Bioimaging of metals by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS).

The distribution analysis of (essential, beneficial, or toxic) metals (e.g., Cu, Fe, Zn, Pb, and others), metalloids, and non-metals in biological tissues is of key interest in life science. Over the past few years, the development and application of several imaging mass spectrometric techniques has been rapidly growing in biology and medicine. Especially, in brain research metalloproteins are in the focus of targeted therapy approaches of neurodegenerative diseases such as Alzheimer's and Parkinson's disease, or stroke, or tumor growth. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) using double-focusing sector field (LA-ICP-SFMS) or quadrupole-based mass spectrometers (LA-ICP-QMS) has been successfully applied as a powerful imaging (mapping) technique to produce quantitative images of detailed regionally specific element distributions in thin tissue sections of human or rodent brain. Imaging LA-ICP-QMS was also applied to investigate metal distributions in plant and animal sections to study, for example, the uptake and transport of nutrient and toxic elements or environmental contamination. The combination of imaging LA-ICP-MS of metals with proteomic studies using biomolecular mass spectrometry identifies metal-containing proteins and also phosphoproteins. Metal-containing proteins were imaged in a two-dimensional gel after electrophoretic separation of proteins (SDS or Blue Native PAGE). Recent progress in LA-ICP-MS imaging as a stand-alone technique and in combination with MALDI/ESI-MS for selected life science applications is summarized.

Evaluation of registration strategies for multi-modality images of rat brain slices.

In neuroscience, small-animal studies frequently involve dealing with series of images from multiple modalities such as histology and autoradiography. The consistent and bias-free restacking of multi-modality image series is obligatory as a starting point for subsequent non-rigid registration procedures and for quantitative comparisons with positron emission tomography (PET) and other in vivo data. Up to now, consistency between 2D slices without cross validation using an inherent 3D modality is frequently presumed to be close to the true morphology due to the smooth appearance of the contours of anatomical structures. However, in multi-modality stacks consistency is difficult to assess. In this work, consistency is defined in terms of smoothness of neighboring slices within a single modality and between different modalities. Registration bias denotes the distortion of the registered stack in comparison to the true 3D morphology and shape. Based on these metrics, different restacking strategies of multi-modality rat brain slices are experimentally evaluated. Experiments based on MRI-simulated and real dual-tracer autoradiograms reveal a clear bias of the restacked volume despite quantitatively high consistency and qualitatively smooth brain structures. However, different registration strategies yield different inter-consistency metrics. If no genuine 3D modality is available, the use of the so-called SOP (slice-order preferred) or MOSOP (modality-and-slice-order preferred) strategy is recommended.

Comparison of 99mTc- and 18F-ubiquicidin autoradiography to anti-Staphylococcus aureus immunofluorescence in rat muscle abscesses.

UNLABELLED: 99mTc-ubiquicidin (UBI) 29-41 is under clinical evaluation for discrimination between bacterial infection and unspecific inflammation. We compared the distribution of 99mTc-UBI 29-41, the potential PET tracers 18F-UBI 29-41 and 18F-UBI 28-41, and 3H-deoxyglucose (DG) in rat muscle abscesses to that of anti-Staphylococcus aureus immunofluorescent imaging. METHODS: Calf abscesses were induced in 15 CDF-Fischer rats after inoculation of Staphylococcus aureus. One to 6 d later, either 18F-UBI 29-41 and 3H-DG (n = 5) or 18F-UBI 28-41 and 3H-DG (n = 6) or 99mTc-UBI 29-41 and 3H-DG (n = 4) were injected simultaneously. Dual-tracer autoradiography of the abscess area was compared with the distribution of bacteria and macrophages. RESULTS: The UBI derivates exhibited increased uptake in the abscess area that partly matched 3H-DG uptake and macrophage infiltration but showed no congruity with areas that were highly positive for bacteria. CONCLUSION: A specific binding of UBI derivatives to Staphylococcus aureus in vivo could not be confirmed in this study.

cis-4-[(18)F]-Fluoro-l-proline fails to detect peripheral tumors in humans.

UNLABELLED: System A amino acid transport is increased in transformed and malignant cells. The amino acid 4-cis[(18)F]fluoro-l-proline (cis-[(18)F]FPro) has been shown to be a substrate of the System A amino acid carrier. In this pilot study, we investigated the diagnostic potential of cis-[(18)F]FPro in patients with various tumors in comparison with [(18)F]fluorodeoxyglucose-positron emission tomography (FDG-PET). METHODS: Eight patients (seven females, one male, age range 43-77 years) with large primary, recurrent or metastatic tumors of different histologies were included in this study. One patient had a recurrent non-Hodgkin lymphoma; two patients, metastatic colon or rectal cancer; one, a metastatic endometrial cancer; one, a multiple myeloma; one, an Ewing sarcoma; one, a metastatic breast cancer and one, a gastrointestinal stromal tumor. PET scans of the trunk were acquired at 1 h after intravenous injection of 400 MBq cis-[(18)F]FPro and compared to PET scans with [(18)F]FDG. RESULTS: None of the tumors or metastatic lesions in this series of patients demonstrated relevant uptake of cis-[(18)F]FPro. In contrast, all tumors with exception of the multiple myeloma showed an intensive uptake of [(18)F]FDG. The mean standardized uptake value of cis-[(18)F]FPro in the tumor or metastases was significantly lower than that of [(18)F]FDG uptake (1.7+/-0.6 vs. 5.7+/-3.0; n=8; P<.01). CONCLUSION: Although other System A-specific tracers have shown relevant tumor uptake, cis-[(18)F]FPro fails to detect most types of human tumors. Based on these results, we cannot recommend a further evaluation of this tracer as a tumor-seeking agent.

Comparison of intravenous and intraperitoneal [123I]IBZM injection for dopamine D2 receptor imaging in mice.

INTRODUCTION: Intraperitoneal (IP) injection represents an attractive alternative route of radiotracer administration for small animal imaging, e.g., for longitudinal studies in transgenic mouse models. We explored the cerebral kinetics of the reversible dopamine D2 receptor ligand [(123)I]IBZM after IP injection in mice. METHODS: Cerebral [(123)I]IBZM kinetics were assessed by ex vivo autoradiography in mice sacrificed between 30 and 200 min after IP or intravenous (IV) injection. The striatum-to-cerebellum (S/C) uptake ratio at 140 min was evaluated in wild-type mice and R6/2 transgenic mice (a Huntington's disease model) in comparison with in vitro autoradiography using [(3)H]raclopride. RESULTS: [(123)I]IBZM uptake was slower and lower after IP injection [maximum uptake in striatum 5.6% injected dose per gram (ID/g) at 60 min] than IV injection (10.5%ID/g at 30 min). Between 60 and 120 min, striatal (cerebellar) uptake after IP injection reached 63% (91%) of the uptake after IV injection. The S/C uptake ratio increased to 15.5 at 200 min after IP injection, which corresponds to 87% of the IV injection value (17.8). Consistent with in vitro [(3)H]raclopride autoradiography, the S/C ratio given by ex vivo [(123)I]IBZM autoradiography (140 min after IP injection) was significantly reduced in R6/2 mice. CONCLUSIONS: Although IP injection resulted in slower kinetics, relevant measures of dopamine D2 receptor availability were comparable. Thus, IP injection represents a promising route of tracer administration for small animal [(123)I]IBZM SPECT. This should considerably simplify the implementation of longitudinal small animal neuroimaging studies, e.g., in transgenic mouse models.

Cerebral kinetics of the dopamine D(2) receptor ligand [(123)I]IBZM in mice.

INTRODUCTION: In vivo small animal imaging of the dopaminergic system is of great interest for basic and applied neurosciences, especially in transgenic mice. Small animal SPECT is particularly attractive because of its superior spatial resolution and tracer availability. We investigated the kinetics of the commercial dopamine D(2) receptor (DZR) ligand [(123)I]IBZM in mice as a prerequisite for an appropriate design of translational SPECT imaging between mice and humans. METHODS: Cerebral kinetics of [(123)I]IBZM under isoflurane anaesthesia were assessed by autoradiography in mice sacrificed at 30, 60, 120 and 200 min after iv injection. To explore the possible effects of isoflurane anaesthesia, an additional mice group was only anaesthetized for 20 min before being sacrificed at 140 min (putative time of single-scan SPECT analysis). RESULTS: Maximum [(123)I]IBZM uptake in the striatum (D(2)R-rich; 10.5+/-2.7 %ID/g) and cerebellum (D(2)R-devoid; 2.4+/-0.7 %ID/g) was observed at 30 min after injection. Thereafter, [(123)I]IBZM uptake decreased slowly in striatum and rapidly in the cerebellum (200 min: 5.3+/-1.9 and 0.4+/-0.2 %ID/g, respectively). The striatum-to-cerebellum (S/C) [(123)I]IBZM uptake ratio increased from 4.6+/-1.2 at 30 min to 11.6+/-2.6 at 120 min. The S/C ratio at 200 min was highly variable (17.8+/-10.1), possibly indicating pseudo-equilibration in some animals. In mice, which were only anaesthetized between 120 and 140 min, a higher S/C ratio of 17.0+/-5.1 was observed. CONCLUSIONS: The present study suggests that [(123)I]IBZM is a suitable ligand for D(2)R-SPECT in mice. Although a single-scan analysis may be a pragmatic semi-quantitative approach, tracer kinetic analyses on dynamic SPECT data should be pursued. The interfering effects of isoflurane anaesthesia need to be considered.

Detection of secondary thalamic degeneration after cortical infarction using cis-4-18F-fluoro-D-proline.

UNLABELLED: The amino acid cis-4-(18)F-fluoro-D-proline (D-cis-(18)F-FPro) exhibits preferential uptake in the brain compared with its L-isomer, but the clinical potential of the tracer is as yet unknown. In this study we explored the cerebral uptake of D-cis-(18)F-FPro in rats with focal cortical infarctions. METHODS: Focal cortical infarctions were induced in different areas of the cortex of 20 Fisher CDF rats by photothrombosis (PT). At variable time points after PT (1 d to 4 wk), the rats were injected intravenously with D-cis-(18)F-FPro. For comparison, 12 rats were injected simultaneously with (3)H-deoxyglucose ((3)H-DG), 3 rats were injected with (3)H-methyl-L-methionine ((3)H-MET), and 2 rats were injected with (3)H-PK11195. Within 2 h after injection of the tracers, coronal cryosections of the brains were produced and evaluated by dual-tracer autoradiography. Lesion-to-brain ratios (L/B ratios) were calculated by dividing the maximal uptake in areas with increased tracer uptake by the mean uptake in normal brain tissue. Histologic slices were stained by toluidine blue and by immunostainings for glial fibrillary acidic protein (GFAP), CD68 for macrophages, and CD11b for microglia. RESULTS: Prominent uptake of D-cis-(18)F-FPro was found in ipsilateral thalamic nuclei (TN) and partially in the corpus striatum starting at 3 d after infarction with increasing L/B ratios up to 4 wk (mean L/B ratio +/- SD, 6.7 +/- 3.5). The involved TN varied with the site of the cortical lesion corresponding to their thalamocortical projections connecting them with their specific target region in the cerebral cortex. The TN were positive for CD11b and GFAP from day 7 onward, whereas uptake of (3)H-DG, (3)H-MET, and (3)H-PK11195 and immunostaining for CD68 were similar to that of normal brain. Furthermore, increased uptake of D-cis-(18)F-FPro was found in the area of the cortical infarctions (mean L/B ratio +/- SD, 12.1 +/- 8.1). From day 5 onward, the pattern of uptake was congruent with that of immunostaining for CD11b and CD68 but was different from that of GFAP. CONCLUSION: D-cis-(18)F-FPro appears to be a sensitive PET tracer for detection of secondary degeneration of TN after cortical injury. The uptake mechanisms of D-cis-(18)F-FPro remain to be elucidated, but the relationship to microglial activation suggests a diagnostic potential in various brain diseases.

Differential uptake of O-(2-18F-fluoroethyl)-L-tyrosine, L-3H-methionine, and 3H-deoxyglucose in brain abscesses.

UNLABELLED: The amino acid O-(2-(18)F-fluoroethyl)-L-tyrosine ((18)F-FET) has been shown to be a useful tracer for brain tumor imaging. Experimental studies demonstrated no uptake of (18)F-FET in inflammatory cells but increased uptake has been reported in single cases of human brain abscesses. To explore this inconsistency, we investigated the uptake of (18)F-FET in comparison with that of L-[methyl-(3)H]methionine ((3)H-MET) and D-(3)H-deoxyglucose ((3)H-DG) in brain and calf abscesses in rats. METHODS: Abscesses were induced in the brain (n = 9) and calf (n = 5) of Fisher CDF rats after inoculation of Staphylococcus aureus. Five days later, (18)F-FET and (3)H-MET (n = 10) or (18)F-FET and (3)H-DG (n = 4) were injected intravenously. One hour after injection the rats were sacrificed, and the brain or calf muscle was investigated using dual-tracer autoradiography. Lesion-to-background ratios (L/B) and standardized uptake values (SUVs) were calculated. The autoradiograms were compared with histology and immunostaining for glial fibrillary acidic protein (GFAP), CD68 for macrophages, and CD11b for microglia. RESULTS: (18)F-FET uptake in the area of macrophage infiltration and activated microglia at the rim of the brain abscesses was low (L/B, 1.5 +/- 0.4). In contrast, high uptake was observed for (3)H-MET as well as for (3)H-DG (L/B, 4.1 +/- 1.1 for (3)H-MET vs. 3.1 +/- 1.5 for (3)H-DG; P < 0.01 vs. (18)F-FET). Results for calf abscesses were similar. In the vicinity of the brain abscesses, slightly increased uptake was noted for (18)F-FET (L/B, 1.8 +/- 0.3) and (3)H-MET (L/B, 1.8 +/- 0.4), whereas (3)H-DG distribution was normal (L/B, 1.2 +/- 0.2). Anti-GFAP immunofluorescence showed a diffuse astrocytosis in those areas. CONCLUSION: Our results demonstrate that there is no accumulation of (18)F-FET in macrophages and activated microglia in experimental brain abscesses, whereas (3)H-MET and (3)H-DG exhibit high uptake in these cells. Thus, the specificity of (18)F-FET for gliomas may be superior to that (3)H-MET and (3)H-DG. Increased (18)F-FET uptake in human brain abscesses appears to be related to reactive astrocytosis.

Differential uptake of [18F]FET and [3H]l-methionine in focal cortical ischemia.

UNLABELLED: Amino acids such as [(11)C-methyl]l-methionine are particularly useful in brain tumor diagnosis, but unspecific uptake (e.g., in cerebral ischemia) has been reported. O-(2-[(18)F]fluoroethyl)-l-tyrosine ([(18)F]FET) shows a clinical potential similar to that of l-methionine (MET) in brain tumor diagnosis but is applicable on a wider clinical scale. The aim of this study was to evaluate the uptake of [(18)F]FET and [(3)H]MET in focal cortical ischemia in rats by dual-tracer autoradiography. METHODS: Focal cortical ischemia was induced in 25 CDF rats using the photothrombosis (PT) model. At different time points up to 6 weeks after the induction of PT, [(18)F]FET and [(3)H]MET were injected intravenously. Additionally, contrast-enhanced magnetic resonance imaging (MRI) was performed in 10 animals. One hour after tracer injection, brains were cut in coronal sections and evaluated by dual-tracer autoradiography. Lesion-to-brain (L/B) ratios were calculated by dividing the maximal uptake in the lesion by the mean uptake in the brain. An L/B ratio of >2.0 was considered indicative of pathological uptake. Histological slices were stained by cresyl violet and supplemented by immunostainings for glial fibrillary acidic protein (GFAP) and CD68 in selected cases. RESULTS: A variably increased uptake of both tracers was observed in the PT lesion and its demarcation zone up to 7 days after PT for [(18)F]FET and up to 6 weeks for [(3)H]MET. The cutoff level of 2.0 was exceeded in 12/25 animals for [(18)F]FET and in 18/25 animals for [(3)H]MET. Focally increased tracer uptake matched contrast enhancement in MRI in 3/10 cases for [(18)F]FET and in 5/10 cases for [(3)H]MET. Immunohistochemical staining in lesions with differential uptake of [(18)F]FET and [(3)H]MET revealed that selective uptake of [(18)F]FET was associated with GFAP-positive astrogliosis while selective [(3)H]MET uptake correlated with CD68-positive macrophage infiltration. CONCLUSIONS: [(18)F]FET, like [(3)H]MET, may exhibit significant uptake in the periphery of cortical infarctions, which has to be considered in the differential diagnosis of unknown brain lesions. There are discrepancies between [(18)F]FET and [(3)H]MET uptake in the area of infarctions that appear to be caused by the preferential uptake of [(18)F]FET in reactive astrocytes versus the preferential uptake of [(3)H]MET in macrophages.

Bioimaging of metals in brain tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and metallomics.

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been developed and established as an emerging technique in the generation of quantitative images of metal distributions in thin tissue sections of brain samples (such as human, rat and mouse brain), with applications in research related to neurodegenerative disorders. A new analytical protocol is described which includes sample preparation by cryo-cutting of thin tissue sections and matrix-matched laboratory standards, mass spectrometric measurements, data acquisition, and quantitative analysis. Specific examples of the bioimaging of metal distributions in normal rodent brains are provided. Differences to the normal were assessed in a Parkinson's disease and a stroke brain model. Furthermore, changes during normal aging were studied. Powerful analytical techniques are also required for the determination and characterization of metal-containing proteins within a large pool of proteins, e.g., after denaturing or non-denaturing electrophoretic separation of proteins in one-dimensional and two-dimensional gels. LA-ICP-MS can be employed to detect metalloproteins in protein bands or spots separated after gel electrophoresis. MALDI-MS can then be used to identify specific metal-containing proteins in these bands or spots. The combination of these techniques is described in the second section.

Comparison of O-(2-18F-fluoroethyl)-L-tyrosine and L-3H-methionine uptake in cerebral hematomas.

UNLABELLED: Radiolabeled amino acids are useful for brain tumor diagnosis, but unspecific uptake near the cerebral hematoma may complicate the differentiation of a neoplastic from a nonneoplastic origin of the hematoma. The aim of this study was to investigate the pattern and time course of O-(2-(18)F-fluorethyl)-l-tyrosine ((18)F-FET) and l-(3)H-methionine ((3)H-MET) uptake in rats with cerebral hematomas. METHODS: Intracerebral hematomas were induced in the striatum of 25 Fischer 344 CDF rats by inoculation of bacterial collagenase. (18)F-FET and (3)H-MET were injected intravenously at different times up to 4 wk after bleeding. One hour after tracer injection, brains were cut in coronal sections and evaluated by dual-tracer autoradiography. Lesion-to-brain (L/B) ratios were calculated by dividing maximal uptake near the hematomas and mean uptake in normal brain tissue. An L/B ratio greater than 1.5 was considered as indicative of pathologic uptake. The autoradiograms were compared with histology and immunostainings for astrogliosis (glial fibrillary acidic protein) and macrophage infiltration (CD68). RESULTS: (18)F-FET exhibited significantly increased uptake near the hematomas between 3 and 14 d after bleeding. The time course of pathologic (3)H-MET uptake was similar, but after 3-4 wk there was still borderline uptake in single animals. The L/B ratios exceeded the cutoff level of 1.5 in 10 of 23 animals for (18)F-FET and in 12 of 22 animals for (3)H-MET but did not exceed a value of 3. Immunostainings indicated that increased uptake of both tracers correlated with reactive astrogliosis, whereas (3)H-MET uptake was additionally increased in areas with macrophage infiltration. CONCLUSION: (18)F-FET, like (3)H-MET, may exhibit significantly increased uptake near cerebral hematomas, especially during the first 2 wk after bleeding, complicating the differentiation between a neoplastic and a nonneoplastic origin of cerebral hematomas.