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.

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.

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.

Functional magnetic resonance microscopy at single-cell resolution in Aplysia californica.

In this work, we show the feasibility of performing functional MRI studies with single-cell resolution. At ultrahigh magnetic field, manganese-enhanced magnetic resonance microscopy allows the identification of most motor neurons in the buccal network of Aplysia at low, nontoxic Mn(2+) concentrations. We establish that Mn(2+) accumulates intracellularly on injection into the living Aplysia and that its concentration increases when the animals are presented with a sensory stimulus. We also show that we can distinguish between neuronal activities elicited by different types of stimuli. This method opens up a new avenue into probing the functional organization and plasticity of neuronal networks involved in goal-directed behaviors with single-cell resolution.

Water diffusion in brain cortex closely tracks underlying neuronal activity.

Neuronal activity results in a local increase in blood flow. This concept serves as the basis for functional MRI. Still, this approach remains indirect and may fail in situations interfering with the neurovascular coupling mechanisms (drugs, anesthesia). Here we establish that water molecular diffusion is directly modulated by underlying neuronal activity using a rat forepaw stimulation model under different conditions of neuronal stimulation and neurovascular coupling. Under nitroprusside infusion, a neurovascular-coupling inhibitor, the diffusion response and local field potentials were maintained, whereas the hemodynamic response was abolished. As diffusion MRI reflects interactions of water molecules with obstacles (e.g., cell membranes), the observed changes point to a dynamic modulation of the neural tissue structure upon activation, which remains to be investigated. These findings represent a significant shift in concept from the current electrochemical and neurovascular coupling principles used for brain imaging, and open unique avenues to investigate mechanisms underlying brain function.

fMRI contrast at high and ultrahigh magnetic fields: insight from complementary methods.

This manuscript examines the origins and nature of the function-derived activation detected by magnetic resonance imaging at ultrahigh fields using different encoding methods. A series of preclinical high field (7 T) and ultra-high field (17.2 T) fMRI experiments were performed using gradient echo EPI, spin echo EPI and spatio-temporally encoded (SPEN) strategies. The dependencies of the fMRI signal change on the strength of the magnetic field and on different acquisition and sequence parameters were investigated. Artifact-free rat brain images with good resolution in all areas, as well as significant localized activation maps upon forepaw stimulation, were obtained in a single scan using fully refocused SPEN sequences devoid of T2* effects. Our results showed that, besides the normal T2-weighted BOLD contribution that arises in spin-echo sequences, fMRI SPEN signals contain a strong component caused by apparent T1-related effects, demonstrating the potential of such technique for exploring functional activation in rodents and on humans at ultrahigh fields.

Relationship between the diffusion time and the diffusion MRI signal observed at 17.2 Tesla in the healthy rat brain cortex.

PURPOSE: To investigate the diffusion time dependency of water diffusion in cortical brain tissue. METHODS: We have combined an oscillating gradient spin-echo (OGSE) and a pulse gradient spin echo (PGSE) spin-echo sequence to acquire diffusion-weighted MRI images in vivo in healthy rat brains over a wide range of diffusion times (1.9-29.2 ms) and estimated the parameters of the biexponential and cumulant expansion diffusion MRI signal models. Diffusion images were obtained at 17.2 Tesla with maximum gradient strength of 1000 mT/m allowing 40 b values up to approximately 4000 s/mm(2). RESULTS: At all diffusion times the log plot of diffusion signal attenuation versus b value was curved, confirming that diffusion is not free, even at very short diffusion times. This suggests that the length scale of obstacles to diffusion must be smaller than the corresponding shortest observed diffusion distance (approximately 1.7 µm). The diffusion MRI signal was also not found in a steady-state, even at our longest diffusion time (29.2 ms), suggesting some degree of segregation of water in pools. CONCLUSION: Overall, the results showed that the parameters derived from the two diffusion models could not well be related to specific tissue features. More specific models must be developed taking into account diffusion signal behavior at high b values and short diffusion times.

Effects of hypotonic stress and ouabain on the apparent diffusion coefficient of water at cellular and tissue levels in Aplysia.

There is evidence that physiological or pathological cell swelling is associated with a decrease of the apparent diffusion coefficient (ADC) of water in tissues, as measured with MRI. However the mechanism remains unclear. Magnetic resonance microscopy, performed on small tissue samples, has the potential to distinguish effects occurring at cellular and tissue levels. A three-dimensional diffusion prepared fast imaging with steady-state free precession sequence for MR microscopy was implemented on a 17.2 T imaging system and used to investigate the effect of two biological challenges known to cause cell swelling, exposure to a hypotonic solution or to ouabain, on Aplysia nervous tissue. The ADC was measured inside isolated neuronal soma and in the region of cell bodies of the buccal ganglia. Both challenges resulted in an ADC increase inside isolated neuronal soma (+31 ± 24% and +30 ± 11%, respectively) and an ADC decrease at tissue level in the buccal ganglia (-12 ± 5% and -18 ± 8%, respectively). A scenario involving a layer of water molecules bound to the inflating cell membrane surface is proposed to reconcile this apparent discrepancy.

Highlighting manganese dynamics in the nervous system of Aplysia californica using MEMRI at ultra-high field.

Exploring the pathways of manganese (Mn(2+)) transport in the nervous system becomes of interest as many recent studies use Mn(2+) as a neural tract tracer in mammals. In this study, we performed manganese enhanced MRI (MEMRI) at 17.2 T on the buccal ganglia of Aplysia californica. The main advantage of this model over mammalian systems is that it contains networks of large identified neurons. Using Mn(2+) retrograde transport along selected nerves, we first validated the mapping of motor neurons' axonal projections into peripheral nerves, previously obtained from optical imaging (Morton et al., 1991). This protocol was found not to alter the functional properties of the neuronal network. Second, we compared the Mn(2+) dynamics inside the ganglia in the presence or absence of chemical stimulation. We found that 2h of stimulation with the modulatory transmitter dopamine increased the extent of areas of intermediate signal enhancement caused by manganese accumulation. In the absence of dopamine, an overall decrease of the enhanced areas in favor of non-enhanced areas was found, as a result of natural Mn(2+) washout. This supports the hypothesis that, upon activation, Mn(2+) is released from labeled neurons and captured by other, initially unlabeled, neurons. However, the latter could not be clearly identified due to lack of sensitivity and multiplicity of possible pathways starting from labeled cells. Nonetheless, the Aplysia buccal ganglia remain a well-suited model for attempting to visualize Mn(2+) transport from neuron to neuron upon activation, as well as for studying dopaminergic modulation in a motor network.

Quantification of microvascular cerebral blood flux and late-stage tumor compartmentalization in 9L gliosarcoma using flow enhanced MRI.

Measurements of tumor microvasculature are important to obtain an understanding of tumor angiogenesis and for the evaluation of therapies. In this work, we characterize the evolution of the microvascular flux at different stages of tumor growth in the 9L rat brain tumor model. The absolute quantification of cerebral blood flux is achieved with MRI at 7 T using the flow enhanced signal intensity (FENSI) method. FENSI flux maps were obtained between 5 and 14 days after glioma cell inoculation. Based on cerebral blood flux maps, we highlighted two main stages of tumor growth, below and above 3 mm, presenting distinct flux patterns and vascular properties. No significant difference emerged from the group analysis performed on the data collected at an early developmental stage (tumor size < 3 mm) when compared with healthy tissue. At a late developmental stage (tumor size > 3 mm), we observed a significant decrease in the cerebral blood flux inside the gliosarcoma (-33%, p < 0.01) and compartmentalization of the tumor (p < 0.05). FENSI flux maps delineated a low-flux tumor core (58 ± 17 µL/min/cm(2) ) and higher vascularized regions around the tumor periphery (85 ± 21 µL/min/cm(2) ). Histology was performed on 11 animals to finely probe the intratumor heterogeneity and microvessel density, and the results were compared with the information derived from FENSI flux maps. The hyper- and hypoperfused tumor regions revealed with FENSI at the late tumor developmental stage correlated well with the ratios of high and low blood vessel density (R(2) = 0.41) and fractional vascular surface (R(2) = 0.67) observed with fluorescence microscopy [cluster of differentiation 31 (CD31) staining].

Diffusion kurtosis imaging and log-normal distribution function imaging enhance the visualisation of lesions in animal stroke models.

In this work, we report a case study of a stroke model in animals using two methods of quantification of the deviations from Gaussian behaviour: diffusion kurtosis imaging (DKI) and log-normal distribution function imaging (LNDFI). The affected regions were predominantly in grey rather than in white matter. The parameter maps were constructed for metrics quantifying the apparent diffusivity (evaluated from conventional diffusion tensor imaging, DKI and LNDFI) and for those quantifying the degree of deviations (mean kurtosis and a parameter σ characterising the width of the distribution). We showed that both DKI and LNDFI were able to dramatically enhance the visualisation of ischaemic lesions in comparison with conventional methods. The largest relative change in the affected versus healthy regions was observed in the mean kurtosis values. The average changes in the mean kurtosis and σ values in the lesions were a factor of two to three larger than the relative changes observed in the mean diffusivity. In conclusion, the applied methods promise valuable perspectives in the assessment of stroke.

The translocator protein ligand [¹8F]DPA-714 images glioma and activated microglia in vivo.

PURPOSE: In recent years there has been an increase in the development of radioligands targeting the 18-kDa translocator protein (TSPO). TSPO expression is well documented in activated microglia and serves as a biomarker for imaging neuroinflammation. In addition, TSPO has also been reported to be overexpressed in a number of cancer cell lines and human tumours including glioma. Here we investigated the use of [(18)F]DPA-714, a new TSPO positron emission tomography (PET) radioligand to image glioma in vivo. METHODS: We studied the uptake of [(18)F]DPA-714 in three different rat strains implanted with 9L rat glioma cells: Fischer (F), Wistar (W) and Sprague Dawley (SD) rats. Dynamic [(18)F]DPA-714 PET imaging, kinetic modelling of PET data and in vivo displacement studies using unlabelled DPA-714 and PK11195 were performed. Validation of TSPO expression in 9L glioma cell lines and intracranial 9L gliomas were investigated using Western blotting and immunohistochemistry of brain tissue sections. RESULTS: All rats showed significant [(18)F]DPA-714 PET accumulation at the site of 9L tumour implantation compared to the contralateral brain hemisphere with a difference in uptake among the three strains (F > W > SD). The radiotracer showed high specificity for TSPO as demonstrated by the significant reduction of [(18)F]DPA-714 binding in the tumour after administration of unlabelled DPA-714 or PK11195. TSPO expression was confirmed by Western blotting in 9L cells in vitro and by immunohistochemistry ex vivo. CONCLUSION: The TSPO radioligand [(18)F]DPA-714 can be used for PET imaging of intracranial 9L glioma in different rat strains. This preclinical study demonstrates the feasibility of employing [(18)F]DPA-714 as an alternative radiotracer to image human glioma.

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.

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.

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.

Post-processing correction of magnetization transfer effects in FENSI perfusion MRI data.

Magnetization transfer effects induced by repetitive saturation pulses employed in flow enhancement of signal intensity imaging sequences currently prevent quantitative, in vivo, cerebral perfusion studies. This study investigates the magnitude of these effects and introduces a post-processing correction protocol. The study shows that the magnetization transfer effect is consistent across individuals, which enables the derivation of a correction factor to be applied in post-acquisition. Our results, obtained for cerebral flux in white and gray matter in rodent brains, are in agreement with cerebral blood flow measurements previously reported in the literature.

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.