Community Survey Results Show that Standardisation of Preclinical Imaging Techniques Remains a Challenge.

PURPOSE: To support acquisition of accurate, reproducible and high-quality preclinical imaging data, various standardisation resources have been developed over the years. However, it is unclear the impact of those efforts in current preclinical imaging practices. To better understand the status quo in the field of preclinical imaging standardisation, the STANDARD group of the European Society of Molecular Imaging (ESMI) put together a community survey and a forum for discussion at the European Molecular Imaging Meeting (EMIM) 2022. This paper reports on the results from the STANDARD survey and the forum discussions that took place at EMIM2022. PROCEDURES: The survey was delivered to the community by the ESMI office and was promoted through the Society channels, email lists and webpages. The survey contained seven sections organised as generic questions and imaging modality-specific questions. The generic questions focused on issues regarding data acquisition, data processing, data storage, publishing and community awareness of international guidelines for animal research. Specific questions on practices in optical imaging, PET, CT, SPECT, MRI and ultrasound were further included. RESULTS: Data from the STANDARD survey showed that 47% of survey participants do not have or do not know if they have QC/QA guidelines at their institutes. Additionally, a large variability exists in the ways data are acquired, processed and reported regarding general aspects as well as modality-specific aspects. Moreover, there is limited awareness of the existence of international guidelines on preclinical (imaging) research practices. CONCLUSIONS: Standardisation of preclinical imaging techniques remains a challenge and hinders the transformative potential of preclinical imaging to augment biomedical research pipelines by serving as an easy vehicle for translation of research findings to the clinic. Data collected in this project show that there is a need to promote and disseminate already available tools to standardise preclinical imaging practices.

Three-Dimensional Iron Oxide Nanoparticle-Based Contrast-Enhanced Magnetic Resonance Imaging for Characterization of Cerebral Arteriogenesis in the Mouse Neocortex.

Purpose: Subsurface blood vessels in the cerebral cortex have been identified as a bottleneck in cerebral perfusion with the potential for collateral remodeling. However, valid techniques for non-invasive, longitudinal characterization of neocortical microvessels are still lacking. In this study, we validated contrast-enhanced magnetic resonance imaging (CE-MRI) for in vivo characterization of vascular changes in a model of spontaneous collateral outgrowth following chronic cerebral hypoperfusion. Methods: C57BL/6J mice were randomly assigned to unilateral internal carotid artery occlusion or sham surgery and after 21 days, CE-MRI based on T2*-weighted imaging was performed using ultra-small superparamagnetic iron oxide nanoparticles to obtain subtraction angiographies and steady-state cerebral blood volume (ss-CBV) maps. First pass dynamic susceptibility contrast MRI (DSC-MRI) was performed for internal validation of ss-CBV. Further validation at the histological level was provided by ex vivo serial two-photon tomography (STP). Results: Qualitatively, an increase in vessel density was observed on CE-MRI subtraction angiographies following occlusion; however, a quantitative vessel tracing analysis was prone to errors in our model. Measurements of ss-CBV reliably identified an increase in cortical vasculature, validated by DSC-MRI and STP. Conclusion: Iron oxide nanoparticle-based ss-CBV serves as a robust, non-invasive imaging surrogate marker for neocortical vessels, with the potential to reduce and refine preclinical models targeting the development and outgrowth of cerebral collateralization.

High-sensitivity detection of optogenetically-induced neural activity with functional ultrasound imaging.

Whole-brain imaging approaches and optogenetic manipulations are powerful tools to map brain-wide neural circuits in vivo. To date, functional magnetic resonance imaging (fMRI) provides the most comprehensive evaluation of such large-scale circuitry. However, functional ultrasound imaging (fUSI) has recently emerged as a complementary imaging modality that can extend such measurements towards the context of diverse behavioral states and tasks. Nevertheless, in order to properly interpret the fUSI signal during these complicated scenarios, it must first be carefully validated against well-established technologies, such as fMRI, in highly controlled experimental settings. Here, to address this need, we compared subsequent fMRI and fUSI recordings in response to direct neuronal activation via optogenetics in the same animals under an identical anesthetic protocol. Specifically, we applied various intensities of light stimulation to the primary motor cortex (M1) of mice and compared the spatiotemporal dynamics of the elicited fMRI and fUSI signals. Overall, our general linear model analysis (t-scores) and time series analysis (z-scores) revealed that fUSI was more sensitive than fMRI for detecting optogenetically-induced neuronal activation. Local field potential recordings in the bilateral M1 and striatum also better co-localized with fUSI activation patterns than those of fMRI. Finally, the fUSI response contained distinct arterial and venous components that provide vascular readouts of neuronal activity with vessel-type specificity.

Dysfunctional Autism Risk Genes Cause Circuit-Specific Connectivity Deficits With Distinct Developmental Trajectories.

Autism spectrum disorders (ASD) are a set of complex neurodevelopmental disorders for which there is currently no targeted therapeutic approach. It is thought that alterations of genes regulating migration and synapse formation during development affect neural circuit formation and result in aberrant connectivity within distinct circuits that underlie abnormal behaviors. However, it is unknown whether deviant developmental trajectories are circuit-specific for a given autism risk-gene. We used MRI to probe changes in functional and structural connectivity from childhood to adulthood in Fragile-X (Fmr1-/y) and contactin-associated (CNTNAP2-/-) knockout mice. Young Fmr1-/y mice (30 days postnatal) presented with a robust hypoconnectivity phenotype in corticocortico and corticostriatal circuits in areas associated with sensory information processing, which was maintained until adulthood. Conversely, only small differences in hippocampal and striatal areas were present during early postnatal development in CNTNAP2-/- mice, while major connectivity deficits in prefrontal and limbic pathways developed between adolescence and adulthood. These findings are supported by viral tracing and electron micrograph approaches and define 2 clearly distinct connectivity endophenotypes within the autism spectrum. We conclude that the genetic background of ASD strongly influences which circuits are most affected, the nature of the phenotype, and the developmental time course of the associated changes.

The role of beta-arrestin2 in shaping fMRI BOLD responses to dopaminergic stimulation.

RATIONALE: The dopamine D2 receptor (D2R) couples to inhibitory Gi/o proteins and is targeted by antipsychotic and antiparkinsonian drugs. Beta-arrestin2 binds to the intracellular regions of the agonist-occupied D2R to terminate G protein activation and promote internalization, but also to initiate downstream signaling cascades which have been implicated in psychosis. Functional magnetic resonance imaging (fMRI) has proven valuable for measuring dopamine receptor-mediated changes in neuronal activity, and might enable beta-arrestin2 function to be studied in vivo. OBJECTIVES: The present study examined fMRI blood oxygenation level dependent (BOLD) signal changes elicited by a dopamine agonist in wild-type (WT) and beta-arrestin2 knockout (KO) mice, to investigate whether genetic deletion of beta-arrestin2 prolongs or otherwise modifies D2R-dependent responses. METHODS: fMRI BOLD data were acquired on a 9.4 T system. During scans, animals received 0.2 mg/kg apomorphine, i.v. In a subset of experiments, animals were pretreated with 2 mg/kg of the D2R antagonist, eticlopride. RESULTS: Following apomorphine administration, BOLD signal decreases were observed in caudate/putamen of WT and KO animals. The time course of response decay in caudate/putamen was significantly slower in KO vs. WT animals. In cingulate cortex, an initial BOLD signal decrease was followed by a positive response component in WT but not in KO animals. Eticlopride pretreatment significantly reduced apomorphine-induced BOLD signal changes. CONCLUSIONS: The prolonged striatal response decay rates in KO animals might reflect impaired D2R desensitization, consistent with the known function of beta-arrestin2. Furthermore, the apomorphine-induced positive response component in cingulate cortex may depend on beta-arrestin2 signaling downstream of D2R.

Quantitative assessment of microvasculopathy in arcAß mice with USPIO-enhanced gradient echo MRI.

Magnetic resonance imaging employing administration of iron oxide-based contrast agents is widely used to visualize cellular and molecular processes in vivo. In this study, we investigated the ability of [Formula: see text] and quantitative susceptibility mapping to quantitatively assess the accumulation of ultrasmall superparamagnetic iron oxide (USPIO) particles in the arcAß mouse model of cerebral amyloidosis. Gradient-echo data of mouse brains were acquired at 9.4 T after injection of USPIO. Focal areas with increased magnetic susceptibility and [Formula: see text] values were discernible across several brain regions in 12-month-old arcAß compared to 6-month-old arcAß mice and to non-transgenic littermates, indicating accumulation of particles after USPIO injection. This was concomitant with higher [Formula: see text] and increased magnetic susceptibility differences relative to cerebrospinal fluid measured in USPIO-injected compared to non-USPIO-injected 12-month-old arcAß mice. No differences in [Formula: see text] and magnetic susceptibility were detected in USPIO-injected compared to non-injected 12-month-old non-transgenic littermates. Histological analysis confirmed focal uptake of USPIO particles in perivascular macrophages adjacent to small caliber cerebral vessels with radii of 2-8 µm that showed no cerebral amyloid angiopathy. USPIO-enhanced [Formula: see text] and quantitative susceptibility mapping constitute quantitative tools to monitor such functional microvasculopathies.

Reconstructing cerebrovascular networks under local physiological constraints by integer programming.

We introduce a probabilistic approach to vessel network extraction that enforces physiological constraints on the vessel structure. The method accounts for both image evidence and geometric relationships between vessels by solving an integer program, which is shown to yield the maximum a posteriori (MAP) estimate to a probabilistic model. Starting from an overconnected network, it is pruning vessel stumps and spurious connections by evaluating the local geometry and the global connectivity of the graph. We utilize a high-resolution micro computed tomography (µCT) dataset of a cerebrovascular corrosion cast to obtain a reference network and learn the prior distributions of our probabilistic model and we perform experiments on in-vivo magnetic resonance microangiography (µMRA) images of mouse brains. We finally discuss properties of the networks obtained under different tracking and pruning approaches.

Magnetic Resonance Q Mapping Reveals a Decrease in Microvessel Density in the arcAß Mouse Model of Cerebral Amyloidosis.

Alterations in density and morphology of the cerebral microvasculature have been reported to occur in Alzheimer's disease patients and animal models of the disease. In this study we compared magnetic resonance imaging (MRI) techniques for their utility to detect age-dependent changes of the cerebral vasculature in the arcAß mouse model of cerebral amyloidosis. Dynamic susceptibility contrast (DSC)-MRI was performed by tracking the passage of a superparamagnetic iron oxide nanoparticle in the brain with dynamic gradient echo planar imaging (EPI). From this measurements relative cerebral blood volume [rCBV(DSC)] and relative cerebral blood flow (rCBF) were estimated. For the same animal maps of the relaxation shift index Q were computed from high resolution gradient echo and spin echo data that were acquired before and after superparamagnetic iron oxide (SPIO) nanoparticle injection. Q-values were used to derive estimates of microvessel density. The change in the relaxation rates [Formula: see text] obtained from pre- and post-contrast gradient echo data was used for the alternative determination of rCBV [rCBV([Formula: see text])]. Linear mixed effects modeling found no significant association between rCBV(DSC), rCBV([Formula: see text]), rCBF, and Q with genotype in 13-month old mice [compared to age-matched non-transgenic littermates (NTLs)] for any of the evaluated brain regions. In 24-month old mice there was a significant association for rCBV(DSC) with genotype in the cerebral cortex, and for rCBV([Formula: see text]) in the cerebral cortex and cerebellum. For rCBF there was a significant association in the cerebellum but not in other brain regions. Q-values in the olfactory bulb, cerebral cortex, striatum, hippocampus, and cerebellum in 24-month old mice were significantly associated with genotype. In those regions Q-values were reduced between 11 and 26% in arcAß mice compared to age-matched NTLs. Vessel staining with CD31 immunohistochemistry confirmed a reduction of microvessel density in the old arcAß mice. We further demonstrated a region-specific association between parenchymal and vascular deposition of ß-amyloid and decreased vascular density, without a correlation with the amount of Aß deposition. We found that Q mapping was more suitable than the hemodynamic read-outs to detect amyloid-related degeneration of the cerebral microvasculature.

Extracting vascular networks under physiological constraints via integer programming.

We introduce an integer programming-based approach to vessel network extraction that enforces global physiological constraints on the vessel structure and learn this prior from a high-resolution reference network. The method accounts for both image evidence and geometric relationships between vessels by formulating and solving an integer programming problem. Starting from an over-connected network, it is pruning vessel stumps and spurious connections by evaluating bifurcation angle and connectivity of the graph. We utilize a high-resolution micro computed tomography (µCT) dataset of a cerebrovascular corrosion cast to obtain a reference network, perform experiments on micro magnetic resonance angiography (µMRA) images of mouse brains and discuss properties of the networks obtained under different tracking and pruning approaches.