15th European Molecular Imaging Meeting
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New Methods & Methodology in Neuroimaging

Session chair: Fabien Chauveau (Lyon, France); Yi Chen (Tuebingen, Germany)
 
Shortcut: PS 06
Date: Wednesday, 25 August, 2021, 2:30 p.m. - 4:00 p.m.
Session type: Parallel Session

Contents

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2:30 p.m. PS 06-01

Introductory Lecture

Cornelius Faber

Münster, Germany

2:54 p.m. PS 06-02

Simultaneous imaging of stimulus-evoked calcium and hemodynamic responses in the mouse brain

Zhenyue Chen1, 2, Quanyu Zhou1, 2, Xosé Luís Deán-Ben1, 2, Ruiqing Ni1, 2, Shy Shoham3, Daniel Razansky1, 2

1 ETH Zurich, Institute of Biomedical Engineering, Zurich, Switzerland
2 University of Zurich, Institute for Biomedical Engineering and Institute of Pharmacology and Toxicology, Zurich, Switzerland
3 NYU Langone Health, Department of Ophthalmology and Tech4Health and Neuroscience Institutes, New York, United States of America

Introduction

Functional neuroimaging has become a primary tool in basic neuroscience and studies of neurodegeneration. Functional magnetic resonance imaging and functional ultrasound can be used to noninvasively map activity in rodent brains. However, the readings mainly represent changes in the blood volume and flow, which may not be sufficient for unravelling the complex processes underlying cerebral activity[1, 2]. We introduce a hybrid epi-fluorescence optoacoustic tomography (FLOT) imaging platform for simultaneous imaging of calcium and hemodynamic brain responses to peripheral stimulation in mice.

Methods

The hybrid FLOT system schematic is shown in Fig. 1a. Briefly, fluorescence excitation was performed with a CW 488 nm laser while volumetric multispectral optoacoustic tomography was achieved with an optical parametric oscillator laser tuned at five wavelengths (700, 730, 755, 800, and 850 nm) on a per-pulse basis at 100 Hz pulse repetition frequency. An electrical paw stimulation paradigm was adopted with the electric pulses width of 5 ms and intensity of 1.0 mA at a stimulus frequency of 4 Hz, onset time of 8 s and burst interval of 82 s (Fig. 1b). A custom data processing pipeline was further developed to facilitate 3D optoacoustic image reconstruction using backprojection and functional data analysis.

Results/Discussion

Concurrent measurement of calcium and hemodynamic responses was achieved non-invasively. Despite the strong scattering of the scalp, localized brain activation upon electrical paw stimulation was still observed with high fidelity in the GCaMP fluorescence (Fig. 1c) while hemodynamic activity is visible as well (Figs. 1d and 1e). Activation maps corresponding to different hemodynamic components, namely HbO, HbR, HbT and sO2, were rendered from volumetric optoacoustic image sequences. Localized responses were clearly observed in cross sections from transverse, sagittal and coronal planes overlaid to Allen mouse brain atlas [3] (Fig. 1f). The averaged activation time courses from selected ROIs (indicated by circles in Fig. 1f) for HbO, HbR, HbT and sO2 components further revealed a clear difference between contralateral and ipsilateral counterparts (Figs. 1g-j). Signal profiles from other regions were also analyzed owing to the large field view captured by the FLOT system (Figs. 1k-l).

Conclusions

Concurrent measurement of calcium and hemodynamic responses revealed a strong correlation between GCaMP signal and associated increases in HbO, HbT, sO2 and decreases in HbR, complementing previously reported observations done with widefield optical mapping and fMRI. The proposed FLOT method expand the capabilities of functional neuroimaging by providing more concurrent and cross-validated multi-modal readings on brain activity.

Acknowledgement

The authors acknowledge grant support from the US National Institutes of Health (UF1 NS107680) and European Research Council (ERC-2015-CoG-682379 to DR).

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Blockley N P, Griffeth V E M, Simon A B, Buxton R B, A review of calibrated blood oxygenation level-dependent (bold) methods for the measurement of task-induced changes in brain oxygen metabolism. Nmr Biomed 2013; 26: 987-1003.
[2] Aydin A, Haselden W D, Goulam Houssen Y, Pouzat C, Rungta R L et al., Transfer functions linking neural calcium to single voxel functional ultrasound signal. Nat Commun 2020; 11: 2954.
[3] Bakker R, Tiesinga P, Kötter R, The scalable brain atlas: instant web-based access to public brain atlases and related content. Neuroinformatics 2015; 13: 353-366.
Figure 1 Hybrid epifluorescence and functional optoacoustic imaging of the mouse brain.

a Schematics of FLOT system. b Electrical stimulation paradigm. c, d GCaMP and hemodynamic activation maps, respectively. e Fluorescence activation curve from circled regions after applying a lowpass filter (< 1 Hz). f Transverse, sagittal and coronal views of the activation maps corresponding to HbO, HbR, HbT and sO2 from optoacoustic images. g-j Averaged activation time courses of HbO, HbR, HbT and sO2 from the circled regions, respectively. k-l Activation time courses of hemodynamic components and lowpass filtered fluorescence signals from ROIs indicated by the stars and squares in c.

Keywords: Functional imaging, optoacoustic tomography, photoacousics, brain activation, dual-modality
3:06 p.m. PS 06-03

A microstructured optical fibre (MOF)–based multifunctional probe for optofluidics and calcium recording in rodent brains

Yi Chen1, 2, Michael H. Frosz2, Azim-Onur Yazici2, Philip S. J. Russell2, Chunqi Qian3, Xin Yu4

1 Max Planck Institute for Biological Cybernetics, Tuebingen, Germany
2 Max Planck Institute for the Science of Light, Erlangen, Germany
3 Michigan State University, Department of Radiology, East Lansing, United States of America
4 Massachusetts General Hospital and Harvard Medical School, Athinoula A. Martinos Center for Biomedical Imaging, Charlestown, United States of America

Introduction

Emerging studies have combined fMRI with fibre optic-based optogenetics, fluorescence Ca2+ recording,and neuropharmacology(i.e.,multi-modal fMRI platform) to decode neural circuitry and decipher brain function1,2.The MOF3 could guide light by a conventional higher-index core modified by the presence of air holes for fluid delivery,providing the possibility to integrate multiple functions in one probe. Here,we developed a pure silica MOF-based probe for optogenetically and sensory-driven single-vessel fMRI4 with simultaneous Ca2+ signals and Mn2+ injection to optimize multi-modal fMRI platform.

Methods

CaMKII.ChR2.mCherry was injected into the right barrel cortex (BC)(Fig.1e) while the GCaMP6f was expressed in the left BC of rats (Fig.2b). The MOF-based probe was inserted in the BC(Fig.1b) to deliver blue light excitation pulses (488 nm) at 3 Hz, 4 s duration, 10 ms width, 15 mW for the optogenetic stimulation and continuously at 5 μW for calcium recording. 0.1mM MnCl2 solution was used as the MR contrast agent with a modified MPRAGE sequence (FOV:1.92×1.92 cm2, 0.7 mm thickness, TR, 4000 ms; Echo TR/TE = 15/1.7 ms;TI,1000 ms;number of segments:4). fMRI block design was 4 s stim on/17.5 s off, 8 epochs for the whole brain EPI:1.5s TR, 400 μm isotropic resolution (Fig.1d,f) and SSFP single vessel BOLD:TR,1.5 s;FOV,9.6×9.6 mm2; 500 μm thickness; 100 μm in-plane resolution (Fig.1g, Fig.2c).

Results/Discussion

First, as proof of concept, a MOF-based optofluidic probe was evaluated in a 14 T scanner for simultaneous optogenetic stimulation and Mn2+ injection (Fig.1b). As Fig.1c shows, the Mn2+ was delivered into the left BC effectively with negligible liquid leaking in the right BC with MOFs of ~230 μm outer diameter. Upon optogentic stimulation, the robust BOLD in the right BC demonstrates sufficient light propagation. In addition, high resolution single-vessel BOLD was acquired (Fig.1e,f,g).
Next, the MOF-based probe was glued to a multi-mode fibre for MRI-compatible calcium recording and Mn2+ injection upon sensory stimulation. The use of a photomultiplier chip makes it MRI-compatible and simplifies the conventional lightpath (Fig.2a). The BOLD for the vessels surrounding the probe and the Ca2+ signals demonstrates its reliability (Fig.2c). Interestingly, the decreased Ca2+ baseline after each injection shows that the extracellular Mn2+ quenches the fluorescence Ca2+ signals5 (Fig.2d,e).

Conclusions

We present a MOF-based fully functional optofluidic probe and a simplified Ca2+ recording lightpath in a 14 T scanner to reduce tissue damage for optogenetic and fluid injection, and simplification of the conventional Ca2+ recording lightpath. The MOF-based probe integrates optogenetic-driven brain fMRI studies with simultaneous Ca2+ recording and drug delivery, which may contribute to uncovering the basis of neuropsychiatric diseases.

Acknowledgement

This research was supported by NIH Brain Initiative funding (RF1NS113278-01, R01 MH111438-01), and the S10 instrument grant (S10 MH124733-01) to Martinos Center, German Research Foundation (DFG) Yu215/3-1, Yu315/2-1, BMBF 01GQ1702, and the internal funding from Max Planck Society. This project has received funding from the European Union Framework Programme for Research and Innovation Horizon 2020 (2014-2020) under the Marie Skłodowska-Curie Grant Agreement No.896245. We thank Dr. R. Pohmann, Dr. J. Engelmann, Mr. M. Vintiloiu, Dr. N. Avdievitch and Ms. H. Schulz for technical support, Dr. P. Douay and Ms. R. König for animal support, the AFNI team for the software support.

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Ferenczi, E. A. et al. Prefrontal cortical regulation of brainwide circuit dynamics and reward-related behavior. Science 351, aac9698, doi:10.1126/science.aac9698 (2016).
[2] He, Y. et al. Ultra-Slow Single-Vessel BOLD and CBV-Based fMRI Spatiotemporal Dynamics and Their Correlation with Neuronal Intracellular Calcium Signals. Neuron 97, 925-939 e925, doi:10.1016/j.neuron.2018.01.025 (2018).
[3] Russell, P. Photonic crystal fibers. Science 299, 358-362, doi:10.1126/science.1079280 (2003).
[4] Yu, X. et al. Sensory and optogenetically driven single-vessel fMRI. Nat Methods 13, 337-340, doi:10.1038/nmeth.3765 (2016).
[5] Petrus, E., Saar, G., Daoust, A., Dodd, S. & Koretsky, A. P. A hierarchy of manganese competition and entry in organotypic hippocampal slice cultures. NMR Biomed 34, e4476, doi:10.1002/nbm.4476 (2021).
Fig.1 MOF-based Mn-injection and optogenetic-driven single vessel fMRI in the BC.

a Electron micrograph cross-section of the MOF.

b Schema and picture of MOF-based probe.

c Left, MOF inserted in the BC in two hemispheres. Right, T1-weighted MPRAGE image showing enhanced signal from Mn-injection site in the left BC.

d A fMRI map of activity during optogenetic stimulation of BC.

e Overview of single vessel mapping, after MnCl2 injection and ChR2-mCherry expression.

f The average BOLD fMRI time courses from BC and individual vessels  for each epoch.

g Single vessel map with venule(dark spot) voxels.

Fig.2 MOF-based Mn-injection and Ca2+ recording with simultaneous single vessel fMRI in the BC.

a Schematic drawing of MOF-based Ca2+ recording setup and fluid injection.

b GCaMP expression in the left BC (upper) and anatomical image for single vessel fMRI setup (lower).

c Single vessel map with venule ROI overlapped with functional BOLD map. Right, the average time courses from the individual vessels upon sensory whisker stimulation.

d A representative activated Ca2+  signal during Mn2+ injection, the black arrow indicates the injection time.

e Ca2+ baseline changes after Mn2+ injection.
Keywords: microstructured optical fibre, optofluidics, calcium recording, single-vessel fMRI, rodent
3:18 p.m. PS 06-04

Humanized Mouse Brain Biomarkers through Transcriptomic Conversion

Roël M. Vrooman1, Judith R. Homberg1, Joanes Grandjean1, 2

1 Radboud University, Donderst Institute for Brain, Cognition, and Behaviour, Nijmegen, Netherlands
2 RadboudUMC, Department of Medical Imaging, Nijmegen, Netherlands

Introduction

The interpretation of whole-brain neuroimaging observations in animal models relies on assumptions of spatial homologies between species. These assumptions have been built upon comparative neuroanatomy, including neuronal composition and axonal projections. To date, these assumptions remain approximate, which in turn impairs our ability to transfer observations between species. The goal of this study is to develop a data-driven approach relying on transcriptomic similarity to seamlessly convert whole-brain biomarkers between mice and humans.

Methods

The fMRI and transcriptomic data were taken from freely available online repositories.1,2 Six brain states were derived for both the human and mouse fMRI data using co-activation patterns (CAPs), which were matched based on spatial homology. Expression data were preprocessed and the mouse data was put into a linear model. Weighting factors from this linear model were then used to estimate a ‘synthetic’ version of the human brain states based on homologous genes. Figure 1 shows that the linear models were created as a linear addition of the expression maps for each gene. The human synthetic brain states were cross-correlated with their ‘biological’ versions to see whether they showed an increased correlation between the matched brain states as compared to the mismatched brain states.

Results/Discussion

Conversion of the mouse brain states, using the transcriptomic homology model, resulted in six synthetic humanized brain states. To compare these synthetic brain states to both the matched biological states and mismatched states, the mean time series for 81 regions were taken from all maps and cross-correlated. These 81 regions were based on the brain parcellations used for the human transcriptomic data.3 Figure 2 shows that synthetic brain states matched to their biological version, indeed show higher correlation compared to mismatched states (difference: 0.235484, 95%CI: 0.018571 - 0.499615, p = 0.045). Furthermore, when comparing the 81 regions from state 3 to its synthetic version, the correlation is highly significant (synthetic 3 ~ state 3: F(1,81) = 37.3, p = 2x10-8)).

Conclusions

Here we demonstrate the possibility to translate between mouse and human data using a data-driven conversion model purely based on homologous gene expression. Although the model shown is currently only validated on fMRI data of co-activation patterns, it should generalize to any type of brain dataset. This model can provide a new way for researchers to translate their mouse brain data to humans, increasing their relevance.

Acknowledgement

RMV is supported by the Dutch Research Council grant OCENW.KLEIN.334 awarded to JG

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Allen Institute for Brain Science. (2004) ‘Allen Mouse Brain Atlas’, Available from: https://mouse.brain-map.org/
[2] Allen Institute for Brain Science. (2010) ‘Allen Human Brain Atlas’, Available from: http://human.brain-map.org/
[3] Desikan, R. S. et al. (2006) ‘An automated labeling system for subdividing the human cerebral cortex on MRI scans into gyral based regions of interest’, NeuroImage, 31(3), pp. 968–980.
Figure 1: Linear models
A 3D mouse brain map (Mouse.mapxyz), e.g. derived from functional MRI group comparison, can be explained as the linear addition of the weighted expression maps (βi * Genei, xyz). The weighting factors (βi) estimated in the previous step, are then transferred to homologous genes expressed in the human brain. The resulting Human.map is a humanized synthetic version of the mouse brain map in standard human coordinates (MNI152). Gene expression information is obtained from the Allen Brain Atlas.
Figure 2: Results

a. Synthetic brain states matched to their biological version show a higher correlation than mismatched brain states (difference: 0.235484, 95%CI: 0.018571 - 0.499615). b. Human state 3 next to its matched humanized synthetic version derived from a mouse brain map. c. Correlation between state 3 and its synthetic version (synthetic 3 ~ state 3: F(1,81) = 37.3, p = 2x10-8).

Keywords: Homology, Transcriptomics, Neuroimaging, Translation, Linear modelling
3:30 p.m. PS 06-05

Bedside monitoring of the dynamic brain connectivity in human neonates using functional Ultrasound

Flora Faure1, Charlie Demené1, Jérôme Baranger1, Alice Frérot2, Catherine Delanoë2, Marianne Alison2, Olivier Baud3, Valérie Biran2, Mickaël Tanter1

1 Physics for Medicine Paris, Inserm, ESPCI Paris, CNRS, PSL University, Paris, France
2 Assistance Publique Hôpitaux de Paris, Robert Debré University Hospital, Paris, France
3 University Hospital of Geneva, Geneva, Switzerland

Introduction

Clinicians have long been interested in neonate functional brain monitoring, as in pathologic patients reversible functional losses often precedes observable irreversible structural insults. By characterizing neonatal functional cerebral networks, resting-state functional connectivity (fC) is envisioned to provide early markers of cognitive impairments. Here we present a pioneering bedside deep brain resting-state fC imaging at 250μm resolution on human neonates using the recently introduced functional ultrasound (fUS), able to identify a pathological neurodevelopmental trajectory.

Methods

A custom ultrasound (US) probe (6.4MHz, 250µm in-plane resolution) was mounted into a custom newborn-adapted headset along with 8 EEG electrodes. An US research system was programmed to acquire 5 minute-long 1s-resolution fUS recordings, giving access to the Cerebral Blood Volume variations (CBV) on a 25x45mm field of view. The probe was fixed facing the anterior fontanel and was rotated with a micro-motor to acquire plane-by-plane scans for automated registration with an MRI neonate atlas [1]. Based on the atlas parcellation, mean CBV time courses for cortical and thalamic structures were extracted before analyzing their relative synchronicity based on instantaneous phase measurements. Those coefficient were gathered in phase matrices, taken for instantaneous connectivity states.

Results/Discussion

The k-mean clustering of the phase matrices of 4 term and 6 preterm neonates revealed that their connectivity dynamically fluctuates between 4 states: 1 state of high synchronicity between bilateral frontal lobes, cingulate gyrus and thalamus, 2 states of progressive thalamo-cortical disconnection, and one state of cingulate-frontal lobe asynchrony. Interestingly, when quantifying the occurrence rate of those 4 states for term vs preterm neonates, they are significantly different (p=0.003) between the 2 groups, preterm neonates spending more time in the 2 states of thalamo-cortical disconnection. This is supported by evidences in the literature that this thalamo-cortical connections matures during the neurodevelopment.

Moreover, when applying this method to a congenital seizure disorder term neonate exhibiting “burst-suppression” EEG pattern, he is found to spend more time in atypical inter-hemispheric cortical disconnection states compared to term neonates.

Conclusions

These results demonstrate that fUS imaging may provide a clinically relevant bedside dynamic fC imaging modality to monitor the emergence of functional networks in the early days of life and to quickly identify and quantify atypical neurodevelopment and connectivity patterns. Its sensitivity in discriminating between term and preterm neonates with low headcount is encouraging for its use as a full-fledged neuroimaging tool for neurodevelopment.

Acknowledgement

This research received funding from the Premup Foundation, the Chiesi Foundation Onlus and the European Union’s Seventh Framework Program (FP7/2007-2013)/ERC Advanced Grant Agreement 339244-FUSIMAGINE.

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Makropoulos et al, 2016,'Regional growth and atlasing of the developing human brain', Neuroimage
Figure 1: fUS bedside monitoring

a. Neonate brain state is monitored using concomitant fUS and EEG measurements. b. Miniaturized motorized probe facing the fontanel of the neonate. c. fUS leverages very high sensitivity to small blood flows, here a neonate brain coronal section. d. 3D imaging trough the anterior fontanel. e. cerebral areas identified on a co-registered reference MRI-base atlas.

Figure 2: Dynamic fC imaging using fUS
a. The synchronicity of the average CBV signal in the 6 in-plane cerebral areas is evaluated to build phase matrices (1 matrix for 1 s of acquisition), before clustering to define frequent brain states (b). c. illustrative 5 min time course showing for a term (top) and a preterm (bottom) neonate: the current phase matrix (thumbnails) and the assigned brain state (color in the strip) d. occurrence of those 4 brain states is different between term and preterm neonates, showing decrease thalamo-cortical connection in the preterm group.
Keywords: Connectivity, Neonates, Functional Ultrasound
3:42 p.m. PS 06-06

Tetrazine-functionalised clearing agent to increase contrast in antibody imaging

Eva Schlein1, Johanna Rokka1, Tobias Gustavsson1, Jonas Eriksson2, 3, Stina Syvänen1, Dag Sehlin1

1 Uppsala Universisty, Department of Public Health and Caring Sciences, Uppsala, Sweden
2 Pet Centre, Uppsala University Hospital, Uppsala, Sweden
3 Uppsala University, Department of Medicinal Chemistry, Uppsala, Sweden

Introduction

Antibodies, designed to enter the brain, reach concentrations up to 80-fold higher than unmodified antibodies, similar to those observed with small radioligands [1]. Due to the long biological half-life, imaging of brain targets with antibody-based ligands must be performed several days post injection. To overcome this issue we used a clearing agent (CA), which induced accelerated peripheral clearance via liver receptors directly after administration [2]. Here, we aimed to combine antibody-based imaging with a CA to allow imaging shortly after administration of an amyloid-β (Aβ) antibody.

Methods

The Aβ antibody RmAb158 [3] was functionalised with trans-Cyclooctene (TCO) for induced clearance. Transgenic (tg-ArcSwe [4], Aβ pathology model) and wildtype mice were administered with [125I]RmAb158-TCO and SPECT/CT scanned 3 days later. Immediately after, CA was administered and additional SPECT/CT scans were taken after 1 h and 1 day post CA injection. The tetrazine-functionalised CA reacts quickly with the TCO-modified antibody (invers electron-demand Diels-Adler reaction) to induce radioligand clearance from blood. Blood was sampled at defined time points after antibody injection for pharmacokinetic analysis. The perfused brain tissue was cryosectioned for subsequent ex vivo autoradiography analysis.

Results/Discussion

Both strategies cleared efficiently, with a decrease of up to 90% of RmAb158. The bispecific RmAb158-scFv8D3 could not be cleared from the blood, likely due to its binding to blood cells. Hence we used RmAb158 for imaging, in combination with the tetrazine-functionalized CA, which was more efficient and has the advantage of inducible clearance. [125I]RmAb158 SPECT/CT images obtained 3 days after antibody injection showed immediate liver accumulation upon CA administration, which substantially reduced antibody concentration in blood (Fig. 1). Contrast of the brain derived signal increased after 1 h CA administration and further improved after 24 h (Fig. 2).

Conclusions

Results indicate that the method works as intended and that the use of clearing agents may be a promising strategy for antibody-based imaging in combination with more short-lived radionuclides.

Acknowledgement

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 813528.

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation

References
[1] Hultqvist, G, et al. 2017, ‘Bivalent Brain Shuttle Increases Antibody Uptake by Monovalent Binding to the Transferrin Receptor’, Theranostics, 7, 308-318
[2] Rossin, R, et al. 2013, ‘Diels–Alder Reaction for Tumor Pretargeting: In Vivo Chemistry Can Boost Tumor Radiation Dose Compared with Directly Labeled Antibody’, The Journal of Nuclear Medicine, 54, 1989-1995
[3] Englund, H, et al. 2007, ‘Sensitive ELISA detection of amyloid-b protofibrils in biological samples’, Journal of Neurochemistry, 103, 334-345
[4] Lord, A, et al. 2006, ‘The Arctic Alzheimer mutation facilitates early intraneuronal A-beta aggregation and senile plaque formation in transgenic mice’, Neurobiology of Aging, 27, 67-77
[125I]-TCO-RmAb158 blood concentration over time.

Blood was sampled after several time points. Highlight parts refer to time points for SPECT/CT imaging.

A. 3 days after injection of [125I] RmAb158;

B. 1 h after CA administration;

C. 24 h after CA injection

SPECT/CT imaging and autoradiograph of tg-ArcSwe mouse.
[125I]-TCO-RmAb158 injected in tg-ArcSwe mouse, scanned 3 days after antibody injection, 1 h after CA and 1 day after CA administration. After CA administration the liver signals initially increased as the antibody was cleared from the blood.
Keywords: amyloid beta, imaging, clearing agent, IEDDA