Click on an contribution preview the abstract content.
Click on an contribution preview the abstract content.
Introductory Talk by Cornelius Faber - Münster, Germany
This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.
In vitro and in vivo validation of a potential PET tracer targeting α-synuclein (#528)
L. Kuebler1, K. Herfert1, S. Buss1, A. Maurer1, R. Stumm1, F. Schmidt4, A. Leonov2, S. Ryazanov2, A. Giese3, C. Grießinger2, B. J. Pichler1
1 Eberhard Karls Univeristy of Tuebingen, Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Tuebingen, Baden-Württemberg, Germany
Imaging α-synuclein (αSYN) pathology to distinguish synucleinopathies from other neurodegenerative disorders is still challenging and relevant PET tracers for an early detection and differential diagnosis are missing. A potential compound, anle138b, was shown to interfere with the pathological aggregation of αSYN . We developed novel lead structures and tested them towards binding affinity and selectivity in a filter binding assay (FBA). One promising candidate was then labeled with C-11 for PET imaging. The brain delivery, clearance rate and metabolites were determined in healthy mice.
In vitro saturation FBAs were performed using recombinant human αSYN, Aß1-42 and tau46 fibrils and a mix of αSYN fibrils, monomers and oligomers. Incubation with increasing concentrations (0.02nM–48nM) of the tritiated compound provided total binding, and non-specific binding was obtained with an excess of the cold compound. KD-values were calculated from non-linear regression. 60 min PET scans were performed after iv injections of 16±3MBq of the C-11-labeled compound in mice. For the metabolite and biodistribution study, two mice were injected with 41±13MBq of the tracer and sacrificed 5 and 15 min after injection. Blood was collected and the mice were perfused with PBS. The right brain hemisphere was homogenized for HPLC. The left hemisphere and other organs were measured in a γ-counter.
Our compound showed high affinity for pure αSYN fibrils (KD < 2 nM) and for αSYN monomeric and oligomeric structures (KD < 1 nM). Binding experiments using Aß1-42 and tau46 fibrils revealed a 100- and 17fold lower affinity. Radiolabeling resulted in radiochemical yields of 35 % and specific activities were 25.8 ± 10.0 GBq/µmol. The dynamic PET data revealed a good blood brain barrier penetration, a fast clearance from the brain and a peak SUV value of 1.5-1.8 in the mouse brain. We further observed two metabolites, one was only present in the plasma and another one was present in the plasma and brain.
Our work provides very encouraging in vitro and in vivo data of a potential new PET tracer to image αSYN depositions in the brain. Future experiments aim to further validate the tracer in human and animal brain tissues with confirmed αSYN pathology and optimize radiosynthesis protocols to increase the specific activity.
 Wagner J, Ryazanov S, Leonov A, Levin J, Shi S, Schmidt F, et al. Anle138b: A novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson’s disease. Acta Neuropathol. 2013;125:795–813.
Keywords: α-synuclein, PET, probe development
Increased functional connectivity in the default mode-like network is related to behavioural outcome in a neurodevelopmental model with relevance for schizophrenia (#390)
S. Missault1, 2, C. Anckaerts2, S. Ahmadoun1, I. Blockx2, K. Bielen3, D. Shah2, S. Kumar-Singh3, A. Van der Linden2, S. Dedeurwaerdere1, M. Verhoye2
1 Experimental Laboratory of Translational Neurosciences, University of Antwerp, Translational Neurosciences, Wilrijk, Belgium
Maternal immune activation (MIA) is an important risk factor for schizophrenia. Functional dysconnectivity and NMDA receptor hypofunction have been postulated to be key features in the pathophysiology of schizophrenia. The aim was to investigate functional connectivity (FC) changes in default mode-like network, and NMDAR function in adult rat offspring prenatally exposed to an immune challenge, and to determine whether they relate to behavioural outcome. Finally, we explored whether MIA offspring exhibit a different pathophysiology depending on the maternal response to the immune stimulus.
Pregnant rats were injected with Poly I:C or saline. 6h post-injection, maternal serum was collected for cytokine analysis and 24h post-injection maternal weight response was recorded. Based on the maternal weight response, offspring were divided into 3 groups: controls (n=11), and offspring of dams that gained and lost weight post-MIA (Poly I:C WG, n=12; Poly I:C WL, n=16). Male adult offspring were subjected to resting-state functional MRI, diffusion tensor imaging (DTI), pharmacological fMRI with 0.2mg/kg NMDAR antagonist MK-801 (7T Bruker PharmaScan), and behavioural testing.
Pregnant dams that lost weight post-MIA displayed a more pronounced increase of the chemokine RANTES vs. controls than rats that gained weight post-MIA. Region of interest- and seed-based analysis of resting-state fMRI data revealed that Poly I:C WL offspring exhibited increased FC in the default mode-like network (Fig.1). Comparison of the BOLD response before and after (20-30 min post) MK-801 administration showed that Poly I:C WG offspring had a much smaller response to the NMDAR antagonist vs. controls (Fig.2). Voxel-based analysis of DTI data revealed no differences in MIA offspring vs. controls. Behavioural deficits were subtle with the most pronounced deficit being an increased anxiety. Hypersynchronicity in the default mode-like network was associated with behavioural outcome in MIA offspring.
MIA offspring displayed a differential pathophysiology dependent on the maternal response to the immune challenge. FC in the default mode-like network was related to the behavioural outcome in MIA offspring.
This study was supported by the Research Foundation Flanders (FWO) (G.0586.12) and Molecular Imaging of Brain Payhology (BRAINPATH) (612360).
Fig.1. Increased functional connectivity (FC) in the default mode-like network (DMN) of Poly I:C WL
A. ROI-based analysis revealed increased FC within the DMN in adult MIA offspring vs. controls, which was most pronounced in Poly I:C WL offspring. B. Average group statistical seed-based FC maps with posterior parietal cortex as seed region (one-sample t-test, FWE corrected, p<0.05, minimal cluster size k=10).
Fig.2. Pharmacological MRI with NMDA receptor antagonist MK-801.
Average group statistical difference maps of BOLD signal pre – post MK-801 administration (uncorrected, p<0.01, minimal cluster size k=10). MIA offspring showed a significantly smaller response to the NMDAR antagonist compared to controls, which was most pronounced in Poly I:C WG offspring.
Keywords: schizophrenia, maternal immune activation, NMDA receptor, resting-state functional MRI, diffusion tensor imaging, pharmacological MRI, behaviour
Longitudinal in vivo MRI assessment of functional connectivity and structural integrity in the TgF344-AD rat model of Alzheimer’s Disease (#395)
C. Anckaerts1, I. Blockx1, P. Summer2, J. Michael2, C. Kreutzer2, H. Boutin3, S. Couillard-Despres2, M. Verhoye1, A. Van der Linden1
1 Bio-Imaging Lab, University of Antwerp, Wilrijk, Belgium
Recently, the TgF344-AD rat model of AD, bearing mutant human amyloid precursor protein (APP) and Presenilin 1 (PS1) genes was first described by Cohen et al1. According to this publication, this model manifests the full spectrum of AD pathology similar to human AD, i.e. progressive cerebral amyloidosis, tauopathy, neuronal loss and age-dependent cognitive decline. Here, we further examined AD-related pathology in this rat model by means of resting state functional MRI (rsfMRI) and diffusion tensor imaging (DTI) to evaluate functional connectivity (FC) and structural integrity, respectively.
Female TgF344-AD rats (NWT=12, NTG=11) were scanned on a 7T MR system in a longitudinal manner (6, 10, 12, 16 and 18 months). Rats were anaesthetized using a mixture of medetomidine (0.05mg/kg s.c. bolus; 0.01mg/kg/h s.c. infusion) and a low dose of isoflurane (0.4%). Each imaging session consisted of a rsfMRI scan (2D GE-EPI; TR 2s; TE 29ms; 20 axial slices; 300 repetitions; (0.234 x 0.234 x 0.8)mm³), a DTI scan (2D DW-SE-EPI; TR 7.5s; TE 26ms; δ 4ms; Δ 12ms; b-factor 800 s/mm²; 60 diffusion directions; 20 slices; (0.234 x 0.234 x 0.8)mm³) and a T2-weighted 3D anatomical scan (RARE, TR 3.185s; TE 44ms; RARE factor 8; (0.11 x 0.25 x 0.20) mm³). Samples for ex vivo analyses were acquired at 10, 16 and 18-20 months.
Analysis of the rsfMRI data revealed significantly reduced FC strength in TG animals (Fig. 1), affecting regions characteristically involved in AD such as the hippocampus and cingulate cortex. These differences in FC between TG and WT were found to be the greatest at 10 months. A voxel-based analysis of the DTI data indicated progressive changes of the structural integrity of the brain (Fig. 2). As such, increased mean diffusivity (MD) was found in the sensory cortex of TG animals, whereas decreased values were present in the ventricular area. Furthermore, TG rats had reduced fractional anisotropy (FA) in the sensory cortex and several white matter structures, whereas increased FA was present in the corpus callosum of TG rats. These DTI changes were exacerbated in advanced stages of the disease (16-18 months). Initial histological examination of the hippocampus indicated progressive gliosis and increasing amyloid plaque load between 10 and 20 months. Further analyses are still ongoing.
Already at early stages, TgF344-AD rats displayed a strong attenuation of the brain’s FC preceding major structural alterations of the brain. Further ex vivo analyses will help in explaining the observed in vivo MRI results.
1. Cohen et al., J Neurosci, 2013
This research was supported by the European Union’s Seventh Framework Programme (grant agreement number 278850; INMiND) and by the Fund for Scientific Research Flanders (FWO) (grant agreements G067515N and G057615N).
Figure 1. Functional connectivity changes in the TgF344-AD rat.
A-E. Average zFC matrices for WT (top) and TG animals (bottom) at each time point. Each square indicates the zFC between each pair of ROIs, with the colour scale indicating the strength of the connectivity.
F. Outcome of the statistical analysis showing significant genotype effects (bottom) and ageing effects (top). The colour scale indicates log-transformed p-values.
Figure 2. Structural alterations in the TgF344-AD rat.
Genotype differences for MD (A) and FA (B), obtained from a voxel-based statistical analysis. Results are shown on a study-specific 3D anatomical template, FWE p<0.05, minimum cluster size of 10 voxels. The colour scale represents t-values with red/yellow indicating higher values in TG animals, whereas blue colours represent lower values in TG animals compared to WT.
Keywords: rsfMRI, DTI, TgF344-AD rat model, Alzheimer's Disease
Cannabinoid-induced changes in intrinsic connectivity of the adult rat brain are mediated by actomyosin contractility (#491)
C. Morisset1, 2, 3, J. Ferrier6, 5, 4, C. Demene1, 2, 3, A. Ricobaraza4, 5, T. Deffieux1, 2, 3, M. Tanter1, 2, 3, Z. Lenkei6, 5, 4
1 INSERM U979, Wave Physics for Medicine Lab, Paris, France
The effects of exogenous cannabinoids on the central nervous system are mediated mainly by CB1R. They interfere with the endocannabinoid system and modify brain connectivity. We have previously described a novel molecular pathway dependent on CB1R signaling and actomyosin contractility. Here we asked whether this pathway may also induce changes in synaptic function and lead to modifications of resting-state functional connectivity in vivo by blocking neuronal actomyosin contractility using Neurelaxin-A in the living rat brain during cannabinoid treatment (CP 55, 940) of adult rats.
Experiments were performed on 12 male Dawley rats, anesthetized with an IP injection of 75 mg.kg-1 ketamine and 1 mg.kg-1 xylazine. Ultrasound imaging was performed in a coronal plane at Bregma–3.6mm. Ultrasound sequences are emitted by an array of 128 elements transducer connected to a modified ultrafast ultrasound scanner. Ultrasensitive Doppler images were acquired every second from plane waves compounding (5 angles, PRF=2500). After 10 minutes recording of baseline functional connectivity (FC), animals were then injected 5µL of either vehicle (n=4) or Neurelaxin-A 5mM (n=4) in the right cerebral ventricle. 15 minutes after the injection, a 10 minutes’ recording were performed then the rats were IP injected with 0.7 mg/kg of CP 55,940. Another 10 minutes’ recording was performed.
In rats receiving the vehicle, the cannabinoid CP 55,940 induced several alterations of the resting state functional connectivity compared to baseline. Overall, the dorsal hippocampus, a brain region particularly rich in CB1R seemed to be the main target of CP 55,940 regarding bilateral connectivity. A seed-based analysis of interhemispheric correlation was performed at each group (Neurelaxin-A or Vehicle-treated rats) for statistical comparison.. While the connectivity patterns remained similar in both conditions at baseline and after pretreatment, the functional connectivity strength between the right and left hippocampi was strikingly reduced after CP 55,940 injection, suggesting that cannabis decreases the interhemispheric connectivity of the hippocampus. Most importantly, actomyosin blockade using Neurelaxin-A pre-treatment was able to prevent this major alteration.
Neurelaxin-A lets us inhibit brain actomyosin contractility in vivo for the first time. This gives preliminary results arguing for a cannabinoid-induced alteration of FC that could be rescued by blocking actomyosin contractility. These results provide new insights into the effects of cannabinoids on brain function and highlight actomyosin contractility as a major effector of cannabis effects. As a strong correlation exists between psychotic pathologies and alteration of FC, it opens new perspectives in the understanding of both cognitive function and the pathogenesis of psychiatric disease.
Keywords: Neuroimaging, ultrasound
BOLD fluctuations under 0.01 Hz detected in resting-state functional MRI (#315)
P. Pais1, Y. Jian1, J. Stelzer1, B. Edlow2, X. Yu1
1 Max Planck Institute - Cybernetics, High Field MRI, Tuebingen, Germany
Spectral analysis in resting-state fMRI (rs-fMRI) studies has mainly focused on the 0.01 to 0.1 Hz frequency range1-6. Frequencies under 0.01 Hz are typically regarded as artifacts from scanner instabilities or physiological noise7, and are routinely excluded from the rs-fMRI analysis. Here, we show robust ultra-slow rs-fMRI signal fluctuations of high regularity in the brain of rats under anesthesia, which seem to be independent from breathing-derived motion8 or cardiovascular oscillations9,10 and possibly indicate a peculiar brain state in the animals.
12 to 15 minutes of rs-fMRI data were acquired from anesthetized rats (under isoflurane, a-chloralose, medetomidine or urethane) using a 3D-EPI sequence with the following parameters: TE, 12.5 ms; TR, 1s; matrix size, 48x48x32; resolution, 400x400x600 µm. All images were acquired with a 12 cm diameter 14.1 T/26 cm magnet interfaced to an Avance III console. Trans-receiver surface coils were used to acquire the whole brain fMRI. Animals were mechanically ventilated, and a low dose of the paralytic agent pancuronium was used to prevent motion artifacts. Frequency decomposition analysis and power estimation of the 0.005-0.012 Hz frequency band were performed to map the ulstra slow oscillations (USO) in the rat brain.
Here we show that frequencies below the classical 0.01 Hz limit can be detected, with high amplitude and rhythmic pattern, in the Blood-Oxygen Level Dependent (BOLD) fMRI signal of animals receiving anesthesia (Fig.1). When present, these slow waves occured predominantly in the hypothalamic area, as confirmed with bandpass power analysis (Fig.2). The features of the reported USO (brain region predominance and apparent absence of correlation with motion or physiological artifacts), suggest that these oscillations might have a neural origin instead of being derived from MR hardware noise. Importantly, infraslow oscillations in a similar range were detected by EEG in brain-injured patients, which appeared related to modulations in the cortical excitability and have been hypothesized to emerge from a deep brain source11.
Our observations suggest that frequency components in the ultra-slow range may contribute to brain function. Future work will aim to clarify the source of these oscillations with neuronal and astrocytic calcium imaging and the utilization of cholinergic modulators to study reversibility of these oscillations in the rat brain.
1. Obrig, H. et al 2000; 2. Mitra, P. P. et al 1997; 3. Biswal, B. et al 1995; 4. Golestani, A. M. et al 2017; 5. Raichle, M. E. et al 2001; 6. Gohel, S. R. & Biswal, B. B. 2015; 7. Smith, A. M. et al 1999; 8. Van de Moortele et al 2002; 9. Cohen, M. A. & Taylor, J. A. 2002; 10. Kato, M. et al 1992; 11. van Putten, M. J. A. M. et al 2015.
Graduate Training Center of Neuroscience Tuebingen.
Keywords: slow oscillations, brain states, rs-fMRI
An in-depth view of the mouse cerebral vascular architecture: a multiscale multimodal imaging approach. (#396)
R. Hinz1, J. R. Detrez2, L. Peeters1, C. Berghmans3, M. Verhoye1, A. Van der Linden1, W. H. De Vos2, G. A. Keliris1
1 Bio-Imaging Lab - University of Antwerp, Biomedical Sciences, Wilrijk, Belgium
Vascular abnormalities are hallmark comorbidities of a variety of neuropathologies. Hence, in-depth knowledge about the cerebral vascular architecture and its alterations in disease are quintessential. Here, we used a multi-scale approach combining, on the same animal, anatomical MRI and time of flight magnetic resonance angiography (TOF-MRA) with high-resolution whole brain microscopic imaging data in order to create a high resolution vascular atlas that could be co-registered to the Allen Brain space providing a link to additional functional, anatomical and genetic information.
Male C57BL/6 (N=6) mice of 12 weeks old were scanned on a 9.4T Biospec. MRI data acquisition included a 3D-T2 anatomical scan followed by three orthogonal orientated 2D-TOF-MRA to assess the macro vasculature. All scans were co-registered to the Allen Brain atlas space using ANTs. Micro vasculature was acquired with 3D microscopic imaging of a cleared mouse brain (iDISCO) with a double labeling of the vasculature using isolectin to stain the vascular wall combined with an albumin staining of the vessel lumen. Cleared samples were imaged on a light sheet microscope with a 1.8x effective magnification and a step-size of 5µm. Each subject’s microscopic imaging data was co-registered to its respective MRI anatomical dataset and subsequently co-registered to the Allen Brain atlas using ANTs.
The developed atlas provides a multi-scale representation of the vasculature of the mouse brain (Fig.1). More specifically, it contains the macro-vasculature acquired with TOF-MRA providing: a) a template of mainly the larger vessels and sinuses (Fig. 1A) and b) a vascular probability map that reflects spatial reliability across subjects with the large vessels presenting high spatial reliability in contrast to further more variable branches (Fig. 2). The atlas also contains a novel double-labeling of the micro-vasculature. Preliminary results show that isolectin staining was superior in labeling capillaries while albumin staining more successfully labeled the bigger vessels and could be used for alignment to the TOF-MRA. By combining both stainings an improved detection of the mouse cerebral vascular architecture can be achieved. Furthermore, the atlas also contains 3D-T2 weighted anatomical information which can be used as an access point for other MRI experiments.
This multi-scale approach provides not only the full vascular tree with unprecedented detail but also a means for co-registration and multivariate analysis of multimodal imaging information via the Allen Brain framework. Future work can further extend this atlas with quantitative computational tools able to identify vascular changes and abnormalities as often observed in neurodegenerative disorders.
This research was supported by Molecular Imaging of Brain Pathophysiology (BRAINPATH) under grant agreement number 612360 within the Marie Curie Actions-Industry-Academia Partnerships and Pathways (IAPP) program, by the Fund of Scientific Research Flanders (FWO G048917N) and Flagship ERA-NET (FLAG-ERA) FUSIMICE (grant agreement G.0D7651N).
Keywords: Atlas, Vasculature, Allen Brain, iDisco, lectine, albumine, T2, TOF-MRA
Multispectral-optoacoustic-tomography imaging of acute cerebral hypoxia and upregulation of matrix-metalloproteinase activity in a mouse model of cerebral ischemia and reperfusion (#11)
R. Ni1, M. Vaas1, W. Ren1, J. Klohs1
1 ETH Zurich & University of Zurich, Institute for Biomedical Engineering, Zurich, Switzerland
Hemodynamic alternations and the subsequent inflammatory responses such as upregulations of matrix metalloproteinases (MMPs) play important roles in the pathophysiology of cerebral ischemia. In this study we aimed to detect in vivo the changes in cerebral tissue oxygenation and MMP activity during and after transient middle cerebral artery (MCA) occlusion (tMCAO) in mice using multispectral optoacoustic tomography (MSOT) co-registered with magnetic resonance imaging (MRI) to derive information on the ischemic lesion.
C57B6L/J mice underwent tMCAO or sham surgery (n = 39) were imaged by MSOT for cerebral hemodynamic changes during 1 h tMCAO or at 48 h after reperfusion. Brain MMP activities were detected by using MSOT with a MMP-activatable probe at 48 h after reperfusion . Diffusion weighted imaging and T2-weighted MRI were performed at 7 T. The MSOT deoxy-, oxyhemoglobin and MMP images were co-registered with structural MR for lesion delineation and anatomical references. Ex vivo near-infrared imaging and triphenyltetrazolium chloride staining were performed with brain slices for visulization of MMP signal and ischemic lesions.
Reduced ipsi/contralateral ratio of tissue oxygen saturation was observed during acute tMCAO compared to sham-operated mice (52.5 ± 23.1 %, vs 98.6 ± 18.3 %, p = 0.0003), which recovered to normal at 48 h after reperfusion (99.9 ± 9.4 %, n = 9). Elevated ipsi-/contralateral MMP signal was detected at 48 h after reperfusion in the ipsi-lesion brain regions of tMCAO (4738.7± 2867.8 MSOT a.u., n = 5) compared to sham-operated mice (1138.4 ± 709.7 MSOT a.u., n = 4, p = 0.0479). The ex vivo near-infrared fluorescence imaging results demonstrated increased MMP signals in the core of the ischemic lesion as defined by triphenyltetrazolium chloride staining.
In conclusion, MSOT constitutes a useful tool for in vivo visualization of hemodynamic alternations and MMP activity. We demonstrated acute cerebral hypoxia and subsequent increase in MMP activity in the mouse brain after focal cerebral ischemia with reperfusion.
 J. Klohs et al., "In vivo near-infrared fluorescence imaging of matrix metalloproteinase activity after cerebral ischemia," J Cereb Blood Flow Metab 29(7), 1284-1292 (2009).
This work was funded by the University of Zurich and the ETH Zurich Foundation through a Seed Grant of "University Medicine Zurich/Hochschulmedizin Zürich" and by funding from the Olga Mayenfisch Stiftung.
Figure 1 In vivo assessment of brain oxygenation and matrix metalloproteinases with MSOT in tMCAO mouse at 48 h after reperfusion (a) Hb, HbO2 images, unmixed signal from HbO2 (red) and Hb (blue), scale 0-2×101 MSOT a.u,; (b) tMCAO mouse injected with MMP-activatable probe 48 h after reperfusion with unmixed signal from MMP (green) overlaid on T2-weighted MR image, scale 0-9×104 MSOT a.u,
Keywords: cerebral ischemia, matrix metalloproteinases, magnetic resonance imaging, hemodynamics, animal model, multispectral optoacoustic imaging