The structure of the cerebral vasculature is immature at birth and undergoes significant postnatal remodeling and expansion. However, how vascularization progresses throughout the brain to integrate volumetric growth with neuronal circuit maturation is unclear. To address this spatiotemporally, we developed a light-sheet-aligned mouse brain annotated developmental atlas (LAMBADA)—a resource for registering and annotating optically cleared developing mouse brains with high temporal resolution, enriched by aligned spatial transcriptomics. Using this resource, we identified three distinct, brain-wide phases of postnatal cerebral vascular development: (1) an isometric expansion phase characterized by canonical transcriptomic signatures; (2) a regional specialization phase coinciding with neuronal maturation and synaptogenesis; and (3) a refinement phase marked by the stabilization of vascular networks and synapses. We delineated the molecular and structural mechanisms underlying these phases by correlating vascular remodeling with spatial transcriptomic gene expression. This atlas provides a foundation for studying developmental neurovascular interactions and serves as a resource for murine postnatal brain development research.

We propose for the first time a direct comparison between brain and endocast characteristics-the number, position, length and proportion of sulci as revealed by the evidence on the brain and their corresponding marks on the endocast that is the internal surface of the skull-in the same living individuals. Using a tailored MRI imaging methodology developed to overcome the limitations inherent to medical imaging on healthy subjects, we compare 3D models of the brain and of the endocast obtained from the same cohort of 75 volunteers. These data were quantified in terms of observation frequency, proportion of dimension for the same structures on the endocast compared to the brain and variation among the analysed sample. We show that identifiable marks on the endocast are often short, discontinuous, and primarily located in the lower regions (particularly in the inferior frontal and temporal lobes areas). This observation contradicts previous practices of drawing long, straight marks on fossil endocasts. An unexpected result of the study is the discovery of endocranial marks unrelated to cerebral sulci, called MNAS (for endocranial Marks Not Associated with Sulci). These marks represent approximately 12% of the depressions observed and pose a challenge for the interpretation of fossil endocasts. Finally, we propose with this work a new standardised approach to studying fossil endocasts, which combines precise guidelines for sulci variation description as well as for inter-individual comparisons, and cross-validation by several researchers. The development of a new approach based on a large quantity of anatomical information presented in the present study, offers new perspectives for reconstructing the anatomy of fossil hominin's brain in a more objective framework, and will have a lasting impact for the study of the evolution of the hominin brain, particularly functional lateralisation and cognitive abilities.

Neuromelanin-sensitive MRI has been proposed as a biomarker of Parkinson's disease pathology. However, the biological and physical origins of this contrast are debated. A recent rodent model of controlled neuromelanin accumulation in the substantia nigra has been developed and recapitulates several features of Parkinson's disease. In this work, we first combined neuromelanin-sensitive-MRI and histology to study neuromelanin accumulation and neurodegeneration in a humanized rat model of Parkinson's disease. Neuromelanin-sensitive-MRI signal changes were biphasic with an initial increase due to the accumulation of neuromelanin in dopaminergic neurons, followed signal decrease due to neurodegeneration. In healthy subjects and patients with isolated rapid eye movement sleep behaviour disorder, neuromelanin-sensitive-MRI signal increased initially and then decreased similarly as in rodents after reaching a similar maximum signal intensity in both groups. In early Parkinson's disease and converted isolated rapid eye movement sleep behaviour disorder patients, neuromelanin-sensitive-MRI signal drop was greater than in healthy individuals. Results in animals and humans show that neuromelanin-sensitive-MRI is a marker of the intracellular neuromelanin accumulation and then of neuronal degeneration and originates mainly from T1 reduction effect of neuromelanin.

Parkinson's disease demonstrates increased iron concentration in the substantia nigra (SN). The progression of iron and its interaction with neuromelanin content and dopaminergic dysregulation from prodromal to early-stage Parkinson's disease remain poorly understood. Using quantitative susceptibility mapping (QSM) and R2* relaxation rate, we investigated brain iron changes in patients with isolated rapid eye movement (REM) sleep behaviour disorder and early-stage Parkinson's disease. Subjects were scanned longitudinally at 3.0 Tesla MRI. QSM and R2* values were calculated in the entire SN and its anterior and posterior dorsal and ventral subdivisions. Baseline and longitudinal group differences were tested using analysis of variance of multiple linear regression models controlling for age and sex and linear mixed-effects modelling respectively. We included 44/36/28 healthy volunteers (HVs), 49/20/11 isolated REM sleep behaviour disorder, 127/88/50 Parkinson's disease at first/second/third visit respectively, separated by a 2-year interval. At baseline, there was a significant increase in QSM and R2* values in Parkinson's disease versus HVs in the posteroventral SN only (QSM: +17.6%%; R2*: +7.1%), which did not reach significance in isolated REM sleep behaviour disorder (QSM: +6.9%, R2*: +3.3%). Longitudinally, only posteroventral SN values demonstrated significant effects for Group and Visit using QSM and R2*. Further, the Group-by-Visit interaction was significant only for QSM. The posteroventral SN iron increased with disease duration and was inversely correlated with the changes in nigral neuromelanin content and striatal DaT levels in Parkinson's disease. The posteroventral nigral iron increased with the progression of the disease as well as dopaminergic denervation in Parkinson's disease. QSM was a stronger quantitative longitudinal marker than R2* in detecting regional nigral iron abnormalities as the disease progressed.

Small-animal diffusion MRI (dMRI) has been used for methodological development and validation, characterizing the biological basis of diffusion phenomena, and comparative anatomy. The steps from animal setup and monitoring, to acquisition, analysis, and interpretation are complex, with many decisions that may ultimately affect what questions can be answered using the resultant data. This work aims to present selected considerations and recommendations from the diffusion community on best practices for preclinical dMRI of in vivo animals. We describe the general considerations and foundational knowledge that must be considered when designing experiments. We briefly describe differences in animal species and disease models and discuss why some may be more or less appropriate for different studies. We, then, give recommendations for in vivo acquisition protocols, including decisions on hardware, animal preparation, and imaging sequences, followed by advice for data processing including preprocessing, model-fitting, and tractography. Finally, we provide an online resource that lists publicly available preclinical dMRI datasets and software packages to promote responsible and reproducible research. In each section, we attempt to provide guides and recommendations, but also highlight areas for which no guidelines exist (and why), and where future work should focus. Although we mainly cover the central nervous system (on which most preclinical dMRI studies are focused), we also provide, where possible and applicable, recommendations for other organs of interest. An overarching goal is to enhance the rigor and reproducibility of small animal dMRI acquisitions and analyses, and thereby advance biomedical knowledge.

The value of preclinical diffusion MRI (dMRI) is substantial. While dMRI enables in vivo non-invasive characterization of tissue, ex vivo dMRI is increasingly used to probe tissue microstructure and brain connectivity. Ex vivo dMRI has several experimental advantages including higher signal-to-noise ratio and spatial resolution compared to in vivo studies, and enabling more advanced diffusion contrasts. Another major advantage of ex vivo dMRI is the direct comparison with histological data as a methodological validation. However, there are a number of considerations that must be made when performing ex vivo experiments. The steps from tissue preparation, image acquisition and processing, and interpretation of results are complex, with decisions that not only differ dramatically from in vivo imaging of small animals, but ultimately affect what questions can be answered using the data. This work represents "Part 2" of a 3-part series of recommendations and considerations for preclinical dMRI. We describe best practices for dMRI of ex vivo tissue, with a focus on the value that ex vivo imaging adds to the field of dMRI and considerations in ex vivo image acquisition. We give general considerations and foundational knowledge that must be considered when designing experiments. We describe differences in specimens and models and discuss why some may be more or less appropriate for different studies. We then give guidelines for ex vivo protocols, including tissue fixation, sample preparation, and MR scanning. In each section, we attempt to provide guidelines and recommendations, but also highlight areas for which no guidelines exist (and why), and where future work should lie. An overarching goal herein is to enhance the rigor and reproducibility of ex vivo dMRI acquisitions and analyses, and thereby advance biomedical knowledge.

Deep Brain Stimulation (DBS) is a well-established approach to treat movement disorders such as Parkinson's Disease, dystonia or essential tremor. For optimal therapy response, accurate electrode placement is critical requiring high signal-to-noise of target areas in preoperative MRI. Currently, imaging protocols vary considerably between DBS centers, making it difficult to compare results or pool data for research purposes. Here, various currently employed MRI sequences from several DBS centers are evaluated regarding their suitability for DBS targeting and a protocol is suggested taking image quality and practical considerations into account. Two healthy subjects (52-year-old female and a 37-year-old male) were each scanned with various sequences (5 T2w, 1 PDw, 4 T2FLAIRw, 2 T2*w, 5 SWI, 2 FGATIR, 1 T1TIR, and 2 QSM techniques) that then were rated by 12 experienced DBS surgeons for their suitability for targeting the subthalamic nucleus (STN), the internal globus pallidus internus (GPi), and the ventrointermediate (VIM) thalamic nucleus. For a subset of sequences, surgeons were asked to identify the optimal DBS target in the STN and GPi. Contrast-to-noise ratios (CNR) were calculated and correlated to intra-rater z-scores and distances of target coordinates. For STN-DBS, surgeons rated T2w, most SWI, QSM, and T2FLAIRw the highest. For GPi-DBS, FGATIR, PDw, and SWI and for VIM-DBS, FGATIR were deemed the most suitable. Higher CNR correlated with higher intra-rater z-scores (R2= 0.29, p < .005) which improved targeting (R2= 0.18, p < .05). Our MRI protocol suggestion is a first step toward standardizing preoperative imaging. All imaging data, MRI sequence parameters, and protocol files are made openly available.

The vascular system regulates brain clearance through arterial blood flow and lymphatic drainage of cerebrospinal fluid (CSF). Idiopathic intracranial hypertension (IIH), characterized by elevated intracranial pressure and dural venous sinus stenoses, can be treated by restoring venous blood flow via venous stenting, suggesting a role for venous blood flow in brain fluid clearance. Using magnetic resonance imaging (MRI) in IIH patients and healthy controls, we identified that dural venous stenoses in IIH were associated with impaired lymphatic drainage, perivenous fluid retention, and brain fluid accumulation. To investigate this further, we developed a mouse model with bilateral jugular vein ligation (JVL), which recapitulated key human findings, including intracranial hypertension, calvarial lymphatic regression, and brain swelling due to impaired clearance. To further dissect the respective roles of dural lymphatics and venous blood flow in brain clearance, we performed JVL in mice with dural lymphatic depletion. These mice exhibited spontaneous elevated intracranial pressure, but JVL did not further exacerbate this effect. Moreover, the synchronous restoration of brain clearance and dural lymphatics observed in mice after JVL was absent in lymphatic-deficient mice.Transcriptomic analyses revealed that lymphatic remodeling induced by JVL was driven by VEGF-C signaling between dural mesenchymal and lymphatic endothelial cells. These findings establish the dural venous sinuses as a critical platform where venous blood flow interacts with mesenchymal cells to preserve dural lymphatic integrity and function, essential for brain fluid clearance. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:MR-guided focused ultrasound (MRgFUS) has been developed to treat essential tremor effectively and noninvasively. Currently, clinical examination is used to identify therapeutic efficacy during treatment, but MRgFUS surgery could benefit from real-time, rater-independent quantitative monitoring of tremor, such as accelerometry data. METHODS:Fourteen patients with medically refractory essential tremor underwent MRgFUS thalamotomy. Patients were instructed to hold postures during treatment. Tremor was monitored during each ultrasonic thermal sonication with MR-compatible accelerometers. Real-time feedback based on tremor amplitude in the 2-20 Hz band was calculated to evaluate the efficacy of each thermal ablation. RESULTS:On average 6 ± 2 ablative sonications only were required to induce improvement in tremor on the clinical rating scale for tremor (CRST) of 89 ± 11% at D + 7, 79 ± 12% at M + 1, 74 ± 19% at M + 3 and 72 ± 23% at M + 12. The overall predictive efficacy measured with accelerometry during the treatment was 70 ± 30%. The tremor amplitude reduction measured with accelerometry was correlated with CRST scores tremor reduction at multiple timepoints (ρ = 0.79 at D + 7, ρ = 0.75 at M + 1, ρ = 0.86 at M + 3, and ρ = 0.63 at M + 12) and accelerometric data gathered during treatment predicted CRST tremor improvement at M + 3 (0.88 area under ROC curve). CONCLUSION:This exploratory study is a proof of concept suggesting that accelerometry measurements can provide real-time feedback on tremor reduction and can complement visual evaluation. In the future, the use of the outcome prediction introduced in this paper may shorten procedure time and limit adverse events by reducing the number of ablative administered sonications.

Research on brain evolution centres mainly on internal mouldings of the skull, known as endocasts; however, the relationship between the size and asymmetry of the brain and endocasts has been poorly investigated in humans. Therefore, the main objective of this study was to investigate whether endocasts can be reliable indicators of brain size and asymmetry. Magnetic Resonance Imaging (MRI) of 75 participants was used to calculate the volume and surface area of the brain, the endocast, and their respective hemispheres. Two asymmetry indices (i.e. directional and absolute asymmetry) were used to assess the differences in volume and surface area between the sides of the brain and endocast. The Pearson correlation coefficient was calculated to assess the relationships between the parameters, and a Monte Carlo simulation for linear regression was performed to generate prediction equations for brain volume. The relationships between the level and direction of asymmetry indices were investigated using the Pearson correlation and McNemar's test, respectively. All correlations were statistically significant; however, correlation coefficients between volumes were stronger (0.894-0.931) than between volumes and surface areas (0.783-0.834). Brain volume can be predicted with high accuracy (ranging between 0.80 and 0.87) using the endocast total volume or the volume of one of the sides. The associations between the levels of asymmetry indices of the brain and endocast were non-significant; however, the McNemar's test indicated that endocasts show the same left- or right-biased asymmetry as the brain. This was the first study conducted on a large sample of brain and endocast data from the same individuals. The results demonstrated that brain volume can be accurately reconstructed using the volume of the endocast or one of its sides. This finding is especially important in the context of reconstructing fossil skulls, which are usually fragmented. Conversely, the asymmetry levels of endocast parameters are not reliable indicators of the actual level of brain volume asymmetry. Future research on fossils should focus on endocast asymmetry direction (left- or right-biased) as this closely corresponds with brain lateralisation.

Preclinical diffusion MRI (dMRI) has proven value in methods development and validation, characterizing the biological basis of diffusion phenomena, and comparative anatomy. While dMRI enables in vivo non-invasive characterization of tissue, ex vivo dMRI is increasingly being used to probe tissue microstructure and brain connectivity. Ex vivo dMRI has several experimental advantages that facilitate high spatial resolution and high SNR images, cutting-edge diffusion contrasts, and direct comparison with histological data as a methodological validation. However, there are a number of considerations that must be made when performing ex vivo experiments. The steps from tissue preparation, image acquisition and processing, and interpretation of results are complex, with many decisions that not only differ dramatically from in vivo imaging of small animals, but ultimately affect what questions can be answered using the data. This work concludes a three-part series of recommendations and considerations for preclinical dMRI. Herein, we describe best practices for dMRI of ex vivo tissue, with a focus on image pre-processing, data processing, and comparisons with microscopy. In each section, we attempt to provide guidelines and recommendations but also highlight areas for which no guidelines exist (and why), and where future work should lie. We end by providing guidelines on code sharing and data sharing and point toward open-source software and databases specific to small animal and ex vivo imaging.

Objective.Magnetic resonance guided transcranial focused ultrasound holds great promises for treating neurological disorders. This technique relies on skull aberration correction which requires computed tomography (CT) scans of the skull of the patients. Recently, ultra-short time-echo (UTE) magnetic resonance (MR) sequences have unleashed the MRI potential to reveal internal bone structures. In this study, we measure the efficacy of transcranial aberration correction using UTE images.Approach.We compare the efficacy of transcranial aberration correction using UTE scans to CT based correction on four skulls and two targets using a clinical device (Exablate Neuro, Insightec, Israel). We also evaluate the performance of a custom ray tracing algorithm using both UTE and CT estimates of acoustic properties and compare these against the performance of the manufacturer's proprietary aberration correction software.Main results.UTE estimated skull maps in Hounsfield units (HU) had a mean absolute error of 242 ± 20 HU (n= 4). The UTE skull maps were sufficiently accurate to improve pressure at the target (no correction: 0.44 ± 0.10, UTE correction: 0.79 ± 0.05, manufacturer CT: 0.80 ± 0.05), pressure confinement ratios (no correction: 0.45 ± 0.10, UTE correction: 0.80 ± 0.05, manufacturer CT: 0.81 ± 0.05), and targeting error (no correction: 1.06 ± 0.42 mm, UTE correction 0.30 ± 0.23 mm, manufacturer CT: 0.32 ± 0.22) (n= 8 for all values). When using CT, our ray tracing algorithm performed slightly better than UTE based correction with pressure at the target (UTE: 0.79 ± 0.05, CT: 0.84 ± 0.04), pressure confinement ratios (UTE: 0.80 ± 0.05, CT: 0.84 ± 0.04), and targeting error (UTE: 0.30 ± 0.23 mm, CT: 0.17 ± 0.15).Significance.These 3D transcranial measurements suggest that UTE sequences could replace CT scans in the case of MR guided focused ultrasound with minimal reduction in performance which will avoid ionizing radiation exposure to the patients and reduce procedure time and cost.

Background: Transcranial ultrasound stimulation (TUS) is a non-invasive brain stimulation technique; when skull aberrations are compensated for, this technique allows, with millimetric accuracy, circumvention of the invasive surgical procedure associated with deep brain stimulation (DBS) and the limited spatial specificity of transcranial magnetic stimulation. Objective: /hypothesis: We hypothesize that MR-guided low-power TUS can induce a sustained decrease of tremor power in patients suffering from medically refractive essential tremor. Methods: The dominant hand only was targeted, and two anatomical sites were sonicated in this exploratory study: the ventral intermediate nucleus of the thalamus (VIM) and the dentato-rubro-thalamic tract (DRT). Patients (N = 9) were equipped with MR-compatible accelerometers attached to their hands to monitor their tremor in real-time during TUS. Results: VIM neurostimulations followed by a low-duty cycle (5 %) DRT stimulation induced a substantial decrease in the tremor power in four patients, with a minimum of 89.9 % reduction when compared with the baseline power a few minutes after the DRT stimulation. The only patient stimulated in the VIM only and with a low duty cycle (5 %) also experienced a sustained reduction of the tremor (up to 93.4 %). Four patients (N = 4) did not respond. The temperature at target was 37.2 +/- 1.4 degrees C compared to 36.8 +/- 1.4 degrees C for a 3 cm away control point. Conclusions: MR-guided low power TUS can induce a substantial and sustained decrease of tremor power. Followup studies need to be conducted to reproduce the effect and better to understand the variability of the response amongst patients. MR thermometry during neurostimulations showed no significant thermal rise, supporting a mechanical effect.

Studies in animal models of Parkinson′s disease (PD) suggested that the accumulation of the neuromelanin (NM), a pigment contained in nigral dopaminergic neurons, could trigger neurodegeneration above a pathogenic threshold. Here we investigated this hypothesis using NM-sensitive MRI in rodents and in patients with isolated rapid eye movement sleep disorders (iRBD) subjects, a prodromal phase of parkinsonism, and early PD. We first combined NM-sensitive MRI and histology to study NM accumulation and neurodegeneration in a humanized rat model of PD. NM-MRI signal changes were biphasic with an initial increase due to the accumulation of NM in dopaminergic neurons, followed signal decrease due to neurodegeneration. In healthy subjects and patients with iRBD, NM-MRI signal increased initially and then decreased similarly as in rodents after reaching a similar maximum signal intensity in both groups. In early PD and converted iRBD patients, NM-MRI signal drop was greater than in healthy individuals. Results in animals and humans show that NM-sensitive MRI is a marker of the intracellular NM accumulation up to a threshold then of neuronal degeneration beyond this threshold and agree with the hypothesis of a pathogenic threshold of NM triggering neurodegeneration. ### Competing Interest Statement The authors have declared no competing interest.