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.

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.

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.

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.

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.

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.

A Longitudinal PET/MRI Study of Colony-Stimulating Factor 1 Receptor-Mediated Microglia Depletion in Experimental Stroke.

Microglia-induced neuroinflammation after stroke contributes to the exacerbation of postischemic damage but also supports neurorestorative events. Longitudinal molecular imaging of microglia-targeted therapies will support the assessment of target engagement, therapy efficacy, and deciphering of the mode of action. We investigated the effects of chronic colony-stimulating factor 1 receptor (CSF-1R) inhibitor-mediated microglia depletion on translocator protein (TSPO)-dependent neuroinflammation and cerebrovascular parameters using PET/MRI. Methods: Forty C57BL/6 mice underwent a 30-min transient occlusion of the middle cerebral artery and were randomly assigned to either a control group or a group treated with CSF-1R inhibitor (PLX5622). Eight mice per group were used for N,N-diethyl-2-(2-(4-(2-18F-fluoroethoxy) phenyl)5,7dimethylpyrazolo[1, 5a]pyrimidin-3-yl)acetamide (18F-DPA-714) (TSPO) PET imaging on days 7, 14, 21, and 30 after ischemia and behavioral tests before and after surgery. An extra group of 8 mice underwent MRI, including T2-weighted (infarct), perfusion-weighted (cerebral blood flow), and diffusion-weighted (water diffusion, cellular density) sequences, on days 1, 3, 7, 14, 21, and 30. Ex vivo analysis (immunoreactivity, gene expression) was performed to characterize the inflammatory environment. Results: We demonstrated that long-term CSF-1R inhibition transiently decreased the TSPO PET signal within the infarct. Residual TSPO activity was partly due to a potentially resistant Iba-1-positive cell populations with low CSF-1R and transmembrane 119 expression. The decrease in selected pro- and antiinflammatory marker expression suggested an apparent global dampening of the neuroinflammatory response. Furthermore, the temporal changes in the MRI parameters highlighted treatment-induced effects on reperfusion and tissue homeostasis, associated with impaired motor function at late stages. Conclusion: Longitudinal TSPO PET/MRI allows the assessment of target engagement and optimization of drug efficiency. PLX5622 has promising immunomodulatory effects, and the optimal therapeutic time window for its application needs to be defined.

Voxel-Based Analysis of the Relation of 3'-Deoxy-3'-[18F]fluorothymidine ([18F]FLT) PET and Diffusion-Weighted (DW) MR Signals in Subcutaneous Tumor Xenografts Does Not Reveal a Direct Spatial Relation of These Two Parameters.

PURPOSE: Multimodal molecular imaging allows a direct coregistration of different images, facilitating analysis of the spatial relation of various imaging parameters. Here, we further explored the relation of proliferation, as measured by [18F]FLT PET, and water diffusion, as an indicator of cellular density and cell death, as measured by diffusion-weighted (DW) MRI, in preclinical tumor models. We expected these parameters to be negatively related, as highly proliferative tissue should have a higher density of cells, hampering free water diffusion. PROCEDURES: Nude mice subcutaneously inoculated with either lung cancer cells (n = 11 A549 tumors, n = 20 H1975 tumors) or colorectal cancer cells (n = 13 Colo205 tumors) were imaged with [18F]FLT PET and DW-MRI using a multimodal bed, which was transferred from one instrument to the other within the same imaging session. Fiducial markers allowed coregistration of the images. An automatic post-processing was developed in MATLAB handling the spatial registration of DW-MRI (measured as apparent diffusion coefficient, ADC) and [18F]FLT image data and subsequent voxel-wise analysis of regions of interest (ROIs) in the tumor. RESULTS: Analyses were conducted on a total of 76 datasets, comprising a median of 2890 data points (ranging from 81 to 13,597). Scatterplots showing [18F]FLT vs. ADC values displayed various grades of relations (Pearson correlation coefficient (PCC) varied from - 0.58 to 0.49, median: -0.07). When relating PCC to tumor volume (median: 46 mm3, range: 3 mm3 to 584 mm3), lung tumors tended to have a more pronounced negative spatial relation of [18F]FLT and ADC with increasing tumor size. However, due to the low number of large tumors (> ~ 200 mm3), this conclusion has to be treated with caution. CONCLUSIONS: A spatial relation of water diffusion, as measured by DW-MRI, and cellular proliferation, as measured by [18F]FLT PET, cannot be detected in the experimental datasets investigated in this study.

Resolving immune cells with patrolling behaviour by magnetic resonance time-lapse single cell tracking.

BACKGROUND: Immune cells show distinct motion patterns that change upon inflammatory stimuli. Monocytes patrol the vasculature to screen for pathogens, thereby exerting an early task of innate immunity. Here, we aimed to non-invasively analyse single patrolling monocyte behaviour upon inflammatory stimuli. METHODS: We used time-lapse Magnetic Resonance Imaging (MRI) of the murine brain to dynamically track single patrolling monocytes within the circulation distant to the actual site of inflammation in different inflammatory conditions, ranging from a subcutaneous pellet model to severe peritonitis and bacteraemia. FINDINGS: Single patrolling immune cells with a velocity of <1 µm/s could be detected and followed dynamically using time-lapse MRI. We show, that due to local and systemic stimuli the slowly patrolling behaviour of monocytes is altered systemically and differs with type, duration and strength of the underlying stimulus. INTERPRETATION: Using time-lapse MRI, it is now possible to investigate the behaviour of single circulating monocytes over the course of the systemic immune response. Monocyte patrolling behaviour is altered systemically even before the onset of clinical symptoms distant to and depending on the underlying inflammatory stimulus. FUNDING: This study was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - CRC 1009 - 194468054 to AZ, CF and - CRC 1450 - 431460824 to MM, SN, HB, AZ, CF, the Joachim Herz Foundation (Add-on Fellowship for Interdisciplinary Life Sciences to MM), the Interdisciplinary Centre for Clinical Research (IZKF, core unit PIX) and the Medical Faculty of the University of Muenster (MEDK fellowship to FF and IF).

Combined resting state-fMRI and calcium recordings show stable brain states for task-induced fMRI in mice under combined ISO/MED anesthesia.

For fMRI in animal models, the combination of low-dose anesthetic, isoflurane (ISO), and the sedative medetomidine (MED) has recently become an advocated regimen to achieve stable neuronal states and brain networks in rats that are required for reliable task-induced BOLD fMRI. However, in mice the temporal stability of neuronal states and networks in resting-state (rs)-fMRI experiments during the combined ISO/MED regimen has not been systematically investigated. Using a multimodal approach with optical calcium (Ca2+) recordings and rs-fMRI, we investigated cortical neuronal/astrocytic Ca2+activity states and brain networks at multiple time points while switching from anesthesia with 1% ISO to a combined ISO/MED regimen. We found that cortical activity states reached a steady-state 45 min following start of MED infusion as indicated by stable Ca2+ transients. Similarly, rs-networks were not statistically different between anesthesia with ISO and the combined ISO/MED regimen 45 and 100 min after start of MED. Importantly, during the transition time we identified changed rs-network signatures that likely reflect the different mode of action of the respective anesthetic; these included a dose-dependent increase in cortico-cortical functional connectivity (FC) presumably caused by reduction of ISO concentration and decreased FC in subcortical arousal nuclei due to MED infusion. Furthermore, we report detection of visual stimulation-induced BOLD fMRI during the stable ISO/MED neuronal state 45 min after induction. Based on our findings, we recommend a 45-minute waiting period after switching from ISO anesthesia to the combined ISO/MED regimen before performing rs- or task-induced fMRI experiments.

Translational value of choroid plexus imaging for tracking neuroinflammation in mice and humans.

Neuroinflammation is a pathophysiological hallmark of multiple sclerosis and has a close mechanistic link to neurodegeneration. Although this link is potentially targetable, robust translatable models to reliably quantify and track neuroinflammation in both mice and humans are lacking. The choroid plexus (ChP) plays a pivotal role in regulating the trafficking of immune cells from the brain parenchyma into the cerebrospinal fluid (CSF) and has recently attracted attention as a key structure in the initiation of inflammatory brain responses. In a translational framework, we here address the integrity and multidimensional characteristics of the ChP under inflammatory conditions and question whether ChP volumes could act as an interspecies marker of neuroinflammation that closely interrelates with functional impairment. Therefore, we explore ChP characteristics in neuroinflammation in patients with multiple sclerosis and in two experimental mouse models, cuprizone diet-related demyelination and experimental autoimmune encephalomyelitis. We demonstrate that ChP enlargement-reconstructed from MRI-is highly associated with acute disease activity, both in the studied mouse models and in humans. A close dependency of ChP integrity and molecular signatures of neuroinflammation is shown in the performed transcriptomic analyses. Moreover, pharmacological modulation of the blood-CSF barrier with natalizumab prevents an increase of the ChP volume. ChP enlargement is strongly linked to emerging functional impairment as depicted in the mouse models and in multiple sclerosis patients. Our findings identify ChP characteristics as robust and translatable hallmarks of acute and ongoing neuroinflammatory activity in mice and humans that could serve as a promising interspecies marker for translational and reverse-translational approaches.

Brain microstructural changes in mice persist in adulthood and are modulated by the palmitoyl acyltransferase ZDHHC7.

For a long time, mice have been classified as adults with completely mature brains at 8 weeks of age, but recent research suggests that developmental brain changes occur for up to 6 months. In particular, adolescence coincides with dramatic changes of neuronal structure and function in the brain that influence the connectivity between areas like hippocampus and medial prefrontal cortex (mPFC). Neuronal development and plasticity are regulated in part by the palmitoyl acyltransferase ZDHHC7, which modulates structural connectivity between hippocampus and mPFC. The aim of the current study was to investigate whether developmental changes take place in hippocampus and mPFC microstructure even after 8 weeks of age and whether deficiency of ZDHHC7 impacts such age-dependent alterations. Altogether, 46 mice at 11, 14 or 17 weeks of age with a genetic Zdhhc7 knockout (KO) or wild type (WT) were analysed with neuroimaging and diffusion tensor-based fibre tractography. The hippocampus and mPFC regions were compared regarding fibre metrics, supplemented by volumetric and immunohistological analyses of the hippocampus. In WT animals, we identified age-dependent changes in hippocampal fibre lengths that followed a U-shaped pattern, whereas in mPFC, changes were linear. In Zdhhc7-deficient animals, the fibre statistics were reduced in both regions, whereas the hippocampus volume and the intensities of myelin and neurofilament were higher in 11-week-old KO mice compared to WTs. Our results confirmed ongoing changes of microstructure in mice up to 17 weeks old and demonstrate that deleting the Zdhhc7 gene impairs fibre development, suggesting that palmitoylation is important in this process.

Retrosplenial Cortex Contributes to Network Changes during Seizures in the GAERS Absence Epilepsy Rat Model.

Resting state-fMRI was performed to explore brain networks in Genetic Absence Epilepsy Rats from Strasbourg and in nonepileptic controls (NEC) during monitoring of the brain state by simultaneous optical Ca2+-recordings. Graph theoretical analysis allowed for the identification of acute and chronic network changes and revealed preserved small world topology before and after seizure onset. The most prominent acute change in network organization during seizures was the segregation of cortical regions from the remaining brain. Stronger connections between thalamic with limbic regions compared with preseizure state indicated network regularization during seizures. When comparing between strains, intrathalamic connections were prominent in NEC, on local level represented by higher thalamic strengths and hub scores. Subtle differences were observed for retrosplenial cortex (RS), forming more connections beyond cortex in epileptic rats, and showing a tendency to lateralization during seizures. A potential role of RS as hub between subcortical and cortical regions in epilepsy was supported by increased numbers of parvalbumin-positive (PV+) interneurons together with enhanced inhibitory synaptic activity and neuronal excitability in pyramidal neurons. By combining multimodal fMRI data, graph theoretical methods, and electrophysiological recordings, we identified the RS as promising target for modulation of seizure activity and/or comorbidities.

Acute stress reveals different impacts in male and female Zdhhc7-deficient mice.

Numerous processes of neuronal development and synaptic plasticity in the brain rely on the palmitoyl acyltransferase ZDHHC7, as it palmitoylates various synaptic and extrasynaptic proteins such as neural cell adhesion molecule (NCAM) or gamma-aminobutyric acid (GABAA) receptors. In addition, ZDHHC7 palmitoylates sex steroid hormone receptors and is, therefore, indirectly linked to mental disorders that often occur because of or in conjunction with stress. In this work, we investigated how ZDHHC7 affects stress responses in mice. For this purpose, genetically modified mice with a knockout of the Zdhhc7 gene (KO) and wild-type (WT) littermates of both sexes were exposed to acute stressors or control conditions and examined with regard to their behavior, brain microstructure, gene expression, and synaptic plasticity. While no behavioral effects of acute stress were found, we did find that acute stress caused reduced mRNA levels of Esr1 and Esr2 coding for estrogen receptor α and ß in the medial prefrontal cortex of male WT and KO mice. Strikingly, after acute stress only male KO mice showed reduced mean fiber lengths of the medioventral hippocampus. Furthermore, Zdhhc7-deficiency impaired synaptic plasticity in mice of both sexes, while acute stress improved it in females, but not in male mice. Taken together, our findings suggest that ZDHHC7 plays a modulatory role in the brain that leads to sex-specific stress responses, possibly due to estrogen receptor-mediated signaling pathways.

Impact of hydroxytyrosol on stroke: tracking therapy response on neuroinflammation and cerebrovascular parameters using PET-MR imaging and on functional outcomes.

Immune cells have been implicated in influencing stroke outcomes depending on their temporal dynamics, number, and spatial distribution after ischemia. Depending on their activation status, immune cells can have detrimental and beneficial properties on tissue outcome after stroke, highlighting the need to modulate inflammation towards beneficial and restorative immune responses. Novel dietary therapies may promote modulation of pro- and anti-inflammatory immune cell functions. Among the dietary interventions inspired by the Mediterranean diet, hydroxytyrosol (HT), the main phenolic component of the extra virgin olive oil (EVOO), has been suggested to have antioxidant and anti-inflammatory properties in vitro. However, immunomodulatory effects of HT have not yet been studied in vivo after stroke. The aim of this project is therefore to monitor the therapeutic effect of a HT-enriched diet in an experimental stroke model using non-invasive in vivo multimodal imaging, behavioural phenotyping and cross-correlation with ex vivo parameters. Methods: A total of N = 22 male C57BL/6 mice were fed with either a standard chow (n = 11) or a HT enriched diet (n = 11) for 35 days, following a 30 min transient middle cerebral artery occlusion (tMCAo). T2-weighted (lesion) and perfusion (cerebral blood flow)-/diffusion (cellular density)-weighted MR images were acquired at days 1, 3, 7, 14, 21 and 30 post ischemia. [18F]DPA-714 (TSPO, neuroinflammation marker) PET-CT scans were acquired at days 7, 14, 21 and 30 post ischemia. Infarct volume (mm3), cerebral blood flow (mL/100g/min), apparent diffusion coefficient (10-4·mm2/s) and percentage of injected tracer dose (%ID/mL) were assessed. Behavioural tests (grip test, rotarod, open field, pole test) were performed prior and after ischemia to access therapy effects on sensorimotor functions. Ex vivo analyses (IHC, IF, WB) were performed to quantify TSPO expression, immune cells including microglia/macrophages (Iba-1, F4/80), astrocytes (GFAP) and peripheral markers in serum such as thiobarbituric acid reactive substances (TBARS) and nitric oxide (NO) 35 days post ischemia. Additionally, gene expression of pro- and anti-inflammatory markers were assessed by rt-qPCR, including tspo, cd163, arg1, tnf and Il-1ß. Results: No treatment effect was observed on temporal [18F]DPA-714 uptake within the ischemic and contralateral region (two-way RM ANOVA, p = 0.71). Quantification of the percentage of TSPO+ area by immunoreactivity indicated a slight 2-fold increase in TSPO expression within the infarct region in HT-fed mice at day 35 post ischemia (p = 0.011) correlating with a 2-3 fold increase in Iba-1+ cell population expressing CD163 as anti-inflammatory marker (R2 = 0.80). Most of the GFAP+ cells were TSPO-. Only few F4/80+ cells were observed at day 35 post ischemia in both groups. No significant treatment effect was observed on global ADC and CBF within the infarct and the contralateral region over time. Behavioural tests indicated improved strength of the forepaws at day 14 post ischemia (p = 0.031). Conclusion: An HT-enriched diet significantly increased the number of Iba-1+ microglia/macrophages in the post-ischemic area, inducing higher expression of anti-inflammatory markers while no clear-cut effect was observed. Also, HT did not affect recovery of the cerebrovascular parameters, including ADC and CBF. Altogether, our data indicated that a prolonged dietary intervention with HT, as a single component of the Mediterranean diet, induces molecular changes that may improve stroke outcomes. Therefore, we support the use of the Mediterranean diet as a multicomponent therapy approach after stroke.