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.

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.

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.

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.

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.

CD8+ T-Lymphocyte-Driven Limbic Encephalitis Results in Temporal Lobe Epilepsy.

OBJECTIVE: Limbic encephalitis (LE) comprises a spectrum of inflammatory changes in affected brain structures including the presence of autoantibodies and lymphoid cells. However, the potential of distinct lymphocyte subsets alone to elicit key clinicopathological sequelae of LE potentially inducing temporal lobe epilepsy (TLE) with chronic spontaneous seizures and hippocampal sclerosis (HS) is unresolved. METHODS: Here, we scrutinized pathogenic consequences emerging from CD8+ T cells targeting hippocampal neurons by recombinant adeno-associated virus-mediated expression of the model-autoantigen ovalbumin (OVA) in CA1 neurons of OT-I/RAG1-/- mice (termed "OVA-CD8+ LE model"). RESULTS: Viral-mediated antigen transfer caused dense CD8+ T cell infiltrates confined to the hippocampal formation starting on day 5 after virus transduction. Flow cytometry indicated priming of CD8+ T cells in brain-draining lymph nodes preceding hippocampal invasion. At the acute model stage, the inflammatory process was accompanied by frequent seizure activity and impairment of hippocampal memory skills. Magnetic resonance imaging scans at day 7 of the OVA-CD8+ LE model revealed hippocampal edema and blood-brain barrier disruption that converted into atrophy until day 40. CD8+ T cells specifically targeted OVA-expressing, SIINFEKL-H-2Kb -positive CA1 neurons and caused segmental apoptotic neurodegeneration, astrogliosis, and microglial activation. At the chronic model stage, mice exhibited spontaneous recurrent seizures and persisting memory deficits, and the sclerotic hippocampus was populated with CD8+ T cells escorted by NK cells. INTERPRETATION: These data indicate that a CD8+ T-cell-initiated attack of distinct hippocampal neurons is sufficient to induce LE converting into TLE-HS. Intriguingly, the role of CD8+ T cells exceeds neurotoxic effects and points to their major pathogenic role in TLE following LE. ANN NEUROL 2021;89:666-685.

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.

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.

A cortical rat hemodynamic response function for improved detection of BOLD activation under common experimental conditions.

For a reliable estimation of neuronal activation based on BOLD fMRI measurements an accurate model of the hemodynamic response is essential. Since a large part of basic neuroscience research is based on small animal data, it is necessary to characterize a hemodynamic response function (HRF) which is optimized for small animals. Therefore, we have determined and investigated the HRFs of rats obtained under a variety of experimental conditions in the primary somatosensory cortex. Measurements were performed on animals of different sex and strain, under different anesthetics, with and without ventilation and using different stimulation modalities. All modalities of stimulation used in this study induced neuronal activity in the primary somatosensory cortex or in subcortical regions. Since the HRFs of the BOLD responses in the primary somatosensory cortex showed a close concordance for the different conditions, we were able to determine a cortical rat HRF. This HRF is based on 143 BOLD measurements of 76 rats and can be used for statistical parametric mapping. It showed substantially faster progression than the human HRF, with a maximum after 2.8 ± 0.8 s, and a following undershoot after 6.1 ± 3.7 s. If the rat HRF was used statistical analysis of rat data showed a significantly improved detection performance in the somatosensory cortex in comparison to the commonly used HRF based on measurements in humans.

Myelination- and immune-mediated MR-based brain network correlates.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS), characterized by inflammatory and neurodegenerative processes. Despite demyelination being a hallmark of the disease, how it relates to neurodegeneration has still not been completely unraveled, and research is still ongoing into how these processes can be tracked non-invasively. Magnetic resonance imaging (MRI) derived brain network characteristics, which closely mirror disease processes and relate to functional impairment, recently became important variables for characterizing immune-mediated neurodegeneration; however, their histopathological basis remains unclear. METHODS: In order to determine the MRI-derived correlates of myelin dynamics and to test if brain network characteristics derived from diffusion tensor imaging reflect microstructural tissue reorganization, we took advantage of the cuprizone model of general demyelination in mice and performed longitudinal histological and imaging analyses with behavioral tests. By introducing cuprizone into the diet, we induced targeted and consistent demyelination of oligodendrocytes, over a period of 5 weeks. Subsequent myelin synthesis was enabled by reintroduction of normal food. RESULTS: Using specific immune-histological markers, we demonstrated that 2 weeks of cuprizone diet induced a 52% reduction of myelin content in the corpus callosum (CC) and a 35% reduction in the neocortex. An extended cuprizone diet increased myelin loss in the CC, while remyelination commenced in the neocortex. These histologically determined dynamics were reflected by MRI measurements from diffusion tensor imaging. Demyelination was associated with decreased fractional anisotropy (FA) values and increased modularity and clustering at the network level. MRI-derived modularization of the brain network and FA reduction in key anatomical regions, including the hippocampus, thalamus, and analyzed cortical areas, were closely related to impaired memory function and anxiety-like behavior. CONCLUSION: Network-specific remyelination, shown by histology and MRI metrics, determined amelioration of functional performance and neuropsychiatric symptoms. Taken together, we illustrate the histological basis for the MRI-driven network responses to demyelination, where increased modularity leads to evolving damage and abnormal behavior in MS. Quantitative information about in vivo myelination processes is mirrored by diffusion-based imaging of microstructural integrity and network characteristics.

Toward precise arterial input functions derived from DCE-MRI through a novel extracorporeal circulation approach in mice.

PURPOSE: Dynamic contrast-enhanced MRI can be used in pharmacokinetic models to quantify functional parameters such as perfusion and permeability. However, precise quantification in preclinical models is challenged by the difficulties to dynamically measure the true arterial blood contrast agent concentration. We propose a novel approach toward a precise and experimentally feasible method to derive the arterial input function from DCE-MRI in mice. METHODS: Arterial blood was surgically shunted from the femoral artery to the tail vein and led through an extracorporeal circulation that resided on the head of brain tumor-bearing mice inside the FOV of a 9.4T MRI scanner. Dynamic 3D-FLASH scanning was performed after injection of gadobutrol with an effective resolution of 0.175 × 0.175 × 1 mm and a temporal resolution of 4 seconds. Pharmacokinetic modeling was performed using the extended Tofts and two-compartment exchange model. RESULTS: Arterial input functions measured inside the extracorporeal circulation showed little noise, small interindividual variance, and typical curve shapes. Ex vivo and mass spectrometry validation measurements documented the influence of shunt flow velocity and hematocrit on estimation of contrast agent concentrations. Modeling of tumors and muscles allowed fitting of the recorded dynamic concentrations, resulting in quantitative plausible parameters. CONCLUSION: The extracorporeal circulation allows deriving the contrast agent dynamics in arterial blood with high robustness and at acceptable experimental effort from DCE-MRI, previously not achievable in mice. It sets the basis for quantitative precise pharmacokinetic modeling in small animals to enhance the translatability of preclinical DCE-MRI measurements to patients.

Introducing Specificity to Iron Oxide Nanoparticle Imaging by Combining 57Fe-Based MRI and Mass Spectrometry.

Iron oxide nanoparticles (ION) are highly sensitive probes for magnetic resonance imaging (MRI) that have previously been used for in vivo cell tracking and have enabled implementation of several diagnostic tools to detect and monitor disease. However, the in vivo MRI signal of ION can overlap with the signal from endogenous iron, resulting in a lack of detection specificity. Therefore, the long-term fate of administered ION remains largely unknown, and possible tissue deposition of iron cannot be assessed with established methods. Herein, we combine nonradioactive 57Fe-ION MRI with ex vivo laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) imaging, enabling unambiguous differentiation between endogenous iron (56Fe) and iron originating from applied ION in mice. We establish 57Fe-ION as an in vivo MRI sensor for cell tracking in a mouse model of subcutaneous inflammation and for assessing the long-term fate of 57Fe-ION. Our approach resolves the lack of detection specificity in ION imaging by unambiguously recording a 57Fe signature.

Anesthesia differentially modulates neuronal and vascular contributions to the BOLD signal.

Most studies involving BOLD fMRI in basic neuroscience research are conducted with anesthetized animals. This study investigates neural and hemodynamic activity through a combination of experiments comprising BOLD fMRI, optical calcium recordings and ASL in vivo. Patch clamp experiments of neurons were conducted to evaluate electrophysiological correlates of neural activity in vitro. Various anesthetic conditions embracing numerous anesthetic depths evoked by different concentrations of isoflurane (ISO) and different degrees of hypercapnia under a constant stimulus were investigated. We observed that different anesthetic conditions had major impact on the results obtained, particularly that anesthesia could cause a massive divergence of different experimental modalities. In ventilated animals, robust BOLD responses were detectable even with relatively deep anesthesia, while in non-ventilated animals, BOLD responses were not detectable under these conditions. This was most likely due to hypercapnia caused by respiratory depression, as in ventilated animals administered CO2 had the same effect. This observation agreed with measurements of perfusion, which showed that inhaled CO2 increased perfusion significantly, while ISO did not. In optical calcium measurements, higher concentrations of ISO decreased spontaneous neural activity, but not stimulus-evoked responses. This observation was explained by a generally lower excitability of neurons under ISO, which suppressed spontaneous activity, and consequently left more neurons available to fire synchronously in response to a stimulus. Interpreting this phenomenon as an integrated signal of independent single neurons was supported by patch clamp experiments as the number of action potentials (APs) per stimulus was decreased by addition of CO2. Addition of ISO on the other hand had no significant effect. Our results provide an explanation on the cellular level for anesthesia-dependent observations in previous studies of task-induced BOLD and resting state connectivity. They further inform selection of the adequate anesthetic regimen for a given combination of modalities.

Probing activation-induced neurochemical changes using optogenetics combined with functional magnetic resonance spectroscopy: a feasibility study in the rat primary somatosensory cortex.

Knowledge about biochemical processes is a prerequisite for a better understanding of mechanisms underlying brain activity. 1 H functional magnetic resonance spectroscopy (1 H-fMRS) at high magnetic fields allows for the non-invasive measurement of metabolic changes during brain activation. Optogenetics, on the other hand, has revolutionized the field of neuroscience. It was previously coupled with functional magnetic resonance imaging (fMRI) techniques in rodents enabling population-specific targeting of cells, investigating brain networks with unprecedented in vivo precision. The coupling of optogenetics and 1 H-fMRS is expected to enhance the specificity of metabolic readouts to validate neuro-energetic theories underlying brain activity. To date, the feasibility of combining optogenetic stimulation with fMRS has not been explored. We used green laser stimulation delivered through an optical fiber implanted superficially above the primary somatosensory forelimb cortex (S1FL) of rats expressing the C1V1 opsin in excitatory neurons. A protocol for the acquisition of functional 1 H MR spectra upon optogenetic stimulation without craniotomy-induced artefacts was established. Quantification of metabolite concentrations in S1FL upon optogenetic and electrical forepaw stimulation demonstrated significantly increased glutamate levels (+8.8% and +9.9%, p < 0.05 respectively), which were compensated by decreased glutamine levels (-17% and -18%, p < 0.05 respectively). Our results demonstrate for the first time the feasibility of combining optogenetic control and functional MR spectroscopy (o-fMRS) in the rat somatosensory cortex, opening new possibilities for monitoring the energetic demands of specific cell populations and for exploring the underpinnings of energy metabolism during brain activity. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Functionalization of Clinically Approved MRI Contrast Agents for the Delivery of VEGF.

In combining the two clinically approved substances ferumoxytol and VEGF-165 via peptide coupling, we propose a straightforward approach to obtain a potentially ready-to-use theranostic contrast agent for specific cardiovascular diseases. Clinical and preclinical magnetic resonance imaging (MRI) studies have shown that intravenously applied superparamagnetic ferumoxytol nanoparticles accumulate in acute ischemic myocardial tissue. On the other hand, growth factors such as VEGF-165 (vascular endothelial growth factor) play a major role during angiogenesis and vasculogenesis. Promising clinical studies with systemic application of VEGF-165 have been performed in the past. However, following untargeted systemic application, the biological half-life of VEGF-165 was too short to develop its full effect. Therefore, we hypothesized that ferumoxytol particles functionalized with VEGF-165 will accumulate in ischemic myocardial regions and can be detected by MRI, while the prolonged retention of VEGF-165 due to ferumoxytol-coupling will help to prevent adverse tissue remodeling. In addition, strategies such as magnetic targeting can be used to enhance targeted local accumulation. As a precondition for further preclinical research, we confirmed the successful coupling between ferumoxytol and VEGF-165 in detail (TEM, XPS, and IR spectroscopy), characterized the functionalized ferumoxytol particles (DLS, TEM, and MRI) and performed in vitro tests that showed their superior effect on cell growth and survival.

Defining mechanisms of neural plasticity after brainstem ischemia in rats.

OBJECTIVE: Neurological recovery after stroke mainly depends on the location of the lesion. A substantial portion of strokes affects the brainstem. However, patterns of neural plasticity following brainstem ischemia are almost unknown. METHODS: Here, we established a rat brainstem ischemia model that resembles key features of the human disease and investigated mechanisms of neural plasticity, including neurogenesis and axonal sprouting as well as secondary neurodegeneration. RESULTS: Spontaneous functional recovery was accompanied by a distinct pattern of axonal sprouting, for example, an increased bilateral fiber outgrowth from the corticorubral tract to the respective contralesional red nucleus suggesting a compensatory role of extrapyramidal pathways after damage to pyramid tracts within the brainstem. Using different markers for DNA replication, we showed that the brainstem displays a remarkable ability to undergo specific plastic cellular changes after injury, highlighting a yet unknown pattern of neurogenesis. Neural progenitor cells proliferated within the dorsal brainstem and migrated toward the lesion, whereas neurogenesis in classic neurogenic niches, the subventricular zone of the lateral ventricle and the hippocampus, remained, in contrast to what is known from hemispheric stroke, unaffected. These beneficial changes were paralleled by long-term degenerative processes, that is, corticospinal fiber loss superior to the lesion, degeneration of spinal tracts, and a decreased neuron density within the ipsilesional substantia nigra and the contralesional red nucleus that might have limited further functional recovery. INTERPRETATION: Our findings provide knowledge of elementary plastic adaptions after brainstem stroke, which is fundamental for understanding the human disease and for the development of new treatments. Ann Neurol 2018;83:1003-1015.

Line scanning fMRI reveals earlier onset of optogenetically evoked BOLD response in rat somatosensory cortex as compared to sensory stimulation.

The combination of optogenetic control and fMRI readout in the brain is increasingly used to assess neuronal networks and underlying signal processing. However, how exactly optogenetic activation or inhibition reproduces normal physiological input has not been fully unraveled. To assess details of temporal dynamics of the hemodynamic response, temporal resolution in rodent fMRI is often not sufficient. Recent advances in human fMRI using faster acquisition schemes cannot be easily translated to small animals due to smaller dimensions, fast physiological motion, and higher sensitivity to artefacts. Here, we applied a one dimensional line scanning acquisition with 50ms temporal resolution in rat somatosensory cortex. We observed that optogenetic activation reproduces the hemodynamic response upon sensory stimulation, but shows a 160 to 340ms earlier onset of the response. This difference is explained by direct activation of all opsin-expressing and illuminated cortical layers, while hemodynamic response to sensory stimulation is delayed during intracortical transmission between cortical layers. Our results confirm that optogenetic activation is a valid model for physiological neuronal input, and that differences in temporal behavior of only a few hundred milliseconds can be resolved in rodent fMRI.