A Novel In Vivo Model for Multiplexed Analysis of Callosal Connections upon Cortical Damage.

Brain damage is the major cause of permanent disability and it is particularly relevant in the elderly. While most studies focused on the immediate phase of neuronal loss upon injury, much less is known about the process of axonal regeneration after damage. The development of new refined preclinical models to investigate neuronal regeneration and the recovery of brain tissue upon injury is a major unmet challenge. Here, we present a novel experimental paradigm in mice that entails the (i) tracing of cortico-callosal connections, (ii) a mechanical lesion of the motor cortex, (iii) the stereological and histological analysis of the damaged tissue, and (iv) the functional characterization of motor deficits. By combining conventional microscopy with semi-automated 3D reconstruction, this approach allows the analysis of fine subcellular structures, such as axonal terminals, with the tridimensional overview of the connectivity and tissue integrity around the lesioned area. Since this 3D reconstruction is performed in serial sections, multiple labeling can be performed by combining diverse histological markers. We provide an example of how this methodology can be used to study cellular interactions. Namely, we show the correlation between active microglial cells and the perineuronal nets that envelop parvalbumin interneurons. In conclusion, this novel experimental paradigm will contribute to a better understanding of the molecular and cellular interactions underpinning the process of cortical regeneration upon brain damage.

Susceptibility of subregions of prefrontal cortex and corpus callosum to damage by high-dose oxytocin-induced labor in male neonatal mice.

Induction and augmentation of labor is one of the most common obstetrical interventions. However, this intervention is not free of risks and could cause adverse events, such as hyperactive uterine contraction, uterine rupture, and amniotic-fluid embolism. Our previous study using a new animal model showed that labor induced with high-dose oxytocin (OXT) in pregnant mice resulted in massive cell death in selective brain regions, specifically in male offspring. The affected brain regions included the prefrontal cortex (PFC), but a detailed study in the PFC subregions has not been performed. In this study, we induced labor in mice using high-dose OXT and investigated neonatal brain damage in detail in the PFC using light and electron microscopy. We found that TUNEL-positive or pyknotic nuclei and Iba-1-positive microglial cells were detected more abundantly in infralimbic (IL) and prelimbic (PL) cortex of the ventromedial PFC (vmPFC) in male pups delivered by OXT-induced labor than in the control male pups. These Iba-1-positive microglial cells were engulfing dying cells. Additionally, we also noticed that in the forceps minor (FMI) of the corpus callosum (CC), the number of TUNEL-positive or pyknotic nuclei and Iba-1-positive microglial cells were largely increased and Iba-1-positive microglial cells phagocytosed massive dying cells in male pups delivered by high-dose OXT-induced labor. In conclusion, IL and PL of the vmPFC and FMI of the CC, were susceptible to brain damage in male neonates after high-dose OXT-induced labor.

Characterisation of early ultrastructural changes in the cerebral white matter of CADASIL small vessel disease using high-pressure freezing/freeze-substitution.

AIMS: The objective of this study was to elucidate the early white matter changes in CADASIL small vessel disease. METHODS: We used high-pressure freezing and freeze substitution (HPF/FS) in combination with high-resolution electron microscopy (EM), immunohistochemistry and confocal microscopy of brain specimens from control and CADASIL (TgNotch3R169C ) mice aged 4-15 months to study white matter lesions in the corpus callosum. RESULTS: We first optimised the HPF/FS protocol in which samples were chemically prefixed, frozen in a sample carrier filled with 20% polyvinylpyrrolidone and freeze-substituted in a cocktail of tannic acid, osmium tetroxide and uranyl acetate dissolved in acetone. EM analysis showed that CADASIL mice exhibit significant splitting of myelin layers and enlargement of the inner tongue of small calibre axons from the age of 6 months, then vesiculation of the inner tongue and myelin sheath thinning at 15 months of age. Immunohistochemistry revealed an increased number of oligodendrocyte precursor cells, although only in older mice, but no reduction in the number of mature oligodendrocytes at any age. The number of Iba1 positive microglial cells was increased in older but not in younger CADASIL mice, but the number of activated microglial cells (Iba1 and CD68 positive) was unchanged at any age. CONCLUSION: We conclude that early WM lesions in CADASIL affect first and foremost the myelin sheath and the inner tongue, suggestive of a primary myelin injury. We propose that those defects are consistent with a hypoxic/ischaemic mechanism.

Machine learning based white matter models with permeability: An experimental study in cuprizone treated in-vivo mouse model of axonal demyelination.

The intra-axonal water exchange time (τi), a parameter associated with axonal permeability, could be an important biomarker for understanding and treating demyelinating pathologies such as Multiple Sclerosis. Diffusion-Weighted MRI (DW-MRI) is sensitive to changes in permeability; however, the parameter has so far remained elusive due to the lack of general biophysical models that incorporate it. Machine learning based computational models can potentially be used to estimate such parameters. Recently, for the first time, a theoretical framework using a random forest (RF) regressor suggests that this is a promising new approach for permeability estimation. In this study, we adopt such an approach and for the first time experimentally investigate it for demyelinating pathologies through direct comparison with histology. We construct a computational model using Monte Carlo simulations and an RF regressor in order to learn a mapping between features derived from DW-MRI signals and ground truth microstructure parameters. We test our model in simulations, and find strong correlations between the predicted and ground truth parameters (intra-axonal volume fraction f: R2 =0.99, τi: R2 =0.84, intrinsic diffusivity d: R2 =0.99). We then apply the model in-vivo, on a controlled cuprizone (CPZ) mouse model of demyelination, comparing the results from two cohorts of mice, CPZ (N=8) and healthy age-matched wild-type (WT, N=8). We find that the RF model estimates sensible microstructure parameters for both groups, matching values found in literature. Furthermore, we perform histology for both groups using electron microscopy (EM), measuring the thickness of the myelin sheath as a surrogate for exchange time. Histology results show that our RF model estimates are very strongly correlated with the EM measurements (ρ = 0.98 for f, ρ = 0.82 for τi). Finally, we find a statistically significant decrease in τi in all three regions of the corpus callosum (splenium/genu/body) of the CPZ cohort (<τi>=310ms/330ms/350ms) compared to the WT group (<τi>=370ms/370ms/380ms). This is in line with our expectations that τi is lower in regions where the myelin sheath is damaged, as axonal membranes become more permeable. Overall, these results demonstrate, for the first time experimentally and in vivo, that a computational model learned from simulations can reliably estimate microstructure parameters, including the axonal permeability .

The effect of microglial ablation and mesenchymal stem cell transplantation on a cuprizone-induced demyelination model.

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system with symptoms such as neuroinflammation, astrocytosis, microgliosis, and axonal degeneration. Mesenchymal stem cells (MSCs) with their immunomodulation, differentiation, and neuroprotection abilities can influence the remyelination process. The goal of this study is to investigate the impact of microglial ablation and MSCs transplantation on remyelination processes in the corpus callosum (CC) of the cuprizone demyelination model. For the induction of a chronic demyelination model, C57BL6 mice were fed with chow containing 0.2% cuprizone (wt/wt) for 12 weeks. For the depletion of microglia, PLX3397 was used as a colony-stimulating factor 1 receptor inhibitor for 21 days. MSCs were injected to the right lateral ventricle and after 2 weeks, the mice were killed. We assessed glial cells using specific markers such as APC, Iba-1, and GFAP using the immunohistochemistry method. Remyelination was evaluated by Luxol fast blue (LFB) staining and transmission electron microscope (TEM). The specific genes of microglia and MSCs were evaluated by a quantitative real-time polymerase chain reaction. According to the results of the study, 21 days of PLX3397 treatment significantly reduced microglial cells, and MSCs transplantation decreased the number of astrocytes, whereas the oligodendrocytes population increased significantly in PLX + MSC group in comparison with the cuprizone mice. Furthermore, PLX and MSC treatment elevated levels of remyelination compared with the cuprizone group, as confirmed by LFB staining and TEM analysis. The molecular results showed that MSC transplantation significantly decreased the number of microglia through the CX3CL1/CX3CR1 axis. These results revealed that PLX3397 treatment and MSCs injection reduced microgliosis and astrocytosis. It also increased the oligodendrocytes population by enhancing remyelination in the CC of the cuprizone model of MS.

Microstructural changes in the brain after long-term post-concussion symptoms: A randomized trial.

A recent randomized controlled trial in young patients with long-term post-concussion symptoms showed that a novel behavioral intervention "Get going After concussIoN" is superior to enhanced usual care in terms of symptom reduction. It is unknown whether these interventional effects are associated with microstructural brain changes. The aim of this study was to examine whether diffusion-weighted MRI indices, which are sensitive to the interactions between cellular structures and water molecules' Brownian motion, respond differently to the interventions of the above-mentioned trial and whether such differences correlate with the improvement of post-concussion symptoms. Twenty-three patients from the intervention group (mean age 22.8, 18 females) and 19 patients from the control group (enhanced usual care) (mean age 23.9, 14 females) were enrolled. The primary outcome measure was the mean kurtosis tensor, which is sensitive to the microscopic complexity of brain tissue. The mean kurtosis tensor was significantly increased in the intervention group (p = 0.003) in the corpus callosum but not in the thalamus (p = 0.78) and the hippocampus (p = 0.34). An increase in mean kurtosis tensor in the corpus callosum tended to be associated with a reduction in symptoms, but this association did not reach significance (p = 0.059). Changes in diffusion tensor imaging metrics did not differ between intervention groups and were not associated with symptoms. The current study found different diffusion-weighted MRI responses from the microscopic cellular structures of the corpus callosum between patients receiving a novel behavioral intervention and patients receiving enhanced usual care. Correlations with improvement of post-concussion symptoms were not evident.

Temporal Changes in In Vivo Glutamate Signal during Demyelination and Remyelination in the Corpus Callosum: A Glutamate-Weighted Chemical Exchange Saturation Transfer Imaging Study.

BACKGROUND: Glutamate-weighted chemical exchange saturation transfer (GluCEST) is a useful imaging tool that can be used to detect changes in glutamate levels in vivo and could also be helpful in the diagnosis of brain myelin changes. We investigated glutamate level changes in the cerebral white matter of a rat model of cuprizone-administered demyelination and remyelination using GluCEST. METHOD: We used a 7 T pre-clinical magnetic resonance imaging (MRI) system. The rats were divided into the normal control (CTRL), cuprizone-administered demyelination (CPZDM), and remyelination (CPZRM) groups. GluCEST data were analyzed using the conventional magnetization transfer ratio asymmetry in the corpus callosum. Immunohistochemistry and transmission electron microscopy analyses were also performed to investigate the myelinated axon changes in each group. RESULTS: The quantified GluCEST signals differed significantly between the CPZDM and CTRL groups (-7.25 ± 1.42% vs. -2.84 ± 1.30%; p = 0.001). The increased GluCEST signals in the CPZDM group decreased after remyelination (-6.52 ± 1.95% in CPZRM) to levels that did not differ significantly from those in the CTRL group (p = 0.734). CONCLUSION: The apparent temporal signal changes in GluCEST imaging during demyelination and remyelination demonstrated the potential usefulness of GluCEST imaging as a tool to monitor the myelination process.

A time-dependent diffusion MRI signature of axon caliber variations and beading.

MRI provides a unique non-invasive window into the brain, yet is limited to millimeter resolution, orders of magnitude coarser than cell dimensions. Here, we show that diffusion MRI is sensitive to the micrometer-scale variations in axon caliber or pathological beading, by identifying a signature power-law diffusion time-dependence of the along-fiber diffusion coefficient. We observe this signature in human brain white matter and identify its origins by Monte Carlo simulations in realistic substrates from 3-dimensional electron microscopy of mouse corpus callosum. Simulations reveal that the time-dependence originates from axon caliber variation, rather than from mitochondria or axonal undulations. We report a decreased amplitude of time-dependence in multiple sclerosis lesions, illustrating the potential sensitivity of our method to axonal beading in a plethora of neurodegenerative disorders. This specificity to microstructure offers an exciting possibility of bridging across scales to image cellular-level pathology with a clinically feasible MRI technique.

Brain microstructure and morphology of very preterm-born infants at term equivalent age: Associations with motor and cognitive outcomes at 1 and 2 years.

Very preterm-born infants are at risk of adverse neurodevelopmental outcomes. Brain magnetic resonance imaging (MRI) at term equivalent age (TEA) can probe tissue microstructure and morphology, and demonstrates potential in the early prediction of outcomes. In this study, we use the recently introduced fixel-based analysis method for diffusion MRI to investigate the association between microstructure and morphology at TEA, and motor and cognitive development at 1 and 2 years corrected age (CA). Eighty infants born <31 weeks' gestation successfully underwent diffusion MRI (3T; 64 directions; b â€‹= â€‹2000s/mm2) at term equivalent age, and had neurodevelopmental follow-up using the Bayley-III motor and cognitive assessments at 1 year (n â€‹= â€‹78) and/or 2 years (n â€‹= â€‹76) CA. Diffusion MRI data were processed using constrained spherical deconvolution (CSD) and aligned to a study-specific fibre orientation distribution template, yielding measures of fibre density (FD), fibre-bundle cross-section (FC), and fibre density and bundle cross-section (FDC). The association between FD, FC, and FDC at TEA, and motor and cognitive composite scores at 1 and 2 years CA, and change in composite scores from 1 to 2 years, was assessed using whole-brain fixel-based analysis. Additionally, the association between diffusion tensor imaging (DTI) metrics (fractional anisotropy FA, mean diffusivity MD, axial diffusivity AD, radial diffusivity RD) and outcomes was investigated. Motor function at 1 and 2 years CA was associated with CSD-based measures of the bilateral corticospinal tracts and corpus callosum. Cognitive function was associated with CSD-based measures of the midbody (1-year outcomes only) and splenium of the corpus callosum, as well as the bilateral corticospinal tracts. The change in motor/cognitive outcomes from 1 to 2 years was associated with CSD-based measures of the splenium of the corpus callosum. Analysis of DTI-based measures showed overall less extensive associations. Post-hoc analysis showed that associations were weaker for 2-year outcomes than they were for 1-year outcomes. Infants with better neurodevelopmental outcomes demonstrated higher FD, FC, and FDC at TEA, indicating better information transfer capacity which may be related to increased number of neurons, increased myelination, thicker bundles, and/or combinations thereof. The fibre bundles identified here may serve as the basis for future studies investigating the predictive ability of these metrics.

Anatomical and ultrastructural sex differences in mean diameter and thickness of myelinated axons in adult rat corpus callosum / Diferencias sexuales anatómicas y ultraestructurales del diámetro medio y grosor de los axones mielinizados en el cuerpo calloso de ratas adultas

Sexual dimorphism exists at all levels of the nervous system. These sex differences could underlie genderrelated differences in behavior and neuropsychological function, as well as the gender differences in the prevalence of various mental disorders such as autism, attention deficit disorders, and schizophrenia. Myelination, on the other hand, is a unique cellular process that can have a dramatic impact on the structure and physiology of an axon and its surrounding tissue. The corpus callosum (CC) is the largest of the brain commissures, which connects the cerebral cortices of the two hemispheres, and provides interhemispheric connectivity for information transfer and processing between cortical regions. Variation in the axonal properties of CC will alter the interhemispheric connectivity. The CC consists of myelinated and unmyelinated axons, glial cells and blood vessels. Several functional studies have reported that the function of CC is associated with its axons density and myelination properties. The sexual dimorphism in the axonal content of the CC has always been controversial; hence, the aim of this study was to analyze the differences in axons' diameter and myelin sheath thickness of the CC between male and female rats. For this purpose, five pairs of adult male and female rats were perfused and the CC were removed and sectioned. Four sections from different subregions of the corpus callosum that represent the genu, anterior body, posterior body, and splenium of the CC were stained and electron microscopic images were captured using stereological guidelines. Later, the axons diameter and myelin sheath thickness for each subregion were calculated and compared between males and females. Our preliminary findings of the present study indicated region specific differences in the myelinated axon thickness and diameter in the CC between male and female rats.

El dimorfismo sexual existe en todos los niveles del sistema nervioso. Estas diferencias de sexo podrían ser la base de las diferencias de comportamiento y función neuropsicológica relacionadas con el sexo, así como las diferencias en la prevalencia de diversos trastornos mentales, como el autismo, los trastornos por déficit de atención y la esquizofrenia. La mielinización, por otro lado, es un proceso celular único que puede tener un impacto dramático en la estructura y fisiología de un axón y su tejido circundante. El cuerpo calloso (CC) es la mayor comisura cerebral, que conecta las cortezas cerebrales de ambos hemisferios, y proporciona la conectividad interhemisférica para la transferencia y el procesamiento de información entre regiones corticales. La variación en las propiedades axonales de CC alterará la conectividad interhemisférica. El CC consiste en axones mielinizados y no mielinizados, células gliales y vasos sanguíneos. Varios estudios funcionales han informado que la función de CC está asociada con la densidad de axones y las propiedades de mielinización. El dimorfismo sexual en el contenido axonal del CC siempre ha sido controvertido; por lo tanto, el objetivo de este estudio fue analizar las diferencias en el diámetro de los axones y el grosor de la vaina de mielina del CC entre ratas macho y hembra. Para este propósito, se perfundieron cinco pares de ratas macho y hembra adultas y se extrajeron y seccionaron las CC. Se tiñeron cuatro secciones de diferentes subregiones del cuerpo calloso que representan el genu, el cuerpo anterior, el cuerpo posterior y el esplenio y se capturaron imágenes de microscopía electrónicas utilizando referencias estereológicas. Posteriormente se calculó el diámetro de los axones y el grosor de la vaina de mielina para cada subregión y se compararon entre machos y hembras. Nuestros hallazgos preliminares del presente estudio indicaron diferencias específicas en el grosor y diámetro del axón mielinizado en el CC entre ratas macho y hembra.

Twenty-first birthday drinking: Extreme-drinking episodes and white matter microstructural changes in the fornix and corpus callosum.

The 21st birthday celebration is characterized by extreme alcohol consumption. Accumulating evidence suggests that high-dose bingeing is related to structural brain changes and cognitive deficits. This is particularly problematic in the transition from adolescence to adulthood when the brain is still maturing, elevating the brain's sensitivity to the acute effects of alcohol intoxication. Heavy drinking is associated with reduced structural integrity in the hippocampus and corpus callosum and is accompanied by cognitive deficits. However, there is little research examining changes in the human brain related to discrete heavy-drinking episodes. The present study investigated whether alcohol exposure during a 21st birthday celebration would result in changes to white matter microstructure by utilizing diffusion tensor imaging measures and a quasi-experimental design. By examining structural changes in the brain from pre- to postcelebration within subjects (N = 49) prospectively, we were able to more directly observe brain changes following an extreme-drinking episode. Region of interest analyses demonstrated increased fractional anisotropy in the posterior fornix (p < .0001) and in the body of the corpus callosum (p = .0029) from pre- to postbirthday celebration. These results suggest acute white matter damage to the fornix and corpus callosum following an extreme-drinking episode, which is especially problematic during continued neurodevelopment. Therefore, 21st birthday drinking may be considered an important target event for preventing acute brain injury in young adults. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Spatio-temporal dynamics of neocortical presynaptic terminal development using multi-photon imaging of the corpus callosum in vivo.

Within the developing central nervous system, the dynamics of synapse formation and elimination are insufficiently understood. It is ideal to study these processes in vivo, where neurons form synapses within appropriate behavioral and anatomical contexts. In vivo analysis is particularly important for long-range connections, since their development cannot be adequately studied in vitro. The corpus callosum (CC) represents a clinically-relevant long-range connection since several neurodevelopmental diseases involve CC defects. Here, we present a novel strategy for in vivo longitudinal and rapid time-lapse imaging of CC presynaptic terminal development. In postnatal mice, the time-course of CC presynaptic terminal formation and elimination was highly variable between axons or groups of axons. Young presynaptic terminals were remarkably dynamic - moving, dividing to generate more boutons, and merging to consolidate small terminals into large boutons. As synaptic networks matured, presynaptic mobility decreased. These rapid dynamics may be important for establishing initial synaptic contacts with postsynaptic partners, refining connectivity patterns or modifying synapse strength during development. Ultimately, this in vivo imaging approach will facilitate investigation of synapse development in other long-range connections and neurodevelopmental disease models.

Gender differences in the rat corpus callosum: An ultrastructure study.

Sexual dimorphism exists at all levels of the nervous system, from genetic, anatomical and system levels. The sexual dimorphism in the axonal content of the corpus callosum (CC) has always been controversial; hence, the aim of this study was to analyse the differences in total, myelinated and unmyelinated axons density of various regions of the CC between male and female rats. To assess that, six pairs of adult male and female rats were perfused and the CC was removed and sectioned. Four sections from different subregions of the corpus callosum that represent the genu, anterior body, posterior body, and splenium, were stained, and electron microscopic images were captured using stereological guidelines. Later, the axons density for each subregion was calculated and compared between males and females. The findings of the present study indicated region-specific differences in the myelinated, unmyelinated or the ratio of myelinated/total axons in the CC between male and female rats.

Distinctive functional deficiencies in axonal conduction associated with two forms of cerebral white matter injury.

AIMS: This study determines whether assessment with compound action potentials (CAPs) can distinguish two different forms of cerebral white matter injury at the functional levels. METHODS: A pure demyelination model was induced in C57/BL6 adult mice by dietary supplementation of cuprizone (0.2%) for 6 weeks. Callosal L-N5-(1-Iminoethyl) ornithine (L-NIO) hydrochloride (27 mg/mL) was injected into the corpus callosum (CC) to induce a focal white matter stroke (WMS), resulting in both demyelination and axonal injury. White matter integrity was assessed by performing CAP recording, electron microscopy, and immunohistological and luxol fast blue (LFB) staining. RESULTS: Immunohistological and electron microscopic analyses confirmed the induction of robust demyelination in CC with cuprizone, and mixed demyelination and axonal damage with L-NIO. Electrophysiologically, cuprizone-induced demyelination significantly reduced the amplitude of negative peak 1 (N1), but increased the amplitude of negative peak 2 (N2), of the CAPs compared to the sham controls. However, cuprizone did not affect the axonal conduction velocity. In contrast, the amplitude and area of both N1 and N2 along with N1 axonal conduction velocity were dramatically decreased in L-NIO-induced WMS. CONCLUSIONS: Concertedly, parameters of the CAPs offer a novel functional assessment strategy for cerebral white matter injury in rodent models.

Mesenchymal stem cell mediated effects on microglial phenotype in cuprizone-induced demyelination model.

Microglial cells have an essential role in neurodegenerative disorders, such as multiple sclerosis. They are divided into two subgroups: M1 and M2 phenotypes. Mesenchymal stem cells (MSC), with neuroprotective and immunomodulating properties, could improve these diseases. We evaluate the immunomodulating effects of MSC on microglial phenotypes and the improvement of demyelination in a cuprizone (CPZ) model of multiple sclerosis (MS). For inducing the chronic demyelination model, C57BL6 mice were given a diet with 0.2% CPZ (w/w) for 12 weeks. In the MSC group, cells were transplanted into the right lateral ventricle of mice. The expression of targeted genes was assessed by real-time polymerase chain reaction. M1 and M2 microglial phenotypes were assessed by immunohistochemistry of inducible nitric oxide synthase (iNOS) and Arg-1, respectively. Remyelination was studied by luxal fast blue (LFB) staining and electron microscopy (EM). We found that MSC transplantation reduced the expression level of M1-specific messenger RNA (mRNA; iNOS and CD86) but increased the expression level of M2 specific genes (CD206, Arg-1, and CX3CR1) in comparison to the CPZ group. Moreover, cell therapy significantly decreased the M1 marker (iNOS+ cells), but M2 marker (Arg-1+ cells) significantly increased in comparison with the CPZ group. In addition, MSC treatment significantly increased the CX3CL1 expression level in comparison with the CPZ group and led to improvement in remyelination, which was confirmed by LFB and EM images. The results showed that MSC transplantation increases the M2 and decreases the M1 phenotype in MS. This change was accompanied by decrease in demyelination and axonal injury and indicated that MSCs have a positive effect on MS by modification of microglia cells.

Substance abuse and white matter: Findings, limitations, and future of diffusion tensor imaging research.

Individuals who abuse substances often differ from nonusers in their brain structure. Substance abuse and addiction is often associated with atrophy and pathology of grey matter, but much less is known about the role of white matter, which constitutes over half of human brain volume. Diffusion tensor imaging (DTI), a method for non-invasively estimating white matter, is increasingly being used to study addiction and substance abuse. Here we review recent DTI studies of major substances of abuse (alcohol, opiates, cocaine, cannabis, and nicotine substance abuse) to examine the relationship, specificity, causality, and permanence of substance-related differences in white matter microstructure. Across substance, users tended to exhibit differences in the microstructure of major fiber pathways, such as the corpus callosum. The direction of these differences, however, appeared substance-dependent. The subsample of longitudinal studies reviewed suggests that substance abuse may cause changes in white matter, though it is unclear to what extent such alterations are permanent. While collectively informative, some studies reviewed were limited by methodological and technical approach. We therefore also provide methodological guidance for future research using DTI to study substance abuse.

Along-axon diameter variation and axonal orientation dispersion revealed with 3D electron microscopy: implications for quantifying brain white matter microstructure with histology and diffusion MRI.

Tissue microstructure modeling of diffusion MRI signal is an active research area striving to bridge the gap between macroscopic MRI resolution and cellular-level tissue architecture. Such modeling in neuronal tissue relies on a number of assumptions about the microstructural features of axonal fiber bundles, such as the axonal shape (e.g., perfect cylinders) and the fiber orientation dispersion. However, these assumptions have not yet been validated by sufficiently high-resolution 3-dimensional histology. Here, we reconstructed sequential scanning electron microscopy images in mouse brain corpus callosum, and introduced a random-walker (RaW)-based algorithm to rapidly segment individual intra-axonal spaces and myelin sheaths of myelinated axons. Confirmed by a segmentation based on human annotations initiated with conventional machine-learning-based carving, our semi-automatic algorithm is reliable and less time-consuming. Based on the segmentation, we calculated MRI-relevant estimates of size-related parameters (inner axonal diameter, its distribution, along-axon variation, and myelin g-ratio), and orientation-related parameters (fiber orientation distribution and its rotational invariants; dispersion angle). The reported dispersion angle is consistent with previous 2-dimensional histology studies and diffusion MRI measurements, while the reported diameter exceeds those in other mouse brain studies. Furthermore, we calculated how these quantities would evolve in actual diffusion MRI experiments as a function of diffusion time, thereby providing a coarse-graining window on the microstructure, and showed that the orientation-related metrics have negligible diffusion time-dependence over clinical and pre-clinical diffusion time ranges. However, the MRI-measured inner axonal diameters, dominated by the widest cross sections, effectively decrease with diffusion time by ~ 17% due to the coarse-graining over axonal caliber variations. Furthermore, our 3d measurement showed that there is significant variation of the diameter along the axon. Hence, fiber orientation dispersion estimated from MRI should be relatively stable, while the "apparent" inner axonal diameters are sensitive to experimental settings, and cannot be modeled by perfectly cylindrical axons.

Cuprizone-induced graded oligodendrocyte vulnerability is regulated by the transcription factor DNA damage-inducible transcript 3.

Oligodendrocytes are integral to efficient neuronal signaling. Loss of myelinating oligodendrocytes is a central feature of many neurological diseases, including multiple sclerosis (MS). The results of neuropathological studies suggest that oligodendrocytes react with differing sensitivity to toxic insults, with some cells dying early during lesion development and some cells being resistant for weeks. This proposed graded vulnerability has never been demonstrated but provides an attractive window for therapeutic interventions. Furthermore, the biochemical pathways associated with graded oligodendrocyte vulnerability have not been well explored. We used immunohistochemistry and serial block-face scanning electron microscopy (3D-SEM) to show that cuprizone-induced metabolic stress results in an "out of phase" degeneration of oligodendrocytes. Although expression induction of stress response transcription factors in oligodendrocytes occurs within days, subsequent oligodendrocyte apoptosis continues for weeks. In line with the idea of an out of phase degeneration of oligodendrocytes, detailed ultrastructural reconstructions of the axon-myelin unit demonstrate demyelination of single internodes. In parallel, genome wide array analyses revealed an active unfolded protein response early after initiation of the cuprizone intoxication. In addition to the cytoprotective pathways, the pro-apoptotic transcription factor DNA damage-inducible transcript 3 (DDIT3) was induced early in oligodendrocytes. In advanced lesions, DDIT3 was as well expressed by activated astrocytes. Toxin-induced oligodendrocyte apoptosis, demyelination, microgliosis, astrocytosis, and acute axonal damage were less intense in the Ddit3-null mutants. This study identifies DDIT3 as an important regulator of graded oligodendrocyte vulnerability in a MS animal model. Interference with this stress cascade might offer a promising therapeutic approach for demyelinating disorders.

Defining Changes in the Spatial Distribution and Composition of Brain Lipids in the Shiverer and Cuprizone Mouse Models of Myelin Disease.

Myelin is composed primarily of lipids and diseases affecting myelin are associated with alterations in its lipid composition. However, correlation of the spatial (in situ) distribution of lipids with the disease-associated compositional and morphological changes is not well defined. Herein we applied high resolution matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS), immunohistochemistry (IHC), and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) to evaluate brain lipid alterations in the dysmyelinating shiverer (Shi) mouse and cuprizone (Cz) mouse model of reversible demyelination. MALDI-IMS revealed a decrease in the spatial distribution of sulfatide (SHexCer) species, SHexCer (d42:2), and a phosphatidylcholine (PC) species, PC (36:1), in white matter regions like corpus callosum (CC) both in the Shi mouse and Cz mouse model. Changes in these lipid species were restored albeit not entirely upon spontaneous remyelination after demyelination in the Cz mouse model. Lipid distribution changes correlated with the local morphological changes as confirmed by IHC. LC-ESI-MS analyses of CC extracts confirmed the MALDI-IMS derived reductions in SHexCer and PC species. These findings highlight the role of SHexCer and PC in preserving the normal myelin architecture and our experimental approaches provide a morphological basis to define lipid abnormalities relevant to myelin diseases.

Psychological stress effects on myelin degradation in the cuprizone-induced model of demyelination.

Multiple sclerosis (MS) is known as the most common demyelinating disease worldwide in which previous studies have shown that stress is a risk factor for the disease's onset and progression. Nevertheless, further studies are needed to investigate the consequences of stress in MS pathology. In this study, after 5 days of exposure to psychological and physical stress as a repetitive distress modality, rats were treated with cuprizone. The demyelination degree was compared in animal groups using Luxol fast blue staining, immunohistochemical staining for myelin basic protein and transmission electron microscopy. Outcomes revealed that animals exposed to stress prior to cuprizone ingestion, elicit more intense demyelination. Continuous psychological distress has more severe effects on myelin sheath destruction in the preclinical stage.