Volume electron microscopy in injured rat brain validates white matter microstructure metrics from diffusion MRI.

Biophysical modeling of diffusion MRI (dMRI) offers the exciting potential of bridging the gap between the macroscopic MRI resolution and microscopic cellular features, effectively turning the MRI scanner into a noninvasive in vivo microscope. In brain white matter, the Standard Model (SM) interprets the dMRI signal in terms of axon dispersion, intra- and extra-axonal water fractions and diffusivities. However, for SM to be fully applicable and correctly interpreted, it needs to be carefully evaluated using histology. Here, we perform a comprehensive histological validation of the SM parameters, by characterizing WM microstructure in sham and injured rat brains using volume (3d) electron microscopy (EM) and ex vivo dMRI. Sensitivity is evaluated by how close each SM metric is to its histological counterpart, and specificity by how independent it is from other, non-corresponding histological features. This comparison reveals that SM is sensitive and specific to microscopic properties, clearing the way for the clinical adoption of in vivo dMRI derived SM parameters as biomarkers for neurological disorders.

A multiscale tissue assessment in a rat model of mild traumatic brain injury.

Diffusion tensor imaging (DTI) has demonstrated the potential to assess the pathophysiology of mild traumatic brain injury (mTBI) but correlations of DTI findings and pathological changes in mTBI are unclear. We evaluated the potential of ex vivo DTI to detect tissue damage in a mild mTBI rat model by exploiting multiscale imaging methods, histology and scanning micro-X-ray diffraction (SµXRD) 35 days after sham-operation (n = 2) or mTBI (n = 3). There were changes in DTI parameters rostral to the injury site. When examined by histology and SµXRD, there was evidence of axonal damage, reduced myelin density, gliosis, and ultrastructural alterations in myelin that were ongoing at the experimental time point of 35 days postinjury. We assessed the relationship between the 3 imaging modalities by multiple linear regression analysis. In this analysis, DTI and histological parameters were moderately related, whereas SµXRD parameters correlated weakly with DTI and histology. These findings suggest that while DTI appears to distinguish tissue changes at the microstructural level related to the loss of myelinated axons and gliosis, its ability to visualize alterations in myelin ultrastructure is limited. The use of several imaging techniques represents a novel approach to reveal tissue damage and provides new insights into mTBI detection.

Histopathological modeling of status epilepticus-induced brain damage based on in vivo diffusion tensor imaging in rats.

Non-invasive magnetic resonance imaging (MRI) methods have proved useful in the diagnosis and prognosis of neurodegenerative diseases. However, the interpretation of imaging outcomes in terms of tissue pathology is still challenging. This study goes beyond the current interpretation of in vivo diffusion tensor imaging (DTI) by constructing multivariate models of quantitative tissue microstructure in status epilepticus (SE)-induced brain damage. We performed in vivo DTI and histology in rats at 79 days after SE and control animals. The analyses focused on the corpus callosum, hippocampal subfield CA3b, and layers V and VI of the parietal cortex. Comparison between control and SE rats indicated that a combination of microstructural tissue changes occurring after SE, such as cellularity, organization of myelinated axons, and/or morphology of astrocytes, affect DTI parameters. Subsequently, we constructed a multivariate regression model for explaining and predicting histological parameters based on DTI. The model revealed that DTI predicted well the organization of myelinated axons (cross-validated R = 0.876) and astrocyte processes (cross-validated R = 0.909) and possessed a predictive value for cell density (CD) (cross-validated R = 0.489). However, the morphology of astrocytes (cross-validated R > 0.05) was not well predicted. The inclusion of parameters from CA3b was necessary for modeling histopathology. Moreover, the multivariate DTI model explained better histological parameters than any univariate model. In conclusion, we demonstrate that combining several analytical and statistical tools can help interpret imaging outcomes to microstructural tissue changes, opening new avenues to improve the non-invasive diagnosis and prognosis of brain tissue damage.

Whole-brain studies of spontaneous behavior in head-fixed rats enabled by zero echo time MB-SWIFT fMRI.

Understanding the link between the brain activity and behavior is a key challenge in modern neuroscience. Behavioral neuroscience, however, lacks tools to record whole-brain activity in complex behavioral settings. Here we demonstrate that a novel Multi-Band SWeep Imaging with Fourier Transformation (MB-SWIFT) functional magnetic resonance imaging (fMRI) approach enables whole-brain studies in spontaneously behaving head-fixed rats. First, we show anatomically relevant functional parcellation. Second, we show sensory, motor, exploration, and stress-related brain activity in relevant networks during corresponding spontaneous behavior. Third, we show odor-induced activation of olfactory system with high correlation between the fMRI and behavioral responses. We conclude that the applied methodology enables novel behavioral study designs in rodents focusing on tasks, cognition, emotions, physical exercise, and social interaction. Importantly, novel zero echo time and large bandwidth approaches, such as MB-SWIFT, can be applied for human behavioral studies, allowing more freedom as body movement is dramatically less restricting factor.

Light sedation with short habituation time for large-scale functional magnetic resonance imaging studies in rats.

Traditionally, preclinical resting state functional magnetic resonance imaging (fMRI) studies have been performed in anesthetized animals. Nevertheless, as anesthesia affects the functional connectivity (FC) in the brain, there has been a growing interest in imaging in the awake state. Obviously, awake imaging requires resource- and time-consuming habituation prior to data acquisition to reduce the stress and motion of the animals. Light sedation has been a less widely exploited alternative for awake imaging, requiring shorter habituation times, while still reducing the effect of anesthesia. Here, we imaged 102 rats under light sedation and 10 awake animals to conduct an FC analysis. We established an automated data-processing pipeline suitable for both groups. Additionally, the same pipeline was used on data obtained from an openly available awake rat database (289 measurements in 90 rats). The FC pattern in the light sedation measurements closely resembled the corresponding patterns in both onsite and offsite awake datasets. However, fewer datasets had to be excluded due to movement in rats with light sedation. The temporal analysis of FC in the lightly sedated group indicated a lingering effect of anesthesia that stabilized after the first 5 min. In summary, our results indicate that the light sedation protocol is a valid alternative for large-scale studies where awake protocols may become prohibitively resource-demanding, as it provides similar results to awake imaging, preserves more scans, and requires shorter habituation times. The large amount of fMRI data obtained in this work are openly available for further analyses.

Infantile status epilepticus disrupts myelin development.

Temporal lobe epilepsy (TLE) is the most prevalent type of epilepsy in adults; it often starts in infancy or early childhood. Although TLE is primarily considered to be a grey matter pathology, a growing body of evidence links this disease with white matter abnormalities. In this study, we explore the impact of TLE onset and progression in the immature brain on white matter integrity and development utilising the rat model of Li-pilocarpine-induced TLE at the 12th postnatal day (P). Diffusion tensor imaging (DTI) and Black-Gold II histology uncovered disruptions in major white matter tracks (corpus callosum, internal and external capsules, and deep cerebral white matter) spreading through the whole brain at P28. These abnormalities were mostly not present any longer at three months after TLE induction, with only limited abnormalities detectable in the external capsule and deep cerebral white matter. Relaxation Along a Fictitious Field in the rotating frame of rank 4 indicated that white matter changes observed at both timepoints, P28 and P72, are consistent with decreased myelin content. The animals affected by TLE-induced white matter abnormalities exhibited increased functional connectivity between the thalamus and medial prefrontal and somatosensory cortex in adulthood. Furthermore, histological analyses of additional animal groups at P15 and P18 showed only mild changes in white matter integrity, suggesting a gradual age-dependent impact of TLE progression. Taken together, TLE progression in the immature brain distorts white matter development with a peak around postnatal day 28, followed by substantial recovery in adulthood. This developmental delay might give rise to cognitive and behavioural comorbidities typical for early-onset TLE.

Microstructural Tissue Changes in a Rat Model of Mild Traumatic Brain Injury.

Our study investigates the potential of diffusion MRI (dMRI), including diffusion tensor imaging (DTI), fixel-based analysis (FBA) and neurite orientation dispersion and density imaging (NODDI), to detect microstructural tissue abnormalities in rats after mild traumatic brain injury (mTBI). The brains of sham-operated and mTBI rats 35 days after lateral fluid percussion injury were imaged ex vivo in a 11.7-T scanner. Voxel-based analyses of DTI-, fixel- and NODDI-based metrics detected extensive tissue changes in directly affected brain areas close to the primary injury, and more importantly, also in distal areas connected to primary injury and indirectly affected by the secondary injury mechanisms. Histology revealed ongoing axonal abnormalities and inflammation, 35 days after the injury, in the brain areas highlighted in the group analyses. Fractional anisotropy (FA), fiber density (FD) and fiber density and fiber bundle cross-section (FDC) showed similar pattern of significant areas throughout the brain; however, FA showed more significant voxels in gray matter areas, while FD and FDC in white matter areas, and orientation dispersion index (ODI) in areas most damage based on histology. Region-of-interest (ROI)-based analyses on dMRI maps and histology in selected brain regions revealed that the changes in MRI parameters could be attributed to both alterations in myelinated fiber bundles and increased cellularity. This study demonstrates that the combination of dMRI methods can provide a more complete insight into the microstructural alterations in white and gray matter after mTBI, which may aid diagnosis and prognosis following a mild brain injury.

Corrigendum: RAFF-4, Magnetization Transfer and Diffusion Tensor MRI of Lysophosphatidylcholine Induced Demyelination and Remyelination in Rats.

[This corrects the article DOI: 10.3389/fnins.2021.625167.].

Spinal cord fMRI with MB-SWIFT for assessing epidural spinal cord stimulation in rats.

PURPOSE: Electrical epidural spinal cord stimulation (SCS) is used as a treatment for chronic pain as well as to partially restore motor function after a spinal cord injury. Monitoring the spinal cord activity during SCS with fMRI could provide important and objective measures of integrative responses to treatment. Unfortunately, spinal cord fMRI is severely challenged by motion and susceptibility artifacts induced by the implanted electrode and bones. This pilot study introduces multi-band sweep imaging with Fourier transformation (MB-SWIFT) technique for spinal cord fMRI during SCS in rats. Given the close to zero acquisition delay and high bandwidth in 3 dimensions, MB-SWIFT is demonstrated to be highly tolerant to motion and susceptibility-induced artifacts and thus holds promise for fMRI during SCS. METHODS: MB-SWIFT with 0.78 × 0.78 × 1.50 mm3 spatial resolution and 3-s temporal resolution was used at 9.4 Tesla in rats undergoing epidural SCS at different frequencies. Its performance was compared with spin echo EPI. The origin of the functional contrast was also explored using suppression bands. RESULTS: MB-SWIFT was tolerant to electrode-induced artifacts and respiratory motion, leading to substantially higher fMRI sensitivity than spin echo fMRI. Clear stimulation frequency-dependent responses to SCS were detected in the rat spinal cord close to the stimulation site. The origin of MB-SWIFT fMRI signals was consistent with dominant inflow effects. CONCLUSION: fMRI of the rat spinal cord during SCS can be consistently achieved with MB-SWIFT, thus providing a valuable experimental framework for assessing the effects of SCS on the central nervous system.

RAFF-4, Magnetization Transfer and Diffusion Tensor MRI of Lysophosphatidylcholine Induced Demyelination and Remyelination in Rats.

Remyelination is a naturally occurring response to demyelination and has a central role in the pathophysiology of multiple sclerosis and traumatic brain injury. Recently we demonstrated that a novel MRI technique entitled Relaxation Along a Fictitious Field (RAFF) in the rotating frame of rank n (RAFFn) achieved exceptional sensitivity in detecting the demyelination processes induced by lysophosphatidylcholine (LPC) in rat brain. In the present work, our aim was to test whether RAFF4, along with magnetization transfer (MT) and diffusion tensor imaging (DTI), would be capable of detecting the changes in the myelin content and microstructure caused by modifications of myelin sheets around axons or by gliosis during the remyelination phase after LPC-induced demyelination in the corpus callosum of rats. We collected MRI data with RAFF4, MT and DTI at 3 days after injection (demyelination stage) and at 38 days after injection (remyelination stage) of LPC (n = 12) or vehicle (n = 9). Cell density and myelin content were assessed by histology. All MRI metrics detected differences between LPC-injected and control groups of animals in the demyelination stage, on day 3. In the remyelination phase (day 38), RAFF4, MT parameters, fractional anisotropy, and axial diffusivity detected signs of a partial recovery consistent with the remyelination evident in histology. Radial diffusivity had undergone a further increase from day 3 to 38 and mean diffusivity revealed a complete recovery correlating with the histological assessment of cell density attributed to gliosis. The combination of RAFF4, MT and DTI has the potential to differentiate between normal, demyelinated and remyelinated axons and gliosis and thus it may be able to provide a more detailed assessment of white matter pathologies in several neurological diseases.

Isoflurane affects brain functional connectivity in rats 1 month after exposure.

Isoflurane, the most commonly used preclinical anesthetic, induces brain plasticity and long-term cellular and molecular changes leading to behavioral and/or cognitive consequences. These changes are most likely associated with network-level changes in brain function. To elucidate the mechanisms underlying long-term effects of isoflurane, we investigated the influence of a single isoflurane exposure on functional connectivity, brain electrical activity, and gene expression. Male Wistar rats (n = 22) were exposed to 1.8% isoflurane for 3 h. Control rats (n = 22) spent 3 h in the same room without exposure to anesthesia. After 1 month, functional connectivity was evaluated with resting-state functional magnetic resonance imaging (fMRI; n = 6 + 6) and local field potential measurements (n = 6 + 6) in anesthetized animals. A whole genome expression analysis (n = 10+10) was also conducted with mRNA-sequencing from cortical and hippocampal tissue samples. Isoflurane treatment strengthened thalamo-cortical and hippocampal-cortical functional connectivity. Cortical low-frequency fMRI power was also significantly increased in response to the isoflurane treatment. The local field potential results indicating strengthened hippocampal-cortical alpha and beta coherence were in good agreement with the fMRI findings. Furthermore, altered expression was found in 20 cortical genes, several of which are involved in neuronal signal transmission, but no gene expression changes were noted in the hippocampus. Isoflurane induced prolonged changes in thalamo-cortical and hippocampal-cortical function and expression of genes contributing to signal transmission in the cortex. Further studies are required to investigate whether these changes are associated with the postoperative behavioral and cognitive symptoms commonly observed in patients and animals.

Assessment of the structural complexity of diffusion MRI voxels using 3D electron microscopy in the rat brain.

Validation and interpretation of diffusion magnetic resonance imaging (dMRI) requires detailed understanding of the actual microstructure restricting the diffusion of water molecules. In this study, we used serial block-face scanning electron microscopy (SBEM), a three-dimensional electron microscopy (3D-EM) technique, to image seven white and grey matter volumes in the rat brain. SBEM shows excellent contrast of cellular membranes, which are the major components restricting the diffusion of water in tissue. Additionally, we performed 3D structure tensor (3D-ST) analysis on the SBEM volumes and parameterised the resulting orientation distributions using Watson and angular central Gaussian (ACG) probability distributions as well as spherical harmonic (SH) decomposition. We analysed how these parameterisations described the underlying orientation distributions and compared their orientation and dispersion with corresponding parameters from two dMRI methods, neurite orientation dispersion and density imaging (NODDI) and constrained spherical deconvolution (CSD). Watson and ACG parameterisations and SH decomposition captured well the 3D-ST orientation distributions, but ACG and SH better represented the distributions due to its ability to model asymmetric dispersion. The dMRI parameters corresponded well with the 3D-ST parameters in the white matter volumes, but the correspondence was less evident in the more complex grey matter. SBEM imaging and 3D-ST analysis also revealed that the orientation distributions were often not axially symmetric, a property neatly captured by the ACG distribution. Overall, the ability of SBEM to image diffusion barriers in intricate detail, combined with 3D-ST analysis and parameterisation, provides a step forward toward interpreting and validating the dMRI signals in complex brain tissue microstructure.

In Vivo Diffusion Tensor Imaging in Acute and Subacute Phases of Mild Traumatic Brain Injury in Rats.

Mild traumatic brain injury (mTBI) is the most common form of TBI with 10-25% of the patients experiencing long-lasting symptoms. The potential of diffusion tensor imaging (DTI) for evaluating microstructural damage after TBI is widely recognized, but the interpretation of DTI changes and their relationship with the underlying tissue damage is unclear. We studied how both axonal damage and gliosis contribute to DTI alterations after mTBI. We induced mTBI using the lateral fluid percussion (LFP) injury model in adult male Sprague Dawley rats and scanned them at 3 and 28 d post-mTBI. To characterize the DTI findings in the tissue, we assessed the histology by performing structure tensor (ST)-based analysis and cell counting on myelin-stained and Nissl-stained sections, respectively. In particular, we studied the contribution of two tissue components, myelinated axons and cellularity, to the DTI changes. Fractional anisotropy (FA), mean diffusivity (MD), and axial diffusivity (AD) were decreased in both white and gray matter areas in the acute phase post-mTBI, mainly at the primary lesion site. In the subacute phase, FA and AD were decreased in the white matter, external capsule, corpus callosum, and internal capsule. Our quantitative histologic assessment revealed axonal damage and gliosis throughout the brain in both white and gray matter, consistent with the FA and AD changes. Our findings suggest that the usefulness of in vivo DTI is limited in its detection of secondary damage distal to the primary lesion, while at the lesion site, DTI detected progressive microstructural damage in the white and gray matter after mTBI.

Multiscale Imaging Approach for Studying the Central Nervous System: Methodology and Perspective.

Non-invasive imaging methods have become essential tools for understanding the central nervous system (CNS) in health and disease. In particular, magnetic resonance imaging (MRI) techniques provide information about the anatomy, microstructure, and function of the brain and spinal cord in vivo non-invasively. However, MRI is limited by its spatial resolution and signal specificity. In order to mitigate these shortcomings, it is crucial to validate MRI with an array of ancillary ex vivo imaging techniques. These techniques include histological methods, such as light and electron microscopy (EM), which can provide specific information on the tissue structure in healthy and diseased brain and spinal cord, at cellular and subcellular level. However, these conventional histological techniques are intrinsically two-dimensional (2D) and, as a result of sectioning, lack volumetric information of the tissue. This limitation can be overcome with genuine three-dimensional (3D) imaging approaches of the tissue. 3D highly resolved information of the CNS achievable by means of other imaging techniques can complement and improve the interpretation of MRI measurements. In this article, we provide an overview of different 3D imaging techniques that can be used to validate MRI. As an example, we introduce an approach of how to combine diffusion MRI and synchrotron X-ray phase contrast tomography (SXRPCT) data. Our approach paves the way for a new multiscale assessment of the CNS allowing to validate and to improve our understanding of in vivo imaging (such as MRI).

Multi-band SWIFT enables quiet and artefact-free EEG-fMRI and awake fMRI studies in rat.

Functional magnetic resonance imaging (fMRI) studies in animal models provide invaluable information regarding normal and abnormal brain function, especially when combined with complementary stimulation and recording techniques. The echo planar imaging (EPI) pulse sequence is the most common choice for fMRI investigations, but it has several shortcomings. EPI is one of the loudest sequences and very prone to movement and susceptibility-induced artefacts, making it suboptimal for awake imaging. Additionally, the fast gradient-switching of EPI induces disrupting currents in simultaneous electrophysiological recordings. Therefore, we investigated whether the unique features of Multi-Band SWeep Imaging with Fourier Transformation (MB-SWIFT) overcome these issues at a high 9.4 T magnetic field, making it a potential alternative to EPI. MB-SWIFT had 32-dB and 20-dB lower peak and average sound pressure levels, respectively, than EPI with typical fMRI parameters. Body movements had little to no effect on MB-SWIFT images or functional connectivity analyses, whereas they severely affected EPI data. The minimal gradient steps of MB-SWIFT induced significantly lower currents in simultaneous electrophysiological recordings than EPI, and there were no electrode-induced distortions in MB-SWIFT images. An independent component analysis of the awake rat functional connectivity data obtained with MB-SWIFT resulted in near whole-brain level functional parcellation, and simultaneous electrophysiological and fMRI measurements in isoflurane-anesthetized rats indicated that MB-SWIFT signal is tightly linked to neuronal resting-state activity. Therefore, we conclude that the MB-SWIFT sequence is a robust preclinical brain mapping tool that can overcome many of the drawbacks of conventional EPI fMRI at high magnetic fields.

Harmonization of pipeline for preclinical multicenter MRI biomarker discovery in a rat model of post-traumatic epileptogenesis.

Preclinical imaging studies of posttraumatic epileptogenesis (PTE) have largely been proof-of-concept studies with limited animal numbers, and thus lack the statistical power for biomarker discovery. Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) is a pioneering multicenter trial investigating preclinical imaging biomarkers of PTE. EpiBios4Rx faced the issue of harmonizing the magnetic resonance imaging (MRI) procedures and imaging data metrics prior to its execution. We present here the harmonization process between three preclinical MRI facilities at the University of Eastern Finland (UEF), the University of Melbourne (Melbourne), and the University of California, Los Angeles (UCLA), and evaluate the uniformity of the obtained MRI data. Adult, male rats underwent a lateral fluid percussion injury (FPI) and were followed by MRI 2 days, 9 days, 1 month, and 5 months post-injury. Ex vivo scans of fixed brains were conducted 7 months post-injury as an end point follow-up. Four MRI modalities were used: T2-weighted imaging, multi-gradient-echo imaging, diffusion-weighted imaging, and magnetization transfer imaging, and acquisition parameters for each modality were tailored to account for the different field strengths (4.7 T and 7 T) and different MR hardwares used at the three participating centers. Pilot data collection resulted in comparable image quality across sites. In interim analysis (of data obtained by April 30, 2018), the within-site variation of the quantified signal properties was low, while some differences between sites remained. In T2-weighted images the signal-to-noise ratios were high at each site, being 35 at UEF, 48 at Melbourne, and 32 at UCLA (p < 0.05). The contrast-to-noise ratios were similar between the sites (9, 10, and 8, respectively). Magnetization transfer ratio maps had identical white matter/ gray matter contrast between the sites, with white matter showing 15% higher MTR than gray matter despite different absolute MTR values (MTR both in white and gray matter was 3% lower in Melbourne than at UEF, p < 0.05). Diffusion-weighting yielded different degrees of signal attenuation across sites, being 83% at UEF, 76% in Melbourne, and 80% at UCLA (p < 0.05). Fractional anisotropy values differed as well, being 0.81 at UEF, 0.73 in Melbourne, and 0.84 at UCLA (p < 0.05). The obtained values in sham animals showed low variation within each site and no change over time, suggesting high repeatability of the measurements. Quality control scans with phantoms demonstrated stable hardware performance over time. Timing of post-TBI scans was designed to target specific phases of the dynamic pathology, and the execution at different centers was highly accurate. Besides a few outliers, the 2-day scans were done within an hour from the target time point. At day 9, most animals were scanned within an hour from the target time point, and all but 2 outliers within 24 h from the target. The 1-month post-TBI scans were done within 31 ± 3 days. MRI procedures and animal physiology during scans were similar between the sites. Taken together, the 10% inter-site difference in FA and 3% difference in MTR values should be included into analysis as a covariate or balanced out in post-processing in order to detect disease-related effects on brain structure at the same scale. However, for a MRI biomarker for post-traumatic epileptogenesis to have realistic chance of being successfully translated to validation in clinical trials, it would need to be a robust TBI-induced structural change which tolerates the inter-site methodological variability described here.

Spontaneous BOLD waves - A novel hemodynamic activity in Sprague-Dawley rat brain detected by functional magnetic resonance imaging.

We report spontaneous hemodynamic activity termed "Spontaneous BOLD Waves" (SBWs) detected by BOLD fMRI in Sprague-Dawley rats under medetomidine anesthesia. These SBWs, which lasted several minutes, were observed in cortex, thalamus and hippocampus. The SBWs' correlates were undetectable in electrophysiological recordings, suggesting an exclusive gliovascular phenomenon dissociated from neuronal activity. SBWs were insensitive to the NMDA receptors antagonist MK-801 but were inhibited by the α1-adrenoceptor blocker prazosin. Since medetomidine is a potent agonist of α2 adrenoceptors, we suggested that imbalance in α1/α2 receptor-mediated signalling pathways alter the vascular reactivity leading to SBWs. The frequency of SBWs increased with intensity of mechanical lung ventilation despite the stable pH levels. In summary, we present a novel type of propagating vascular brain activity without easily detectable underlying neuronal activity, which can be utilized to study the mechanisms of vascular reactivity in functional and pharmacological MRI and has practical implications for designing fMRI experiments in anesthetized animals.

Awake Rat Brain Functional Magnetic Resonance Imaging Using Standard Radio Frequency Coils and a 3D Printed Restraint Kit.

Functional magnetic resonance imaging (fMRI) is a powerful noninvasive tool for studying spontaneous resting state functional connectivity (RSFC) in laboratory animals. Brain function can be significantly affected by generally used anesthetics, however, rendering the need for awake imaging. Only a few different awake animal habituation protocols have been presented, and there is a critical need for practical and improved low-stress techniques. Here we demonstrate a novel restraint approach for awake rat RSFC studies. Our custom-made 3D printed restraint kit is compatible with a standard Bruker Biospin MRI rat bed, rat brain receiver coil, and volume transmitter coil. We also implemented a progressive habituation protocol aiming to minimize the stress experienced by the rats, and compared RSFC between awake, lightly sedated, and isoflurane-anesthetized rats. Our results demonstrated that the 3D printed restraint kit was suitable for RSFC studies of awake rats. During the short 4-day habituation period, the plasma corticosterone concentration, movement, and heart rate, which were measured as stress indicators, decreased significantly, indicating adaptation to the restraint protocol. Additionally, 10 days after the awake MRI session, rats exhibited no signs of depression or anxiety based on open-field and sucrose preference behavioral tests. The RSFC data revealed significant changes in the thalamo-cortical and cortico-cortical networks between the awake, lightly sedated, and anesthetized groups, emphasizing the need for awake imaging. The present work demonstrates the feasibility of our custom-made 3D printed restraint kit. Using this kit, we found that isoflurane markedly affected brain connectivity compared with that in awake rats, and that the effect was less pronounced, but still significant, when light isoflurane sedation was used instead.

Quantification of anisotropy and orientation in 3D electron microscopy and diffusion tensor imaging in injured rat brain.

Diffusion tensor imaging (DTI) reveals microstructural features of grey and white matter non-invasively. The contrast produced by DTI, however, is not fully understood and requires further validation. We used serial block-face scanning electron microscopy (SBEM) to acquire tissue metrics, i.e., anisotropy and orientation, using three-dimensional Fourier transform-based (3D-FT) analysis, to correlate with fractional anisotropy and orientation in DTI. SBEM produces high-resolution 3D data at the mesoscopic scale with good contrast of cellular membranes. We analysed selected samples from cingulum, corpus callosum, and perilesional cortex of sham-operated and traumatic brain injury (TBI) rats. Principal orientations produced by DTI and 3D-FT in all samples were in good agreement. Anisotropy values showed similar patterns of change in corresponding DTI and 3D-FT parameters in sham-operated and TBI rats. While DTI and 3D-FT anisotropy values were similar in grey matter, 3D-FT anisotropy values were consistently lower than fractional anisotropy values from DTI in white matter. We also evaluated the effect of resolution in 3D-FT analysis. Despite small angular differences in grey matter samples, lower resolution datasets provided reliable results, allowing for analysis of larger fields of view. Overall, 3D SBEM allows for more sophisticated validation studies of diffusion imaging contrast from a tissue microstructural perspective.

Functional connectivity under six anesthesia protocols and the awake condition in rat brain.

Resting-state functional magnetic resonance imaging (rsfMRI) is a translational imaging method with great potential in several neurobiologic applications. Most preclinical rsfMRI studies are performed in anesthetized animals, but the confounding effects of anesthesia on the measured functional connectivity (FC) are poorly understood. Therefore, we measured FC under six commonly used anesthesia protocols and compared the findings with data obtained from awake rats. The results demonstrated that each anesthesia protocol uniquely modulated FC. Connectivity patterns obtained under propofol and urethane anesthesia were most similar to that observed in awake rats. FC patterns in the α-chloralose and isoflurane-medetomidine combination groups had moderate to good correspondence with that in the awake group. The FC patterns in the isoflurane and medetomidine groups differed most from that in the awake rats. These results can be directly exploited in rsfMRI study designs to improve the data quality, comparability, and interpretation.