Multiparametric MRI for characterization of the tumour microenvironment.

Our understanding of tumour biology has evolved over the past decades and cancer is now viewed as a complex ecosystem with interactions between various cellular and non-cellular components within the tumour microenvironment (TME) at multiple scales. However, morphological imaging remains the mainstay of tumour staging and assessment of response to therapy, and the characterization of the TME with non-invasive imaging has not yet entered routine clinical practice. By combining multiple MRI sequences, each providing different but complementary information about the TME, multiparametric MRI (mpMRI) enables non-invasive assessment of molecular and cellular features within the TME, including their spatial and temporal heterogeneity. With an increasing number of advanced MRI techniques bridging the gap between preclinical and clinical applications, mpMRI could ultimately guide the selection of treatment approaches, precisely tailored to each individual patient, tumour and therapeutic modality. In this Review, we describe the evolving role of mpMRI in the non-invasive characterization of the TME, outline its applications for cancer detection, staging and assessment of response to therapy, and discuss considerations and challenges for its use in future medical applications, including personalized integrated diagnostics.

Epilepsy-related functional brain network alterations are already present at an early age in the GAERS rat model of genetic absence epilepsy.

Introduction: Genetic Absence Epilepsy Rats from Strasbourg (GAERS) represent a model of genetic generalized epilepsy. The present longitudinal study in GAERS and age-matched non-epileptic controls (NEC) aimed to characterize the epileptic brain network using two functional measures, resting state-functional magnetic resonance imaging (rs-fMRI) and manganese-enhanced MRI (MEMRI) combined with morphometry, and to investigate potential brain network alterations, following long-term seizure activity. Methods: Repeated rs-fMRI measurements at 9.4 T between 3 and 8 months of age were combined with MEMRI at the final time point of the study. We used graph theory analysis to infer community structure and global and local network parameters from rs-fMRI data and compared them to brain region-wise manganese accumulation patterns and deformation-based morphometry (DBM). Results: Functional connectivity (FC) was generally higher in GAERS when compared to NEC. Global network parameters and community structure were similar in NEC and GAERS, suggesting efficiently functioning networks in both strains. No progressive FC changes were observed in epileptic animals. Network-based statistics (NBS) revealed stronger FC within the cortical community, including regions of association and sensorimotor cortex, and with basal ganglia and limbic regions in GAERS, irrespective of age. Higher manganese accumulation in GAERS than in NEC was observed at 8 months of age, consistent with higher overall rs-FC, particularly in sensorimotor cortex and association cortex regions. Functional measures showed less similarity in subcortical regions. Whole brain volumes of 8 months-old GAERS were higher when compared to age-matched NEC, and DBM revealed increased volumes of several association and sensorimotor cortex regions and of the thalamus. Discussion: rs-fMRI, MEMRI, and volumetric data collectively suggest the significance of cortical networks in GAERS, which correlates with an increased fronto-central connectivity in childhood absence epilepsy (CAE). Our findings also verify involvement of basal ganglia and limbic regions. Epilepsy-related network alterations are already present in juvenile animals. Consequently, this early condition seems to play a greater role in dynamic brain function than chronic absence seizures.

Radial compressed sensing imaging improves the velocity detection limit of single cell tracking time-lapse MRI.

PURPOSE: Time-lapse MRI enables tracking of single iron-labeled cells. Yet, due to temporal blurring, only slowly moving cells can be resolved. To study faster cells for example during inflammatory processes, accelerated acquisition is needed. METHODS: A rotating phantom system was developed to quantitatively measure the current maximum detectable speed of cells in time-lapse MRI. For accelerated cell tracking, an interleaved radial acquisition scheme was applied to phantom and murine brain in vivo time-lapse MRI experiments at 9.4 T. Detection of iron-labeled cells was evaluated in fully sampled and undersampled reconstructions with and without compressed sensing. RESULTS: The rotating phantom system enabled ultra-slow rotation of phantoms and a velocity detection limit of full-brain Cartesian time-lapse MRI of up to 172 µm/min was determined. Both phantom and in vivo measurements showed that single cells can be followed dynamically using radial time-lapse MRI. Higher temporal resolution of undersampled reconstructions reduced geometric distortion, the velocity detection limit was increased to 1.1 mm/min in vitro, and previously hidden fast-moving cells were recovered. In the mouse brain after in vivo labeling, a total of 42 ± 4 cells were counted in fully sampled, but only 7 ± 1 in undersampled images due to streaking artifacts. Using compressed sensing 33 ± 4 cells were detected. CONCLUSION: Interleaved radial time-lapse MRI permits retrospective reconstruction of both fully sampled and accelerated images, enables single cell tracking at higher temporal resolution and recovers cells hidden before due to blurring. The velocity detection limit as determined with the rotating phantom system increased two- to three-fold compared to previous results.

Corrections for Rabi oscillations in cardiac chemical exchange saturation transfer MRI under the influence of very short preparation pulses.

Very short chemical exchange saturation transfer (CEST) pulses are beneficial in cardiac continuous wave (cw) CEST MRI, especially in small animals because of their rapid heartbeat; however, they result in signal modulations caused by Rabi oscillations. Therefore, we implemented two different filter techniques, DOwnsampling by SEparation of CEST spectrum into two parts (DOSE) and time domain (TD)-based filtering, to correct for these signal corruptions, allowing a reliable quantification of glucose-weighted CEST (glucoCEST) MRI contrast. In our study, cw CEST measurements were performed on a 9.4-T small animal BioSpec system using CEST pulses in the range of 10 to 200 ms. Experimental dependencies of Rabi oscillations on key MRI parameters were validated by Bloch-McConnell (BM) simulations. Filter efficiency was explored in a glucose concentration series as well as in the myocardium of healthy mice (n = 8), and glucoCEST contrast was subsequently quantified. The experimental results showed that the impact of Rabi oscillations on CEST spectra increased with decreasing CEST pulse length, optimized B0 homogeneity, and shorter T2 relaxation time, in accordance with results from BM simulations. Both investigated filter techniques reduced these signal modulations significantly, with DOSE filtering preserving the amplitude and TD filtering the spectral information of CEST data more accurately. Upon filter application, a significant decrease in glucoCEST contrast in the myocardium of healthy mice was observed after glucose infusion (pTD = 0.0079, pDOSE = 0.0044). To conclude, this study offers comprehensive experimental insights into Rabi oscillations within CEST MRI data along with methodological considerations that could be further advanced into a robust and precise cardiac cw CEST protocol by integrating DOSE and TD filtering into the standard CEST analysis pipeline.

Generation of a whole-brain hemodynamic response function and sex-specific differences in cerebral processing of mechano-sensation in mice detected by BOLD fMRI.

BOLD fMRI has become a prevalent method to study cerebral sensory processing in rodent disease models, including pain and mechanical hypersensitivity. fMRI data analysis is frequently combined with a general-linear-model (GLM) -based analysis, which uses the convolution of a hemodynamic response function (HRF) with the stimulus paradigm. However, several studies indicated that the HRF differs across species, sexes, brain structures, and experimental factors, including stimulation modalities or anesthesia, and hence might strongly affect the outcome of BOLD analyzes. While considerable work has been done in humans and rats to understand the HRF, much less is known in mice. As a prerequisite to investigate mechano-sensory processing and BOLD fMRI data in male and female mice, we (1) designed a rotating stimulator that allows application of two different mechanical modalities, including innocuous von Frey and noxious pinprick stimuli and (2) determined and statistically compared HRFs across 30 brain structures and experimental conditions, including sex and, stimulus modalities. We found that mechanical stimulation lead to brain-wide BOLD signal changes thereby allowing extraction of HRFs from multiple brain structures. However, we did not find differences in HRFs across all brain structures and experimental conditions. Hence, we computed a whole-brain mouse HRF, which is based on 88 functional scans from 30 mice. A comparison of this mouse-specific HRF with our previously reported rat-derived HRF showed significantly slower kinetics in mice. Finally, we detected pronounced differences in cerebral BOLD activation between male and female mice with mechanical stimulation, thereby exposing divergent processing of noxious and innocuous stimuli in both sexes.

Multiparametric chemical exchange saturation transfer MRI detects metabolic changes in breast cancer following immunotherapy.

BACKGROUND: With metabolic alterations of the tumor microenvironment (TME) contributing to cancer progression, metastatic spread and response to targeted therapies, non-invasive and repetitive imaging of tumor metabolism is of major importance. The purpose of this study was to investigate whether multiparametric chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI) allows to detect differences in the metabolic profiles of the TME in murine breast cancer models with divergent degrees of malignancy and to assess their response to immunotherapy. METHODS: Tumor characteristics of highly malignant 4T1 and low malignant 67NR murine breast cancer models were investigated, and their changes during tumor progression and immune checkpoint inhibitor (ICI) treatment were evaluated. For simultaneous analysis of different metabolites, multiparametric CEST-MRI with calculation of asymmetric magnetization transfer ratio (MTRasym) at 1.2 to 2.0 ppm for glucose-weighted, 2.0 ppm for creatine-weighted and 3.2 to 3.6 ppm for amide proton transfer- (APT-) weighted CEST contrast was conducted. Ex vivo validation of MRI results was achieved by 1H nuclear magnetic resonance spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry imaging with laser postionization and immunohistochemistry. RESULTS: During tumor progression, the two tumor models showed divergent trends for all examined CEST contrasts: While glucose- and APT-weighted CEST contrast decreased and creatine-weighted CEST contrast increased over time in the 4T1 model, 67NR tumors exhibited increased glucose- and APT-weighted CEST contrast during disease progression, accompanied by decreased creatine-weighted CEST contrast. Already three days after treatment initiation, CEST contrasts captured response to ICI therapy in both tumor models. CONCLUSION: Multiparametric CEST-MRI enables non-invasive assessment of metabolic signatures of the TME, allowing both for estimation of the degree of tumor malignancy and for assessment of early response to immune checkpoint inhibition.

Dynamic cell tracking using time-lapse MRI with variable temporal resolution Cartesian sampling.

PURPOSE: Temporal resolution of time-lapse MRI to track individual iron-labeled cells is limited by the required data-acquisition time to fill k-space and to reach sufficient SNR. Although motion of slowly patrolling monocytes can be resolved, detection of fast-moving immune cells requires improved acquisition and reconstruction strategies. THEORY AND METHODS: For accelerated MRI cell tracking, a Cartesian sampling scheme was designed, in which the fully sampled and undersampled k-space data for different acceleration factors were acquired simultaneously, and multiple undersampling ratios could be chosen retrospectively. Compressed-sensing reconstruction was applied using dictionary learning and low-rank constraints. Detection of iron-labeled monocytes was evaluated with simulations, rotating phantom experiments and in vivo mouse brain measurements at 9.4 T. RESULTS: Fully sampled and 2.4-times and 4.8-times accelerated images were reconstructed and had sufficient contrast-to-noise ratio (CNR) for single cells to be resolved and followed dynamically. The phantom experiments showed an improvement in CNR of 6.1% per µm/s in the 4.8-times undersampled images. Geometric distortion of cells caused by motion was visibly reduced in the accelerated images, which enabled detection of moving cells with velocities of up to 7.0 µm/s. In vivo, additional cells were resolved in the accelerated images due to the improved temporal resolution. CONCLUSION: The easy-to-implement flexible Cartesian sampling scheme with compressed-sensing reconstruction permits simultaneous acquisition of both fully sampled and high temporal resolution images. The CNR of moving cells is effectively improved, enabling the recovery of high velocity cells with sufficient contrast at virtually no cost.

Disentangling the impact of cerebrospinal fluid formation and neuronal activity on solute clearance from the brain.

BACKGROUND: Despite recent attention, pathways and mechanisms of fluid transposition in the brain are still a matter of intense discussion and driving forces underlying waste clearance in the brain remain elusive. Consensus exists that net solute transport is a prerequisite for efficient clearance. The individual impact of neuronal activity and cerebrospinal fluid (CSF) formation, which both vary with brain state and anesthesia, remain unclear. METHODS: To separate conditions with high and low neuronal activity and high and low CSF formation, different anesthetic regimens in naive rat were established, using Isoflurane (ISO), Medetomidine (MED), acetazolamide or combinations thereof. With dynamic contrast-enhanced MRI, after application of low molecular weight contrast agent (CA) Gadobutrol to cisterna magna, tracer distribution was monitored as surrogate for solute clearance. Simultaneous fiber-based Ca2+-recordings informed about the state of neuronal activity under different anesthetic regimen. T2-weighted MRI and diffusion-weighted MRI (DWI) provided size of subarachnoidal space and aqueductal flow as surrogates for CSF formation. Finally, a pathway and mechanism-independent two-compartment model was introduced to provide a measure of efficiency for solute clearance from the brain. RESULTS: Anatomical imaging, DWI and Ca2+-recordings confirmed that conditions with distinct levels of neuronal activity and CSF formation were achieved. A sleep-resembling condition, with reduced neuronal activity and enhanced CSF formation was achieved using ISO+MED and an awake-like condition with high neuronal activity using MED alone. CA distribution in the brain correlated with the rate of CSF formation. The cortical brain state had major influence on tracer diffusion. Under conditions with low neuronal activity, higher diffusivity suggested enlargement of extracellular space, facilitating a deeper permeation of solutes into brain parenchyma. Under conditions with high neuronal activity, diffusion of solutes into parenchyma was hindered and clearance along paravascular pathways facilitated. Exclusively based on the measured time signal curves, the two-compartment model provided net exchange ratios, which were significantly larger for the sleep-resembling condition than for the awake-like condition. CONCLUSIONS: Efficiency of solute clearance in brain changes with alterations in both state of neuronal activity and CSF formation. Our clearance pathway and mechanism agnostic kinetic model informs about net solute transport, solely based on the measured time signal curves. This rather simplifying approach largely accords with preclinical and clinical findings.

Translational imaging of TSPO reveals pronounced innate inflammation in human and murine CD8 T cell-mediated limbic encephalitis.

Autoimmune limbic encephalitis (ALE) presents with new-onset mesial temporal lobe seizures, progressive memory disturbance, and other behavioral and cognitive changes. CD8 T cells are considered to play a key role in those cases where autoantibodies (ABs) target intracellular antigens or no ABs were found. Assessment of such patients presents a clinical challenge, and novel noninvasive imaging biomarkers are urgently needed. Here, we demonstrate that visualization of the translocator protein (TSPO) with [18F]DPA-714-PET-MRI reveals pronounced microglia activation and reactive gliosis in the hippocampus and amygdala of patients suspected with CD8 T cell ALE, which correlates with FLAIR-MRI and EEG alterations. Back-translation into a preclinical mouse model of neuronal antigen-specific CD8 T cell-mediated ALE allowed us to corroborate our preliminary clinical findings. These translational data underline the potential of [18F]DPA-714-PET-MRI as a clinical molecular imaging method for the direct assessment of innate immunity in CD8 T cell-mediated ALE.

EXERTION: a pilot trial on the effect of aerobic, smartwatch-controlled exercise on stroke recovery: effects on motor function, structural repair, cognition, mental well-being, and the immune system.

INTRODUCTION: Motor impairments are the objectively most striking sequelae after stroke, but non-motor consequences represent a high burden for stroke survivors as well. Depression is reported in one third of patients, the fatigue prevalence ranges from 23 to 75% due to heterogenous definitions and assessments. Cognitive impairment is found in one third of stroke patients 3-12 months after stroke and the risk for dementia is doubled by the event. Aerobic exercise has been shown to reduce depressive symptoms, counteract fatigue, and improve cognitive functions in non-stroke patients. Furthermore, exercise is known to strengthen the immune system. It is unknown, though, if aerobic exercise can counteract poststroke depression, fatigue, poststroke dementia and poststroke immunosuppression. Therefore, we aim to analyse the effect of aerobic exercise on functional recovery, cognition, emotional well-being, and the immune system. Reorganization of topological networks of the brain shall be visualized by diffusion MRI fibre tracking. METHODS: Adults with mild to moderate stroke impairment (initial NIHSS or NIHSS determined at the moment of maximal deterioration 1-18) are recruited within two weeks of stroke onset. Study participants must be able to walk independently without risk of falling. All patients are equipped with wearable devices (smartwatches) measuring the heart rate and daily step count. The optimal heart rate zone is determined by lactate ergometry at baseline. Patients are randomized to the control or the intervention group, the latter performing a heart rate-controlled walking training on own initiative 5 times a week for 45 min. All patients receive medical care and stroke rehabilitation to the usual standard of care. The following assessments are conducted at baseline and after 90 days: Fugl Meyer-assessment for the upper and lower extremity, 6 min-walk test, neuropsychological assessment (cognition: MoCA, SDMT; fatigue and depression: FSMC, HADS-D, participation: WHODAS 2.0 12-items), blood testing (i.e. immune profiling to obtain insights into phenotype and functional features of distinct immune-cell subsets) and cranial magnetic resonance imaging (MRI) with grid-sampled diffusion weighted imaging, white matter fibre tracking and MR spectroscopy. PERSPECTIVE: This study investigates the effect of smartwatch-controlled aerobic exercise on functional recovery, cognition, emotional well-being, the immune system, and neuronal network reorganization in stroke patients. Trial registration ClinicalTrials.gov NCT Number: NCT05690165. First posted19 January 2023. Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT05690165.

Vascular response patterns to targeted therapies in murine breast cancer models with divergent degrees of malignancy.

BACKGROUND: Response assessment of targeted cancer therapies is becoming increasingly challenging, as it is not adequately assessable with conventional morphological and volumetric analyses of tumor lesions. The tumor microenvironment is particularly constituted by tumor vasculature which is altered by various targeted therapies. The aim of this study was to noninvasively assess changes in tumor perfusion and vessel permeability after targeted therapy in murine models of breast cancer with divergent degrees of malignancy. METHODS: Low malignant 67NR or highly malignant 4T1 tumor-bearing mice were treated with either the multi-kinase inhibitor sorafenib or immune checkpoint inhibitors (ICI, combination of anti-PD1 and anti-CTLA4). Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with i.v. injection of albumin-binding gadofosveset was conducted on a 9.4 T small animal MRI. Ex vivo validation of MRI results was achieved by transmission electron microscopy, immunohistochemistry and laser ablation-inductively coupled plasma-mass spectrometry. RESULTS: Therapy-induced changes in tumor vasculature differed between low and highly malignant tumors. Sorafenib treatment led to decreased tumor perfusion and endothelial permeability in low malignant 67NR tumors. In contrast, highly malignant 4T1 tumors demonstrated characteristics of a transient window of vascular normalization with an increase in tumor perfusion and permeability early after therapy initiation, followed by decreased perfusion and permeability parameters. In the low malignant 67NR model, ICI treatment also mediated vessel-stabilizing effects with decreased tumor perfusion and permeability, while ICI-treated 4T1 tumors exhibited increasing tumor perfusion with excessive vascular leakage. CONCLUSION: DCE-MRI enables noninvasive assessment of early changes in tumor vasculature after targeted therapies, revealing different response patterns between tumors with divergent degrees of malignancy. DCE-derived tumor perfusion and permeability parameters may serve as vascular biomarkers that allow for repetitive examination of response to antiangiogenic treatment or immunotherapy.

A Carboxy-terminal Smarcb1 Point Mutation Induces Hydrocephalus Formation and Affects AP-1 and Neuronal Signalling Pathways in Mice.

The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal differentiation. Mutations of its core subunit SMARCB1 result in a broad spectrum of pathologies, including aggressive rhabdoid tumours or neurodevelopmental disorders. Other mouse models have addressed the influence of a homo- or heterozygous loss of Smarcb1, yet the impact of specific non-truncating mutations remains poorly understood. Here, we have established a new mouse model for the carboxy-terminal Smarcb1 c.1148del point mutation, which leads to the synthesis of elongated SMARCB1 proteins. We have investigated its impact on brain development in mice using magnetic resonance imaging, histology, and single-cell RNA sequencing. During adolescence, Smarcb11148del/1148del mice demonstrated rather slow weight gain and frequently developed hydrocephalus including enlarged lateral ventricles. In embryonic and neonatal stages, mutant brains did not differ anatomically and histologically from wild-type controls. Single-cell RNA sequencing of brains from newborn mutant mice revealed that a complete brain including all cell types of a physiologic mouse brain is formed despite the SMARCB1 mutation. However, neuronal signalling appeared disturbed in newborn mice, since genes of the AP-1 transcription factor family and neurite outgrowth-related transcripts were downregulated. These findings support the important role of SMARCB1 in neurodevelopment and extend the knowledge of different Smarcb1 mutations and their associated phenotypes.

A preclinical randomized controlled multi-centre trial of anti-interleukin-17A treatment for acute ischaemic stroke.

Multiple consensus statements have called for preclinical randomized controlled trials to improve translation in stroke research. We investigated the efficacy of an interleukin-17A neutralizing antibody in a multi-centre preclinical randomized controlled trial using a murine ischaemia reperfusion stroke model. Twelve-week-old male C57BL/6 mice were subjected to 45 min of transient middle cerebral artery occlusion in four centres. Mice were randomly assigned (1:1) to receive either an anti-interleukin-17A (500 µg) or isotype antibody (500 µg) intravenously 1 h after reperfusion. The primary endpoint was infarct volume measured by magnetic resonance imaging three days after transient middle cerebral artery occlusion. Secondary analysis included mortality, neurological score, neutrophil infiltration and the impact of the gut microbiome on treatment effects. Out of 136 mice, 109 mice were included in the analysis of the primary endpoint. Mixed model analysis revealed that interleukin-17A neutralization significantly reduced infarct sizes (anti-interleukin-17A: 61.77 ± 31.04 mm3; IgG control: 75.66 ± 34.79 mm3; P = 0.01). Secondary outcome measures showed a decrease in mortality (hazard ratio = 3.43, 95% confidence interval = 1.157-10.18; P = 0.04) and neutrophil invasion into ischaemic cortices (anti-interleukin-17A: 7222 ± 6108 cells; IgG control: 28 153 ± 23 206 cells; P < 0.01). There was no difference in Bederson score. The analysis of the gut microbiome showed significant heterogeneity between centres (R = 0.78, P < 0.001, n = 40). Taken together, neutralization of interleukin-17A in a therapeutic time window resulted in a significant reduction of infarct sizes and mortality compared with isotype control. It suggests interleukin-17A neutralization as a potential therapeutic target in stroke.

The impact of vasomotion on analysis of rodent fMRI data.

Introduction: Small animal fMRI is an essential part of translational research in the cognitive neurosciences. Due to small dimensions and animal physiology preclinical fMRI is prone to artifacts that may lead to misinterpretation of the data. To reach unbiased translational conclusions, it is, therefore, crucial to identify potential sources of experimental noise and to develop correction methods for contributions that cannot be avoided such as physiological noise. Aim of this study was to assess origin and prevalence of hemodynamic oscillations (HDO) in preclinical fMRI in rat, as well as their impact on data analysis. Methods: Following the development of algorithms for HDO detection and suppression, HDO prevalence in fMRI measurements was investigated for different anesthetic regimens, comprising isoflurane and medetomidine, and for both gradient echo and spin echo fMRI sequences. In addition to assessing the effect of vasodilation on HDO, it was studied if HDO have a direct neuronal correlate using local field potential (LFP) recordings. Finally, the impact of HDO on analysis of fMRI data was assessed, studying both the impact on calculation of activation maps as well as the impact on brain network analysis. Overall, 303 fMRI measurements and 32 LFP recordings were performed in 71 rats. Results: In total, 62% of the fMRI measurements showed HDO with a frequency of (0.20 ± 0.02) Hz. This frequent occurrence indicated that HDO cannot be generally neglected in fMRI experiments. Using the developed algorithms, HDO were detected with a specificity of 95%, and removed efficiently from the signal time courses. HDO occurred brain-wide under vasoconstrictive conditions in both small and large blood vessels. Vasodilation immediately interrupted HDO, which, however, returned within 1 h under vasoconstrictive conditions. No direct neuronal correlate of HDO was observed in LFP recordings. HDO significantly impacted analysis of fMRI data, leading to altered cluster sizes and F-values for activated voxels, as well as altered brain networks, when comparing data with and without HDO. Discussion: We therefore conclude that HDO are caused by vasomotion under certain anesthetic conditions and should be corrected during fMRI data analysis to avoid bias.

Profiling specific cell populations within the inflammatory tumor microenvironment by oscillating-gradient diffusion-weighted MRI.

BACKGROUND: The inflammatory tumor microenvironment (TME) is formed by various immune cells, being closely associated with tumorigenesis. Especially, the interaction between tumor-infiltrating T-cells and macrophages has a crucial impact on tumor progression and metastatic spread. The purpose of this study was to investigate whether oscillating-gradient diffusion-weighted MRI (OGSE-DWI) enables a cell size-based discrimination between different cell populations of the TME. METHODS: Sine-shaped OGSE-DWI was combined with the Imaging Microstructural Parameters Using Limited Spectrally Edited Diffusion (IMPULSED) approach to measure microscale diffusion distances, here relating to cell sizes. The accuracy of IMPULSED-derived cell radii was evaluated using in vitro spheroid models, consisting of either pure cancer cells, macrophages, or T-cells. Subsequently, in vivo experiments aimed to assess changes within the TME and its specific immune cell composition in syngeneic murine breast cancer models with divergent degrees of malignancy (4T1, 67NR) during tumor progression, clodronate liposome-mediated depletion of macrophages, and immune checkpoint inhibitor (ICI) treatment. Ex vivo analysis of IMPULSED-derived cell radii was conducted by immunohistochemical wheat germ agglutinin staining of cell membranes, while intratumoral immune cell composition was analyzed by CD3 and F4/80 co-staining. RESULTS: OGSE-DWI detected mean cell radii of 8.8±1.3 µm for 4T1, 8.2±1.4 µm for 67NR, 13.0±1.7 for macrophage, and 3.8±1.8 µm for T-cell spheroids. While T-cell infiltration during progression of 4T1 tumors was observed by decreasing mean cell radii from 9.7±1.0 to 5.0±1.5 µm, increasing amount of intratumoral macrophages during progression of 67NR tumors resulted in increasing mean cell radii from 8.9±1.2 to 12.5±1.1 µm. After macrophage depletion, mean cell radii decreased from 6.3±1.7 to 4.4±0.5 µm. T-cell infiltration after ICI treatment was captured by decreasing mean cell radii in both tumor models, with more pronounced effects in the 67NR tumor model. CONCLUSIONS: OGSE-DWI provides a versatile tool for non-invasive profiling of the inflammatory TME by assessing the dominating cell type T-cells or macrophages.

Pathophysiological Mechanisms of Cardiac Dysfunction in Transgenic Mice with Viral Myocarditis.

Viral myocarditis is pathologically associated with RNA viruses such as coxsackievirus B3 (CVB3), or more recently, with SARS-CoV-2, but despite intensive research, clinically proven treatment is limited. Here, by use of a transgenic mouse strain (TG) containing a CVB3ΔVP0 genome we unravel virus-mediated cardiac pathophysiological processes in vivo and in vitro. Cardiac function, pathologic ECG alterations, calcium homeostasis, intracellular organization and gene expression were significantly altered in transgenic mice. A marked alteration of mitochondrial structure and gene expression indicates mitochondrial impairment potentially contributing to cardiac contractile dysfunction. An extended picture on viral myocarditis emerges that may help to develop new treatment strategies and to counter cardiac failure.

Multiparametric MRI enables for differentiation of different degrees of malignancy in two murine models of breast cancer.

Objective: The objective of this study was to non-invasively differentiate the degree of malignancy in two murine breast cancer models based on identification of distinct tissue characteristics in a metastatic and non-metastatic tumor model using a multiparametric Magnetic Resonance Imaging (MRI) approach. Methods: The highly metastatic 4T1 breast cancer model was compared to the non-metastatic 67NR model. Imaging was conducted on a 9.4 T small animal MRI. The protocol was used to characterize tumors regarding their structural composition, including heterogeneity, intratumoral edema and hemorrhage, as well as endothelial permeability using apparent diffusion coefficient (ADC), T1/T2 mapping and dynamic contrast-enhanced (DCE) imaging. Mice were assessed on either day three, six or nine, with an i.v. injection of the albumin-binding contrast agent gadofosveset. Ex vivo validation of the results was performed with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), histology, immunhistochemistry and electron microscopy. Results: Significant differences in tumor composition were observed over time and between 4T1 and 67NR tumors. 4T1 tumors showed distorted blood vessels with a thin endothelial layer, resulting in a slower increase in signal intensity after injection of the contrast agent. Higher permeability was further reflected in higher Ktrans values, with consecutive retention of gadolinium in the tumor interstitium visible in MRI. 67NR tumors exhibited blood vessels with a thicker and more intact endothelial layer, resulting in higher peak enhancement, as well as higher maximum slope and area under the curve, but also a visible wash-out of the contrast agent and thus lower Ktrans values. A decreasing accumulation of gadolinium during tumor progression was also visible in both models in LA-ICP-MS. Tissue composition of 4T1 tumors was more heterogeneous, with intratumoral hemorrhage and necrosis and corresponding higher T1 and T2 relaxation times, while 67NR tumors mainly consisted of densely packed tumor cells. Histogram analysis of ADC showed higher values of mean ADC, histogram kurtosis, range and the 90th percentile (p90), as markers for the heterogenous structural composition of 4T1 tumors. Principal component analysis (PCA) discriminated well between the two tumor models. Conclusions: Multiparametric MRI as presented in this study enables for the estimation of malignant potential in the two studied tumor models via the assessment of certain tumor features over time.

Ultrafast CEST line scanning as a method to quantify mutarotation kinetics.

The process of mutarotation of sugars caused by a balanced reaction between their corresponding α and ß isomers, has been known for almost 200 years. Still, it remains essential in modern biochemical research, as enzymatic reactions catalyzed by mutarotases are crucial for various pathways in the energy metabolism. In our study a fast magnetic resonance technique based on chemical exchange saturation transfer (CEST) line scanning (LS) was implemented as a method to measure mutarotation kinetics on a 9.4 T small animal MRI scanner. As proof of concept, the isomeric conversion of two hexoses (glucose and galactose) and pentoses (xylose and arabinose) was investigated in an aqueous solution over time. The technique allowed for ultrafast data acquisition without the implementation of complicated encoding schemes and acceleration procedures. Thus, CEST LS provided complete CEST spectra with a frequency step size of 19.6 Hz in less than one minute. For the mutarotation analysis, CEST spectra were acquired over a time duration of four hours and analyzed with four established CEST quantification approaches - based on either asymmetry of CEST spectra or a multi-pool Lorentzian fit. The isomer ratios of the different sugars at equilibrium were determined with an overall accuracy of 94 %, using an adapted 2-side chemical exchange (CE) model. The estimated mutarotation rate constants at 22 °C were in good agreement with conventionally measured reference values, derived from optical and spectroscopic techniques.

Fiber-based lactate recordings with fluorescence resonance energy transfer sensors by applying an magnetic resonance-informed correction of hemodynamic artifacts.

Significance: Fluorescence resonance energy transfer (FRET) sensors offer enormous benefits when studying neurophysiology through confocal microscopy. Yet, their use for fiber-based in vivo recordings is hampered by massive confounding effects and has therefore been scarcely reported. Aim: We aim to investigate whether in vivo fiber-based lactate recordings in the rodent brain are feasible with FRET sensors and implement a correction algorithm for the predominant hemodynamic artifact. Approach: We performed fiber-based FRET recordings of lactate (Laconic) and calcium (Twitch-2B) simultaneously with functional MRI and pharmacological MRI. MR-derived parameters were applied to correct hemodynamic artifacts. Results of FRET measurements were validated by local field potential, magnetic resonance spectroscopy, and blood analysis. Results: Hemodynamic artifacts dominated fiber-based in vivo FRET measurements with both Laconic and Twitch-2B. Our MR-based correction algorithm enabled to remove the artifacts and detect lactate and calcium changes during sensory stimulation or intravenous lactate injections. Conclusions: In vivo fiber-based lactate recordings are feasible using FRET-based sensors. However, signal corrections are required. MR-derived hemodynamic parameters can successfully be applied for artifact correction.

Short-Term Colony-Stimulating Factor 1 Receptor Inhibition-Induced Repopulation After Stroke Assessed by Longitudinal 18F-DPA-714 PET Imaging.

Studies on colony-stimulating factor 1 receptor (CSF-1R) inhibition-induced microglia depletion indicated that inhibitor withdrawal allowed the renewal of the microglia compartment via repopulation and resolved the inflammatory imbalance. Therefore, we investigated for the first time (to our knowledge) the effects of microglia repopulation on inflammation and functional outcomes in an ischemic mouse model using translocator protein (TSPO)-PET/CT and MR imaging, ex vivo characterization, and behavioral tests. Methods: Eight C57BL/6 mice per group underwent a 30-min transient occlusion of the middle cerebral artery. The treatment group received CSF-1R inhibitor in 1,200 ppm PLX5622 chow (Plexxikon Inc.) from days 3 to 7 to induce microglia/macrophage depletion and then went back to a control diet to allow repopulation. The mice underwent T2-weighted MRI on day 1 after ischemia and 18F-labeled N,N-diethyl-2-(2-[4-(2-fluoroethoxy)phenyl]-5,7-dimethylpyrazolo[1,5-α]pyrimidine-3-yl)acetamide (18F-DPA-714) (TSPO) PET/CT on days 7, 14, 21, and 30. The percentage injected tracer dose per milliliter within the infarct, contralateral striatum, and spleen was assessed. Behavioral tests were performed to assess motor function recovery. Brains were harvested on days 14 and 35 after ischemia for ex vivo analyses (immunoreactivity and real-time quantitative polymerase chain reaction) of microglia- and macrophage-related markers. Results: Repopulation significantly increased 18F-DPA-714 uptake within the infarct on days 14 (P < 0.001) and 21 (P = 0.002) after ischemia. On day 14, the ionized calcium binding adaptor molecule 1 (Iba-1)-positive cell population showed significantly higher expression of TSPO, CSF-1R, and CD68, in line with microglia repopulation. Gene expression analyses on day 14 indicated a significant increase in microglia-related markers (csf-1r, aif1, and p2ry12) with repopulation, whereas peripheral cell recruitment-related gene expression decreased (cx3cr1 and ccr2), indicative of peripheral recruitment during CSF-1R inhibition. Similarly, uncorrected spleen uptake was significantly higher on day 7 after ischemia with treatment (P = 0.001) and decreased after drug withdrawal. PLX5622-treated mice walked a longer distance (P < 0.001) and more quickly (P = 0.009), and showed greater forelimb strength (P < 0.001), than control mice on day 14. Conclusion: This study highlighted the potential of 18F-DPA-714 PET/CT imaging to track microglia and macrophage repopulation after short-term CSF-1R inhibition in stroke.