamic chest planar imaging.

BACKGROUND: Cerebral blood flow (CBF) quantitation using technetium-99m hexamethylpropyleneamine oxime (99mTc-HMPAO) generally requires assessment of input function by arterial blood sampling, which would be invasive for small animals. We therefore performed chest dynamic planar imaging, instead of arterial blood sampling, to estimate the input function and establish noninvasive quantitation method of rat CBF using the image-derived input function. RESULTS: Integrated radioactivity concentration in the heart-blood pool on planar images (AUCBlood-planar) was identical to that in arterial blood samples (AUCBlood-sampling). Radioactivity concentration in the brain determined by SPECT imaging (CBrain-SPECT) was identical to that using brain sampling (CBrain-sampling). Noninvasively calculated CBF obtained by dividing CBrain-SPECT by AUCBlood-planar was well correlated with conventionally estimated CBF obtained by dividing CBrain-sampling by AUCBlood-sampling. CONCLUSION: Rat CBF could be noninvasively quantitated using 99mTc-HMPAO chest dynamic planar imaging and head SPECT imaging without arterial blood sampling.

Quantitation of rat cerebral blood flow using 99mTc-HMPAO.

INTRODUCTION: Technetium-99m-hexamethylpropyleneamine oxime (99mTc-HMPAO) is potentially useful for the assessment of cerebral blood flow (CBF) in small animals. In this paper, a procedure for quantitation of rat CBF using 99mTc-HMPAO was determined. METHODS: Biodistribution of 99mTc-radioactivity in normal rats was determined after intravenous administration of 99mTc-HMPAO. Acetazolamide treated rats were intravenously administered with the mixture of 99mTc-HMPAO and N-isopropyl-[125I]iodoamphetamine ([125I]IMP), and arterial blood was then collected for 5min. After blood sampling, the brain radioactivity concentration was measured with the auto-well γ counter. RESULTS: The brain radioactivity concentration after intravenous administration of 99mTc-HMPAO was steady from 14s to 60min post-injection. A double tracer experiment using 99mTc-HMPAO and [125I]IMP showed that 19s was the average of the optimal integration interval of arterial blood 99mTc-radioactivity concentration to obtain CBF values measured by 99mTc-HMPAO identical to those determined by [125I]IMP. The CBF value determined by 99mTc-HMPAO, calculated by dividing the brain radioactivity concentration at 5min post-injection by the integrated arterial blood radioactivity concentration until 19s post-injection, was well correlated with CBF as determined by [125I]IMP. CONCLUSION: These results suggest that the CBF quantitation procedure described in this paper could be useful for rat CBF assessment.

PEG modification on (111)In-labeled phosphatidyl serine liposomes for imaging of atherosclerotic plaques.

INTRODUCTION: Previously, we reported a probe for imaging of atherosclerotic plaques: (111)In-labeled liposomes. Liposomes were modified with phosphatidylserine (PS) because macrophages recognize PS and phagocytize apoptotic cells in plaques. PS modification was successful and we could visualize atherosclerotic plaques by single-photon emission computed tomography (SPECT). However, too-rapid blood clearance reduced accumulation of PS-liposomes in plaques in vivo. Therefore, in the present study, PS-liposomes were modified with polyethylene glycol (PEG) to retard the rate of blood clearance. METHODS: PS-liposomes (size, 100 nm or 200 nm) were PEGylated with PEG2000 or PEG5000 at 1 or 5 mol%, and radiolabeled with (111)In. For the study of uptake in vitro, liposomes were incubated with mouse peritoneal macrophages. Biodistribution studies in vivo were carried out in ddY mice. En face autoradiograms were obtained with apoE(-/-) mice upon intravenous injection of (111)In-liposomes. RESULTS: Uptake was decreased significantly at 5 mol% PEGylation in 100-nm PS-liposomes (*P<0.05 vs. 0 mol%). All the PEGylated liposomes tested showed significantly lower uptake than the non-PEGylated control in 200-nm liposomes. In vivo results showed slower blood clearance in PEGylated liposomes. Autoradiograms in apoE(-/-) mice were well matched with Oil Red O staining. Additionally, 200-nm PS-liposomes modified with 5%PEG2000 ([(111)In]5%PEG2000PS200) showed the highest uptake to the region in vivo. CONCLUSIONS: As expected, PEGylation retarded the rate of blood clearance. In addition, it affected liposome uptake by macrophages in vitro. These results suggest that the balance between the rate of blood clearance and macrophage recognition is important, and [(111)In]5%PEG2000PS200 showed the best results in our investigation.

Development of 111In-labeled liposomes for vulnerable atherosclerotic plaque imaging.

UNLABELLED: Macrophage infiltration is a common characteristic feature of atherosclerotic-vulnerable plaques. Macrophages recognize phosphatidylserine (PS) exposed on the surface of apoptotic cells, which triggers the engulfment of the apoptotic cells by macrophages through phagocytosis. In this study, we prepared radiolabeled PS liposomes for detection of vulnerable plaques. METHODS: PS liposomes were prepared by lipid film hydration. Phosphatidylcholine (PC) liposomes were prepared as controls. Liposomes (100 or 200 nm) were generated by an extruder to produce PS100, PS200, PC100, and PC200 liposomes. These were then radiolabeled by encapsulating (111)In-nitrilotriacetic acid using an active-loading method. (111)In liposomes were incubated with cultured macrophages for 2 h, and the uptake level was measured. For biodistribution studies, the (111)In liposomes were injected intravenously into ddY mice. In addition, the (111)In liposomes were injected into apolipoprotein E-deficient (apoE-/-) mice, and the aortas were harvested for autoradiography and oil red O staining. For SPECT imaging, (111)In liposomes were injected intravenously into Watanabe heritable hyperlipidemic rabbits and scanned 48 h after injection. RESULTS: The radiochemical yields were greater than 95% for all the prepared (111)In liposomes. The level of in vitro uptake by macrophages was 60.5, 14.7, 32.0, and 14.4 percentage injected dose per milligram of protein for (111)In-PS100, (111)In-PC100, (111)In-PS200, and (111)In-PC200, respectively. In biodistribution studies, high spleen uptake was seen with PC liposomes. Liver uptake was high for all liposomes but was lowest with (111)In-PS200. The blood half-lives were 3.2, 22.0, 3.6, and 7.4 min for (111)In-PS100, (111)In-PC100, (111)In-PS200, and (111)In-PC200, respectively. The distribution of (111)In-labeled PS liposomes into atherosclerotic regions determined by autoradiography was well matched with the results of oil red O staining in apoE-/- mice. The target-to-nontarget ratios were 2.62, 2.23, 3.27, and 2.51 for (111)In-PS100, (111)In-PC100, (111)In-PS200, and (111)In-PC200, respectively. The aorta was successfully visualized by SPECT at 48 h after (111)In-labeled PS liposome injection; however, high liver uptake was also observed. DISCUSSION: From the in vitro uptake study, it has been demonstrated that macrophage targeting was accomplished by PS modification. Also, an atherosclerotic region was successfully detected by (111)In-PS200 in apoE-/- mice and Watanabe heritable hyperlipidemic rabbits in vivo. Liposome modification to obtain slower blood clearance and lower liver uptake would be required to improve the SPECT images.

What can be seen by 18F-FDG PET in atherosclerosis imaging? The effect of foam cell formation on 18F-FDG uptake to macrophages in vitro.

UNLABELLED: (18)F-FDG PET is a promising tool for detecting vulnerable plaques, depending on the extent of macrophage infiltration; however, it is still not clear which stage of the lesion can be detected by (18)F-FDG PET. METHODS: In this study, we investigated the effect of foam cell formation on (18)F-FDG uptake using cultured mouse peritoneal macrophages. RESULTS: (18)F-FDG accumulation was increased by foam cell formation, but the uptake was decreased to the control level after complete differentiation to foam cells. Changes in hexokinase activity tended to accompany changes in (18)F-FDG uptake. In contrast, changes in glucose-6-phosphatase activity and glucose transporter 1 expression did not parallel (18)F-FDG uptake. CONCLUSION: Our results suggest that (18)F-FDG PET detects the early stage of foam cell formation in atherosclerosis.