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Multimodal ex vivo Mouse Ischemic Brain Lesion Visualization: Quest for a Cross-compatible Fixation Protocol (#434)
S. Škokić1, M. Dobrivojević Radmilović1, C. Dullin2, 3, G. Tromba2, S. Gajović1
1 University of Zagreb School of Medicine, Croatian Institute for Brain Research, Zagreb, Croatia
The assessment of mouse ischemic brain lesion relies greatly on in vivo imaging. However, ex vivo imaging is preferred for observing tissue architecture at higher resolutions, for more accurate mapping of events across the brain. Tissue fixation involves dehydration and structural changes which often reduce MRI signal-to-noise (SNR) and contrast-to-noise (CNR) ratios. Another issue is the incompatibility of tissue preparation protocols for different imaging modalities, such as the use of ethanol in MRI. In this study, we evaluate the cross-compatibility of tissue preparation protocols.
C57Bl/6 albino mice (2 groups, N = 3) underwent cerebral ischemia induced by 60-minute middle cerebral artery occlusion. In control mice, the filament was not completely inserted. In group 1, the isolated brains were prepared by Evaporation-of-Organic-Solvent (EOS) method, yielding water- and lipid-free specimens for high-resolution synchrotron phase-contrast CT imaging (SRµCT) and afterward rehydrated for MRI. In group 2, the isolated brains were first fixated with 4% PFA for MRI imaging and subsequently treated by EOS to be imaged with SRµCT.
SRµCT images were reconstructed using standard filtered backprojection algorithm (SYRMEP Tomo Project). MRI imaging was done on a Bruker BioSpec 70/20 USR system, using fast spin echo T2 weighted sequences with a spatial resolution of 0.1x0.1x0.4 mm.
The contrast between the lesion area and surrounding healthy tissue in MRI images was higher in group 1 than in group 2. Conversely, the morphological features of the brain tissue were better preserved in group 2 than in group 1. This can be attributed to EOS tissue preparation, which removes both water and lipids from specimens and affects predominantly the myelin sheath structure. The result can be observed with MRI as a change in T2 response, affecting mostly the myelin-abundant regions such as the Corpus callosum.
Furthermore, we observed that the rehydration of EOS-prepared samples (group 1) did not reproduce the same level of SNR as in PFA-fixated samples (group 2) nor did the rehydrated brains regain their initial size after rehydration, the effect of which obscures direct volumetric comparisons.
EOS tissue preparation preserves sufficient contrast between the ischemic lesion and surrounding tissue both on MRI and SRµCT images. However, rehydration does not increase SNR sufficiently for subsequent MRI imaging.
This work has been supported in part by Croatian Science Foundation under the project IP-06-2016-1892 RepairStroke and in part by Synchrotron Light Source ‘Elettra’ grant n.20170140. The MRI scans were performed at the Laboratory for Regenerative Neuroscience – GlowLab, University of Zagreb, Croatia.
Keywords: MRI, synchrotron, ex vivo, brain, stroke
Evaluation of the novel TSPO radiotracer [18F] VUIIS1008 in a preclinical model of cerebral ischemia in rats (#273)
K. R. Pulagam1, L. Colás2, D. Padro3, S. Plaza-García3, V. Gómez-Vallejo1, M. Higuchi4, J. Llop1, A. Martin2
1 CIC biomaGUNE, Radiochemistry and Nuclear imaging Lab, San Sebastian, Spain
Cerebral ischemia is accompanied by a marked inflammatory reaction resulting in a strong activation of the resident glial cells and leukocyte infiltration.1 In vivo positron-emission tomography (PET) imaging of transporter protein (TSPO) expression is an attractive and indispensable tool for the diagnosis and therapy evaluation of neuroinflammation after cerebral ischemia. Despite several radiotracers have shown an excellent capacity to image neuroinflammation, novel radiotracers such as [18F] VUIIS1008 have shown promising properties to visualize and quantify the in vivo expression of TSPO.
Longitudinal in vivo magnetic resonance (MRI) and PET imaging studies with the novel TSPO radiotracer 2-(5,7-diethyl-2-(4-(2-[18F] fluoroethoxy) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl)-N, N-diethylacetamide ([18F] VUIIS1008), and (N, N-diethyl-2-(2-[4-(2-fluoroethoxy)-phenyl]-5,7-dimethyl-pyrazolo [1,5-a] yrimidin-3-yl)-acetamide ([18F] DPA-714) were carried out before and at days 1, 3, 7, 14, 21, and 28 following the transient middle cerebral artery occlusion (MCAO) in rats.
MRI images showed the extension and evolution of the brain infarction after ischemic stroke in rats. PET imaging with [18F] VUIIS1008 and [18F] DPA714 showed a progressive increase in the ischemic brain hemisphere during the first week, peaking at day 7 and followed by a decline from days 14 to 28 after cerebral ischemia. [18F] DPA714 uptake showed a mild uptake increase compared to [18F] VUIIS1008 in TSPO-rich ischemic brain regions. In vivo [18F] VUIIS1008 binding displacement with VUIIS1008 was more efficient than DPA714. Finally, immunohistochemistry confirmed a high expression of TSPO in microglial cells at day 7 after the MCAO in rats.
We report here the PET imaging of TSPO with both [18F] VUIIS1008 and [18F] DPA-714 in a rat model of cerebral ischemia. Our results confirmed the progressive binding increase of [18F] VUIIS1008 in the ischemic hemisphere during the first week after cerebral ischemia, followed by a decline later on.These findings are consistent with the PET signal uptake pattern of [18F] DPA-714, a well-known radiotracer for TSPO. Altogether, these findings suggest that [18F] VUIIS1008 could become a valuable tool for the diagnosis and treatment evaluation of neuroinflammation following ischemic stroke.
1. Kim JY, Kawabori M, Yenari MA. Curr Med Chem. 2014;21(18): 2076–97.
This study was funded by Spanish Ministry of Economy and Competitiveness (SAF2014-54070-JIN) and the project FATENANO (PCIN-2015-116 and ERA-NET SIINN – 2014, ID.46).
Keywords: T2W-MRI, PET, Cerebral ischemia
Construction of an flow-phantom for MRI based on MR imaging data of a rat’s head (#485)
F. Euchner1, C. Bruns1, R. Ringleb1, D. Müller1, U. Bommerich1, T. Herrmann1, M. Plaumann1, J. Bernarding1
1 Otto-von-Guericke University, Institute for Biometrics and Medical Informatics, Magdeburg, Saxony-Anhalt, Germany
Imaging of blood vessels is important e.g. is case of stroke. Nearly every third person in Germany has problem with calcification. Therefore, it is important to find narrowings of the blood vessels as early as possible. Especially perfluorinated contrast agents have excellent properties for angiography examinations. The low solubility in aqueous solution enables the detection of a bolus in 19F MRI studies.[1,2] To reduce the number of animals experiments,in the first basic research step, we synthesize a new MR flow phantom which allows e.g. an estimation of concentration of the contrast agent.
In the first step 1H anatomical MR images of a rat’s head were measured on a Bruker 4.7 T animal scanner. Afterwards 1H MR angiography experiments show the position and size of the blood vessels. All measurements occur with the same Doty volume coil. With these imaging data segmentations of the vessels were done and a 3D model of the head was printed out by a 3D-printer (Ultimaker2 Extended). For recycling of the phantom a flexible hoses, which can easily cleaned by flushing, is placed in the right position. This allows a reproducable injection of a contrast agent. To minimize trouble with air, the phantom was filled with agarose gel (1%). For first comparable measurements the artificial vessels were filled with a) water and b) trifluoroethanol dissolved in water.
The flowchart (Figure 1) shows step by step the contruction of the new phantom. Exemplarily, slice number 20 (of 40) of the basic data set of the anatomical measurements (RARE-sequence, axial) is shown in Figure 2 a). The corresponding slice of the angiography (FLASH-TOF-2D-flow-comp) is presented in Figure 2b). As it can be observed in Figure 2c), which shows a slice of the 19F images of the trifluoroethanol filled phantom, the main vessels in rat brain have the same diameter and position as in the phantom. The printing procedure and the 3D print (including the hoses) are shown in Figure 2d) and e). The new phantom allows the first determination of necessary concentration of new MR contrast agents. Furthermore, parameters of novel pulse sequencies can be tested and optimized using this model.
In conclusion, we present the construction of a new phantom, which allows first MR-measurements to optimize experimental parameters, such as concentration, flow rate or pulse sequencies. The 3D printed phantom based on measured rat head parameters. A fast repetition of an experiment is is possible and a change of a contrast agent of interest can be realized very fast.
 Ojima I. Fluorine in Medical Chemistry and Chemical Biology, 1. Ed., Wiley-Blackwell: Chichester, 2009.
 Ruiz-Cabello J, et al. Fluorine 19F MRS and MRI in biomedicine. NMR Biomed. 2011;24(2):114-129.
This work was supported by the Deutsche Forschungsgemeinschaft (DFG BE 1824/8-1)
Keywords: MR-phantom, 19F imaging, contrast agent, 3D print, MR angiography
Nuclear imaging of neuroinflammation with nanobodies targeting Cell Adhesion Molecules (#415)
M. Vandesquille1, D. Jasim2, D. Bochicchio1, K. Kostarelos2, M. Fairclough1, N. Devoogdt3, C. Vincke4, S. Muyldermans4, L. Parkes5, H. Boutin1
1 University of Manchester, Wolfson Molecular Imaging Centre, Faculty of Biology, Medicine and Health, School of Health Sciences, Division of Informatics, Imaging and Data Sciences, Manchester, United Kingdom
Neuroinflammation is a critical feature in many disorders including stroke, diabetes, hypertension or neurodegenerative diseases. This process includes early expression of Cell Adhesion Molecules (CAMs) at the luminal surface of endothelial cells1. Thus, specific imaging probes for CAMs appear to be promising tools to detect brain inflammation earlier, quantify disease activity and guide specific therapy. In this study, we describe the screening and selection by SPECT/CT of nanobodies (Nbs) targeting three different CAMs: P-selectin, ICAM-1, and VCAM-1, in a rat model of neuroinflammation.
Ten Nbs were studied: 3 per targeted CAM, and 1 irrelevant Nb (Bcll10) design with no specific target. These 6xHis-tag-Nbs were radiolabelled with 99mTc as previously described2. Neuroinflammation was induced in Wistar rats by injection of TNF-α (2×1 µL, 0.5 µg/µL in PBS) in the right striatum. PBS was injected in the left striatum as control. After 24h, rats were injected in the tail vein with 99mTc radiolabelled Nbs and imaged 3h later by SPECT/CT (NanoScan, Mediso). Analyses of the Signal to Noise Ratio (SNR) were done by quantification of the Standard Uptake Value (SUV) in the ipsi- to contralateral striatum (BrainVisa, Anatomist).
The screening of 9 Nbs targeting CAMs in a rat model of neuroinflammation led us to the selection of one Nb specific for VCAM-1: Nb3873. Representative SPECT/CT images obtained after iv injection of irrelevant (Bcll10) and specific Nb3873 Nbs are illustrated in Figure 1 A and B respectively. As shown in Figure 2, comparison of the mean SUV per striatum in these two groups revealed: 1) no specific Nb uptake in the PBS-injected striata (p>0.999); 2) no significant difference between ipsi- and contralateral sides in the irrelevant Bcll10 group (p=0.088), suggesting that the injection of TNF-α do not significantly modify the diffusion of Nbs; 3) a significant 25-fold SUV increase in ipsi- to contralateral sides in the VCAM-1 group (p<0.001), and a 10-fold SUV increase between Nb3873 vs. Bcll10 in ipsilateral sides (p<0.001).
Results demonstrated a high specific ipsi- to contralateral uptake of this 99mTc-Nb3873 tracer. Ongoing experiments will bring information about the biodistribution of Nb3873 and increase the number of animals per group. The next step will be to evaluate these molecular tracers in animal models of hypertension (Spontaneous Hypertensive rats) and Alzheimer’s disease (TgF344-AD rats). Moreover, radiolabelling of the Nbs with 18F is in development to extend the application to PET imaging.
Man, S., et al. (2007). Brain Pathol 17(2): 243-250.
Xavier, C., et al. (2012). Methods Mol Biol 911: 485-490.
This work was funded by the Engineering and Physical Sciences Research Council (grant EP/M005909/1).
Representative SPECT/CT images of 99mTc-Nbs in a rat model of neuroinflammation
Figure 1: Representative SPECT/CT images obtained 3 hours after iv injection of 99mTc-Nbs in a rat model of neuroinflammation. A: Images obtained with the irrelevant 99mTc-Nb (Bcll10) showing the absence of specific binding in both PBS and TNF-α injected striata. B: Images obtained with a 99mTc-Nb3873 targeting VCAM-1 demonstrate the high binding of the tracer in the ipsilateral striatum only.
In vivo quantification of SPECT images
Figure 2: In vivo quantification of SPECT images: binding of both the irrelevant (blue, n=4) and the VCAM-1 (red, n=2) 99mTc-Nbs in contra- and ipsilateral sides. Results are expressed as the mean SUV per striatum ± SEM (*** shows significant differences, p<0.001).
Keywords: Cell adhesion molecules, VCAM-1, nanobody, SPECT, neuroinflammation
Preclinical evaluation of myelin radiotracers in a rat model of focal demyelination (#212)
M. Zhang1, 2, G. Hugon1, T. Billard3, 4, C. Bouillot4, R. Bolbos4, J. - B. Langlois4, B. Li2, L. Zimmer1, 4, F. Chauveau1
1 Univ. Lyon, Lyon Neuroscience Research Center (CRNL); CNRS UMR5292; INSERM U1028, Univ. Lyon 1, Lyon, France
The formation of focal demyelinated lesions and progressive failure of remyelination is the main characteristic of multiple sclerosis (MS). While several remyelination strategies are currently under clinical investigation , there is no consensus on which imaging technique can offer the best reliable measure of remyelination . Positron Emission Tomography (PET) may provide a direct and quantitative detection of myelin content. Building on pioneer [11C]PiB studies , the main objective of this work is to progress towards a second generation of fluorine-18 labelled myelin radiotracers.
[18F]BF227 (n=10), [18F]AV-45 (n=5) and [11C]PiB (n=5) were evaluated in a rat model of focal demyelination induced by a stereotactic injection of lyso-phosphatidyl-choline (LPC, or lysolecithin) in the right corpus callosum, vs saline in the contralateral site. MRI (Bruker Biospec 7T) was used to confirm demyelination, measure diffusion parameters, and exclude animals with necrosis or hydrocephalus. In vivo PET (Inveon, Siemens) was immediately followed by ex vivo autoradiography (BAS-5000, Fujifilm) to get high resolution images. Brain sections were subsequently stained with Sudan Black B (SBB).
In vivo, [18F]BF227 and [11C]PiB showed a similar ratio of WM to GM uptake in the whole brain (1.2 at 30min) while [18F]AV-45 showed an early, increased ratio (1.3 at 10min). Despite extended areas of demyelination observed on MRI, focal demyelination was hardly detected on PET images (Fig. 1), whatever the radiotracer: corpus callosum ipsi-to-contralateral uptake ratios were comprised between 0.95 and 1. Reliable autoradiograms were obtained for F-18 radiotracers only. [18F]BF227 and [18F]AV-45 showed similar WM to GM uptake ratios, and similar corpus callosum ipsi-to-contralateral uptake ratios (Fig. 2A). Only [18F]AV-45 showed a signiﬁcant correlation with the optical density of SBB-stained myelin measured in the same sections (Fig. 2B).
In contrast to previous reports , LPC-induced demyelination was not detected in vivo. MRI highlighted pitfalls of the animal model that might lead to false-negative PET detection. Nevertheless, high-resolution ex vivo autoradiography after MRI-confirmed demyelination suggests that [18F]AV-45 provides a better myelin biomarker than [18F]BF227 and deserve further investigation in MS models or patients.
(A) Autoradiography yielded similar results for [18F]BF227 and [18F]AV-45, in terms of i) white matter (WM, green ROI) to gray matter (GM, violet ROI) uptake ratio, and ii) ipsilateral (red ROI) to contralateral (green ROI) uptake ratio. (B) Only [18F]AV-45 correlated significantly with optical density ratio after Sudan Black B staining of myelin (*p<0.05).
Keywords: Multiple Sclerosis, myelin, radiotracer
Toolboxes for Allen brain atlas registration of MR and histology images to study functional recovery after stroke in the mouse (#38)
S. P. Koch1, 2, F. Knab1, U. Grittner1, R. Bernard1, S. Mueller1, 2, M. Foddis1, J. Lips1, P. Euskirchen1, N. Zerbe3, P. Hufnagl3, A. Rex1, U. Dirnagl1, T. D. Farr4, C. Harms1, P. Boehm-Sturm1, 2
1 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Experimental Neurology and Center for Stroke Research Berlin, Berlin, Berlin, Germany
Atlas registration is a prerequisite for many advanced postprocessing techniques such as voxel-wise group statistics. We have previously developed a MATLAB toolbox for registration of MR images to the Allen brain atlas (ABA).1,2 Here, using techniques modified from voxel-based lesion symptom mapping3, we introduce correlative methods for mapping of behavioral data on MR images to identify anatomical regions whose damage best explain a functional deficit. Furthermore, a toolbox for image registration of the ABA to stained tissue sections is presented in order to validate the imaging results.
60 C57/Bl6 mice were trained in the staircase test4, and randomized to undergo 45min middle cerebral artery occlusion (MCAO, n=17), sham surgery (n=17) or photothrombosis (PT, n=9). Experimenters were blinded to the condition of animals. MR images acquired 24 h post stroke (RARE, 32 0.5 mm thick slices, FOV=(25.6 mm)2, MTX=2562, TR/TE=4.2 s/36 ms, ETL=4, 6:43 min) at 7 T (Bruker, Germany) were registered to the ABA.2 The mean number of reached pellets at 2-6 d was correlated i) region-wise with lesion volume or ii) voxel-wise with image intensity. Animals were sacrificed at 21 d and NeuN+ cells were automatically detected (http://cellprofiler.org/) on tissue sections. Corresponding slices of the ABA were warped to each tissue slice using a custom ELASTIX-based MATLAB toolbox.
PT expectedly produced more cortically located strokes. PT mice had smaller deficits than in the MCAO group, indicating relevance of subcortex. For MCAO, a trivial correlation of performance of the left paw and total stroke volume (r=-0.73) was found. Atlas- and voxel-based symptom mapping further resolved areas involved in somatosensory and fear processing but also more caudal structures, e.g. thalamus and midbrain (Fig. 1). A limitation was the high number of excluded animals, strategies to statistically deal with this need to be explored. ABA region-wise correlation analysis of histologically determined neuronal densities with the behavioral deficit further highlighted importance of the left CA3 for the performance of the right paw (Fig. 2). Although automatic cell counting on tissue sections is subject to error and presence of a stroke lesion requires highly reproducible sectioning, ABA registration using nonlinear algorithms could surprisingly well compensate this in our hands.
We have developed atlas registration toolboxes for region- and voxel-wise analysis of the interaction of tissue damage and functional recovery after stroke in the mouse. In order to gain a more mechanistic insight, we aim to modulate the function of regions with highest correlation, e.g. the stratum lucidum or layer 6 of the somatosensory cortex, with pharmacogenetic approaches in future studies.
1 Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, Bernard A et al. Genome-wide atlas of gene expression in the adult mouse brain. Nature 2007; 445: 168–76.
2 Koch S, Mueller S, Foddis M, Bienert T, von Elverfeldt D, Knab F et al. Atlas registration for edema-corrected MRI lesion volume in mouse stroke models. J Cereb Blood Flow Metab 2017; : 0271678X1772663. https://github.com/philippboehmsturm/antx
3 Bates E, Wilson SM, Saygin AP, Dick F, Sereno MI, Knight RT et al. Voxel-based lesion-symptom mapping. Nat Neurosci 2003; 6: 448–50.
4 Emmrich J V, Neher JJ, Boehm-Sturm P, Endres M, Dirnagl U, Harms C. Stage 1 Registered Report: Effect of deficient phagocytosis on neuronal survival and neurological outcome after temporary middle cerebral artery occlusion (tMCAo) [version 1; referees: 2 approved]. F1000Research 2017; 6. doi:10.12688/f1000research.12537.1.
This work was supported by the Federal Ministry of Education and Research (BMBF) (grant number 01EO0801, Center for Stroke Research Berlin) and Deutsche Forschungsgemeinschaft (DFG) (Excellence Cluster NeuroCure, Excellence Cluster BrainLinks-BrainTools).
Fig. 1: Voxel-based symptom mapping.
A) Voxels with significant correlation of T2w image intensity (z-scored based on mean/std of contralateral hemisphere) and performance (permutation test, FDR corrected, cluster thresholded). B) Correlation of T2w z-score and performance from one example cluster (each dot represents a mouse). C) Anatomical regions in which the peaks of the ten statistically strongest surviving clusters are located.
Fig. 2: Region-wise correlation of histological neuronal cell densities with performance.
A) Neuronal nuclei (red) are automatically detected on DAB-NeuN histology images. A corresponding ABA slice is warped to the image, allowing group correlation of cell density to performance over ABA regions. B) Example for one region, each dot represents one mouse. C) Regions ordered by correlation with deficit. L/R are ipsi/contralateral to lesion, respectively.
Keywords: Allen brain atlas, stroke, mouse, image registration, histopathology, functional recovery
Prominent vessels on quantitative susceptibility maps indicate microvascular pathology after experimental cerebral ischemia and reperfusion (#19)
M. Vaas1, A. Deistung2, 3, 4, J. R. Reichenbach2, 5, A. Keller6, A. Kipar7, J. Klohs1
1 University of Zurich and ETH, Institute for Biomedical Engineering, Zurich, Switzerland
Prominent vessels in the brain of patients with ischemic stroke have been observed on susceptibility weighted images and quantitative susceptibility maps (QSM).1-2 However, the occurrence of prominent vessels after restoration of reperfusion has so far not been evaluated. The goal of the current study was to perform QSM in the middle cerebral artery occlusion (MCAO) model of cerebral ischemia and to quantitatively assess the occurrence of prominent vessels. Moreover, immunohistochemistry was used to assess underlying vessel pathology in brain sections.
Mice underwent 1h of MCAO followed by reperfusion using the intraluminal filament technique. A Bruker PharmaScan 47/16 operating at 200 MHz and equipped with a cryogenic transmit-receive coil was used for MRI. During acquisition mice were spontaneously breathing under isoflurane anesthesia (1.5%). A 3D multi-echo gradient recalled echo sequence was applied using a FOV=25.6 mm×25.6 mm×8 mm and an acquisition matrix=256×256×80, resulting in an effectively isotropic spatial resolution of 100 μm×100 μm×100 μm. Four echoes were recorded (TE1-4=4.5/10.5/16.5/22.5 ms) with TR=100 ms, flip angle=15°. Data was postprocessed for the generation of susceptibility maps and volume-of-interest (VOI) analysis was performed. Brains sections were prepared and stained with anti-mouse collagen IV.
Prominent vessels with high magnetic susceptibility were seen on magnetic susceptibility maps of the ischemic hemisphere on all time points. Prominent vessels appeared larger in diameter than comparable vessels on the contralateral side. Furthermore, an increased number of prominent vessels were found in the ischemic hemisphere of mice imaged at 12h, 24h and 48h after reperfusion compared to mice imaged at 2h, 4h and 6h after reperfusion. Significantly higher differences in magnetic susceptibility were found in prominent vessels of the ischemic ipsilateral side compared to the contralateral hemisphere at 2h and 4h after reperfusion. Immunohistological examination demonstrated dilated larger vessels appeared and capillaries with swollen endothelial cells and narrowing of the vessel lumen.
Microvascular pathology hampers reperfusion of ischemic tissue and promotes secondary tissue injury. Thus, prominent vessels are an important indicator of underlying microvascular pathology and may by pivotal for diagnosis and therapeutic decision making in stroke patients.
This study was by the Swiss National Science Foundation (Grant PZ00P3_136822) and the Hartmann-Müller Foundation.
Figure 1 (a) Quantitative susceptibility maps of the ischemic hemisphere after MCAO. Prominent vessels with higher magnetic susceptibilities are seen (white arrows). (b) Differences in magnetic susceptibility in vessels. Immunohistochemistry showed (c) dilated larger vessels (*). Bar=100 µm. and (d) swelling of endothelial cells and narrowing of the vessel lumen of capillaries. Bar=20 µm.
Keywords: MRI, quantitative susceptibility mapping, mice, cerebral ischemia