Microglia maintain structural integrity during fetal brain morphogenesis.

Microglia (MG), the brain-resident macrophages, play major roles in health and disease via a diversity of cellular states. While embryonic MG display a large heterogeneity of cellular distribution and transcriptomic states, their functions remain poorly characterized. Here, we uncovered a role for MG in the maintenance of structural integrity at two fetal cortical boundaries. At these boundaries between structures that grow in distinct directions, embryonic MG accumulate, display a state resembling post-natal axon-tract-associated microglia (ATM) and prevent the progression of microcavities into large cavitary lesions, in part via a mechanism involving the ATM-factor Spp1. MG and Spp1 furthermore contribute to the rapid repair of lesions, collectively highlighting protective functions that preserve the fetal brain from physiological morphogenetic stress and injury. Our study thus highlights key major roles for embryonic MG and Spp1 in maintaining structural integrity during morphogenesis, with major implications for our understanding of MG functions and brain development.

Efficient optimization of chemical exchange saturation transfer MRI at 7 T using universal pulses and virtual observation points.

PURPOSE: To optimize the homogeneity of the presaturation module in a Chemical Exchange Saturation Transfer (CEST) acquisition at 7 T using parallel transmission (pTx). THEORY AND METHODS: An optimized pTx-CEST presaturation scheme based on precomputed universal pulses was designed. The optimization was performed by minimizing the L2-norm between the effective B 1 , RMS + $$ {B}_{1,\mathrm{RMS}}^{+} $$ and a given target while imposing energy constraints under virtual observation points (VOPs) supervision. The proposed method was evaluated through simulations and experimentally, both in vitro, on a realistic human head phantom, and in vivo, on healthy volunteers. The results were compared with circular polarization (CP) presaturation and other pTx approaches previously proposed. All experiments were conducted on a 7 T MRI scanner using a commercial 8Tx/32Rx head coil. RESULTS: The simulations show that the proposed pTx strategy boosted with VOPs is superior to the CP mode and existent pTx approaches. While the best results are obtained with subject specific pulses, the gain provided by the use of VOPs renders the universal pulses superior to tailored pulses optimized under vendor provided Specific Absorption Rate (SAR) management. In the phantom, the glucose MTR asym $$ {\mathrm{MTR}}_{\mathrm{asym}} $$ map was significantly more homogeneous than with CP (root mean square error [RMSE] 17% vs. 30%). The efficiency of the method for in vivo hydroxyl, glutamate and rNOE weighted CEST acquisitions was also demonstrated. CONCLUSION: The use of a pTx presaturation scheme based on universal pulses optimized under VOP SAR management is significantly benefiting CEST imaging at high magnetic field.

Cranial window for longitudinal and multimodal imaging of the whole mouse cortex.

Significance: All functional brain imaging methods have technical drawbacks and specific spatial and temporal resolution limitations. Unraveling brain function requires bridging the data acquired with cellular and mesoscopic functional imaging. This imposes the access to animal preparations, allowing longitudinal and multiscale investigations of brain function in anesthetized and awake animals. Such preparations are optimal to study normal and pathological brain functions while reducing the number of animals used. Aim: To fulfill these needs, we developed a chronic and stable preparation for a broad set of imaging modalities and experimental design. Approach: We describe the detailed protocol for a chronic cranial window, transparent to light and ultrasound, devoid of BOLD functional magnetic resonance imaging (fMRI) artifact and allowing stable and longitudinal multimodal imaging of the entire mouse cortex. Results: The inexpensive, transparent, and curved polymethylpentene cranial window preparation gives access to the entire mouse cortex. It is compatible with standard microscopic and mesoscopic neuroimaging methods. We present examples of data on the neurovascular unit and its activation using two-photon, functional ultrasound imaging, and BOLD fMRI. Conclusion: This preparation is ideal for multimodal imaging in the same animal.

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.

In vivo detection of carnosine and its derivatives using chemical exchange saturation transfer.

PURPOSE: To detect carnosine, anserine and homocarnosine in vivo with chemical exchange saturation transfer (CEST) at 17.2 T. METHODS: CEST MR acquisitions were performed using a CEST-linescan sequence developed in-house and optimized for carnosine detection. In vivo CEST data were collected from three different regions of interest (the lower leg muscle, the olfactory bulb and the neocortex) of eight rats. RESULTS: The CEST effect for carnosine, anserine and homocarnosine was characterized in phantoms, demonstrating the possibility to separate individual contributions by employing high spectral resolution (0.005 ppm) and low CEST saturation power (0.15 µ$$ \mu $$ T). The CEST signature of these peptides was evidenced, in vivo, in the rat brain and skeletal muscle. The presence of carnosine and anserine in the muscle was corroborated by in vivo localized spectroscopy (MRS). However, the sensitivity of MRS was insufficient for carnosine and homocarnosine detection in the brain. The absolute amounts of carnosine and derivatives in the investigated tissues were determined by liquid chromatography-electrospray ionization-tandem mass spectrometry using isotopic dilution standard methods and were in agreement with the CEST results. CONCLUSION: The robustness of the CEST-linescan approach and the favorable conditions for CEST at ultra-high magnetic field allowed the in vivo CEST MR detection of carnosine and related peptides. This approach could be useful to investigate noninvasively the (patho)-physiological roles of these molecules.

PEAKIT: A Gaussian Process regression analysis tool for chemical exchange saturation transfer spectra.

Chemical Exchange Saturation Transfer (CEST) is a powerful technique for metabolic imaging, capable of exploring concentrations in the µM to mM range. However, extracting quantitative information from Z-spectra can be challenging due to the non-CEST contributions present and the limited knowledge about the exchanging pools. The PEAKIT tool is proposed as an alternative approach to quantifying CEST peaks, which requires no prior assumptions about the frequency offset or the underlying shape of the baseline. Specifically, the tool takes as input an experimental Z-spectrum and proceeds to identify peak candidates. After a baseline estimation based on Gaussian Process regression, PEAKIT outputs the chemical shift offsets, the areas, the heights and the statistical significance of the detected peaks. The performance and limitations of the PEAKIT tool are discussed for in vitro and in vivo applications.

Imaging of two samples with a single transmit/receive channel using coupled ceramic resonators for MR microscopy at 17.2 T.

In this paper we address the possibility to perform imaging of two samples within the same acquisition time using coupled ceramic resonators and one transmit/receive channel. We theoretically and experimentally compare the operation of our ceramic dual-resonator probe with a wire-wound solenoid probe, which is the standard probe used in ultrahigh-field magnetic resonance microscopy. We show that due to the low-loss ceramics used to fabricate the resonators, and a favorable distribution of the electric field within the conducting sample, a dual probe, which contains two samples, achieves an SNR enhancement by a factor close to the square root of 2 compared with a solenoid optimized for one sample.

Differential effects of aquaporin-4 channel inhibition on BOLD fMRI and diffusion fMRI responses in mouse visual cortex.

The contribution of astrocytes to the BOLD fMRI and DfMRI responses in visual cortex of mice following visual stimulation was investigated using TGN-020, an aquaporin 4 (AQP4) channel blocker, acting as an astrocyte function perturbator. Under TGN-020 injection the amplitude of the BOLD fMRI response became significantly higher. In contrast no significant changes in the DfMRI responses and the electrophysiological responses were observed. Those results further confirm the implications of astrocytes in the neurovascular coupling mechanism underlying BOLD fMRI, but not in the DfMRI responses which remained unsensitive to astrocyte function perturbation.

Diffusion MRI reveals in vivo and non-invasively changes in astrocyte function induced by an aquaporin-4 inhibitor.

The Glymphatic System (GS) has been proposed as a mechanism to clear brain tissue from waste. Its dysfunction might lead to several brain pathologies, including the Alzheimer's disease. A key component of the GS and brain tissue water circulation is the astrocyte which is regulated by acquaporin-4 (AQP4), a membrane-bound water channel on the astrocytic end-feet. Here we investigated the potential of diffusion MRI to monitor astrocyte activity in a mouse brain model through the inhibition of AQP4 channels with TGN-020. Upon TGN-020 injection, we observed a significant decrease in the Sindex, a diffusion marker of tissue microstructure, and a significant increase of the water diffusion coefficient (sADC) in cerebral cortex and hippocampus compared to saline injection. These results indicate the suitability of diffusion MRI to monitor astrocytic activity in vivo and non-invasively.

A New Tool for In Vivo Study of Astrocyte Connexin 43 in Brain.

Astrocytes are glial cells organized in dynamic and structured networks in the brain. These plastic networks, involving key proteins such as connexin 43 (Cx43), are engaged in fine neuronal tuning and have recently been considered as emerging therapeutic targets in central nervous system disorders. We developed and validated a new application of the manganese-enhanced magnetic resonance imaging (MEMRI) technique allowing in vivo investigations of astrocyte-neuron interactions through quantification of brain Cx43 functional activity. The proof of concept has been achieved by quantification of MEMRI signals in brain after either local astrocyte-specific Cx43 knockdown with shRNA or systemic administration of Cx43 blockers. Unilateral hippocampal Cx43 genetical silencing was associated with an ipsilateral local increase of MEMRI signal. Furthermore, Cx43 blockers also enhanced MEMRI signal responses in hippocampus. Altogether, these data reveal the MEMRI technique as a tool for quantitative imaging of in vivo Cx43-dependent function in astrocytes under physiological and pathological conditions.

Enhancing surface coil sensitive volume with hybridized electric dipoles at 17.2 T.

Preclinical MR applications at 17.2 T can require field of views on the order of a few square centimeters. This is a challenging task as the proton Larmor frequency reaches 730 MHz. Most of the protocols at such frequencies are performed with surface transceiver coils for which the sensitive volume and the signal to noise ratio (SNR) is given by their size. Here we propose an approach based on metamaterials in order to enhance the sensitive volume of a commercial surface coil for small animal imaging at 17.2 T. We designed a passive resonator composed of four hybridized electric dipoles placed onto the floor of the MRI bed. Combining numerical and experimental results on a phantom and in vivo, we demonstrate a 20% increase of the sensitive volume in depth and 25% along the rostro-caudal axis while maintaining more than 85% of the local SNR right beneath the surface coil plane. Moreover, our solution gives the ability to double the average SNR in the region between 1.2 and 2 cm away from the loop using a single layer of 1 mm thick metallic wires easy to design and manufacture.

Systematic Analysis of the Improvements in Magnetic Resonance Microscopy with Ferroelectric Composite Ceramics.

The spatial resolution and signal-to-noise ratio (SNR) attainable in magnetic resonance microscopy (MRM) are limited by intrinsic probe losses and probe-sample interactions. In this work, the possibility to exceed the SNR of a standard solenoid coil by more than a factor-of-two is demonstrated theoretically and experimentally. This improvement is achieved by exciting the first transverse electric mode of a low-loss ceramic resonator instead of using the quasi-static field of the metal-wire solenoid coil. Based on theoretical considerations, a new probe for microscopy at 17 T is developed as a dielectric ring resonator made of ferroelectric/dielectric low-loss composite ceramics precisely tunable via temperature control. Besides the twofold increase in SNR, compared with the solenoid probe, the proposed ceramic probe does not cause static-field inhomogeneity and related image distortion.

Spatial contribution of hippocampal BOLD activation in high-resolution fMRI.

While the vascular origin of the BOLD-fMRI signal is established, the exact neurovascular coupling events contributing to this signal are still incompletely understood. Furthermore, the hippocampal spatial properties of the BOLD activation are not elucidated, although electrophysiology approaches have already revealed the precise spatial patterns of neural activity. High magnetic field fMRI offers improved contrast and allows for a better correlation with the underlying neuronal activity because of the increased contribution to the BOLD signal of small blood vessels. Here, we take advantage of these two benefits to investigate the spatial characteristics of the hippocampal activation in a rat model before and after changing the hippocampal plasticity by long-term potentiation (LTP). We found that the hippocampal BOLD signals evoked by electrical stimulation at the perforant pathway increased more at the radiatum layer of the hippocampal CA1 region than at the pyramidal cell layer. The return to the baseline of the hippocampal BOLD activation was prolonged after LTP induction compared with that before most likely due vascular or neurovascular coupling changes. Based on these results, we conclude that high resolution BOLD-fMRI allows the segregation of hippocampal subfields probably based on their underlying vascular or neurovascular coupling features.

Mesoscopic and microscopic imaging of sensory responses in the same animal.

Imaging based on blood flow dynamics is widely used to study sensory processing. Here we investigated the extent to which local neuronal and capillary responses (two-photon microscopy) are correlated to mesoscopic responses detected with fast ultrasound (fUS) and BOLD-fMRI. Using a specialized chronic olfactory bulb preparation, we report that sequential imaging of the same mouse allows quantitative comparison of odour responses, imaged at both microscopic and mesoscopic scales. Under these conditions, functional hyperaemia occurred at the threshold of neuronal activation and fUS-CBV signals could be detected at the level of single voxels with activation maps varying according to blood velocity. Both neuronal and vascular responses increase non-linearly as a function of odour concentration, whereas both microscopic and mesoscopic vascular responses are linearly correlated to local neuronal calcium. These data establish strengths and limits of mesoscopic imaging techniques to report neural activity.

Brain sugar consumption during neuronal activation detected by CEST functional MRI at ultra-high magnetic fields.

Blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) indirectly measures brain activity based on neurovascular coupling, a reporter that limits both the spatial and temporal resolution of the technique as well as the cellular and metabolic specificity. Emerging methods using functional spectroscopy (fMRS) and diffusion-weighted fMRI suggest that metabolic and structural modifications are also taking place in the activated cells. This paper explores an alternative metabolic imaging approach based on Chemical Exchange Saturation Transfer (CEST) to assess potential metabolic changes induced by neuronal stimulation in rat brains at 17.2 T. An optimized CEST-fMRI data acquisition and processing protocol was developed and used to experimentally assess the feasibility of glucoCEST-based fMRI. Images acquired under glucose-sensitizing conditions showed a substantial negative contrast that highlighted the same brain regions as those activated with BOLD-fMRI. We ascribe this novel fMRI contrast to CEST's ability to monitor changes in the local concentration of glucose, a metabolite closely coupled to neuronal activity. Our findings are in good agreement with literature employing other modalities. The use of CEST-based techniques for fMRI is not limited to glucose detection; other metabolic pathways involved in neuronal activation could be potentially probed. Moreover, being non invasive, it is conceivable that the same approach can be used for human studies.

Intracellular manganese enhanced MRI signals reflect the frequency of action potentials in Aplysia neurons.

BACKGROUND: Manganese-enhanced magnetic resonance imaging (MEMRI) is an increasingly popular alternative to standard functional MRI methods in animal studies. The contrast in MEMRI images is based on the accumulation of Mn2+ ions inside neurons, and, since manganese can serve as calcium analogue, this accumulation reflects calcium dynamics providing versatile information about brain neuroarchitecture and functionality. However, despite its use as a functional imaging tool, the exact relationship between the MEMRI signal and neuronal activity remains elusive. NEW METHOD: In order to better understand the mechanisms underlying Mn2+ accumulation resulting in MEMRI signal enhancement we investigated single neuron responses of isolated Aplysia buccal ganglia subjected to chemical (dopamine) or electrical stimulation of an input nerve (oesophageal nerve). The elicited electrical activity that represents a fictive feeding was recorded with electrophysiological methods and the Mn2+ uptake in individual neurons was evaluated with MEMRI at 17.2T. RESULTS & COMPARISON WITH EXISTING METHOD(S): We show a positive correlation between bursts of electrical activity and MEMRI signal intensity in identified neurons and demonstrate that the MEMRI signal reflects mainly fast and high membrane depolarization processes such as action potentials, and it is not sensitive to slow and small membrane depolarizations, such as post-synaptic potentials.

Modulation of water diffusion by activation-induced neural cell swelling in Aplysia Californica.

Diffusion functional magnetic resonance imaging (DfMRI) has been proposed as a method for functional neuroimaging studies, as an alternative to blood oxygenation level dependent (BOLD)-fMRI. DfMRI is thought to more directly reflect neural activation, but its exact mechanism remains unclear. It has been hypothesized that the water apparent diffusion coefficient (ADC) decrease observed upon neural activation results from swelling of neurons or neuron parts. To elucidate the origin of the DfMRI response at cellular level we performed diffusion MR microscopy at 17.2 T in Aplysia californica buccal ganglia and compared the water ADCs at cellular and ganglia levels before and after neuronal activation induced by perfusion with a solution containing dopamine. Neural cell swelling, evidenced from optical microscopy imaging, resulted in an intracellular ADC increase and an ADC decrease at ganglia level. Furthermore, the intracellular ADC increase was found to have a significant positive correlation with the increase in cell size. Our results strongly support the hypothesis that the ADC decrease observed with DfMRI upon neuronal activation at tissue level reflects activation-induced neural cell swelling.

Quantitative DLA-based compressed sensing for T1-weighted acquisitions.

High resolution Manganese Enhanced Magnetic Resonance Imaging (MEMRI), which uses manganese as a T1 contrast agent, has great potential for functional imaging of live neuronal tissue at single neuron scale. However, reaching high resolutions often requires long acquisition times which can lead to reduced image quality due to sample deterioration and hardware instability. Compressed Sensing (CS) techniques offer the opportunity to significantly reduce the imaging time. The purpose of this work is to test the feasibility of CS acquisitions based on Diffusion Limited Aggregation (DLA) sampling patterns for high resolution quantitative T1-weighted imaging. Fully encoded and DLA-CS T1-weighted images of Aplysia californica neural tissue were acquired on a 17.2T MRI system. The MR signal corresponding to single, identified neurons was quantified for both versions of the T1 weighted images. For a 50% undersampling, DLA-CS can accurately quantify signal intensities in T1-weighted acquisitions leading to only 1.37% differences when compared to the fully encoded data, with minimal impact on image spatial resolution. In addition, we compared the conventional polynomial undersampling scheme with the DLA and showed that, for the data at hand, the latter performs better. Depending on the image signal to noise ratio, higher undersampling ratios can be used to further reduce the acquisition time in MEMRI based functional studies of living tissues.

A two-pool model to describe the IVIM cerebral perfusion.

IntraVoxel Incoherent Motion (IVIM) is a magnetic resonance imaging (MRI) technique capable of measuring perfusion-related parameters. In this manuscript, we show that the mono-exponential model commonly used to process IVIM data might be challenged, especially at short diffusion times. Eleven rat datasets were acquired at 7T using a diffusion-weighted pulsed gradient spin echo sequence with b-values ranging from 7 to 2500 s/mm2 at three diffusion times. The IVIM signals, obtained by removing the diffusion component from the raw MR signal, were fitted to the standard mono-exponential model, a bi-exponential model and the Kennan model. The Akaike information criterion used to find the best model to fit the data demonstrates that, at short diffusion times, the bi-exponential IVIM model is most appropriate. The results obtained by comparing the experimental data to a dictionary of numerical simulations of the IVIM signal in microvascular networks support the hypothesis that such a bi-exponential behavior can be explained by considering the contribution of two vascular pools: capillaries and somewhat larger vessels.

In vivo online magnetic resonance quantification of absolute metabolite concentrations in microdialysate.

In order to study metabolic processes in animal models of diseases and in patients, microdialysis probes have evolved as powerful tools that are minimally invasive. However, analyses of microdialysate, performed remotely, do not provide real-time monitoring of microdialysate composition. Microdialysate solutions can theoretically be analyzed online inside a preclicinal or clinical MRI scanner using MRS techniques. Due to low NMR sensitivity, acquisitions of real-time NMR spectra on very small solution volumes (µL) with low metabolite concentrations (mM range) represent a major issue. To address this challenge we introduce the approach of combining a microdialysis probe with a custom-built magnetic resonance microprobe that allows for online metabolic analysis (1H and 13C) with high sensitivity under continuous flow conditions. This system is mounted inside an MRI scanner and allows performing simultaneously MRI experiments and rapid MRS metabolic analysis of the microdialysate. The feasibility of this approach is demonstrated by analyzing extracellular brain cancer cells (glioma) in vitro and brain metabolites in an animal model in vivo. We expect that our approach is readily translatable into clinical settings and can be used for a better and precise understanding of diseases linked to metabolic dysfunction.