Sex chromosomes and hormones independently influence healthy brain development but act similarly after cranial radiation.

The course of normal development and response to pathology are strongly influenced by biological sex. For instance, female childhood cancer survivors who have undergone cranial radiation therapy (CRT) tend to display more pronounced cognitive deficits than their male counterparts. Sex effects can be the result of sex chromosome complement (XX vs. XY) and/or gonadal hormone influence. The contributions of each can be separated using the four-core genotype mouse model (FCG), where sex chromosome complement and gonadal sex are decoupled. While studies of FCG mice have evaluated brain differences in adulthood, it is still unclear how sex chromosome and sex hormone effects emerge through development in both healthy and pathological contexts. Our study utilizes longitudinal MRI with the FCG model to investigate sex effects in healthy development and after CRT in wildtype and immune-modified Ccl2-knockout mice. Our findings in normally developing mice reveal a relatively prominent chromosome effect prepubertally, compared to sex hormone effects which largely emerge later. Spatially, sex chromosome and hormone influences were independent of one another. After CRT in Ccl2-knockout mice, both male chromosomes and male hormones similarly improved brain outcomes but did so more separately than in combination. Our findings highlight the crucial role of sex chromosomes in early development and identify roles for sex chromosomes and hormones after CRT-induced inflammation, highlighting the influences of biological sex in both normal brain development and pathology.

Differential effects of early or late exposure to prenatal maternal immune activation on mouse embryonic neurodevelopment.

Exposure to maternal immune activation (MIA) in utero is a risk factor for neurodevelopmental and psychiatric disorders. MIA-induced deficits in adolescent and adult offspring have been well characterized; however, less is known about the effects of MIA exposure on embryo development. To address this gap, we performed high-resolution ex vivo MRI to investigate the effects of early (gestational day [GD]9) and late (GD17) MIA exposure on embryo (GD18) brain structure. We identify striking neuroanatomical changes in the embryo brain, particularly in the late-exposed offspring. We further examined the putative neuroanatomical underpinnings of MIA timing in the hippocampus using electron microscopy and identified differential effects due to MIA timing. An increase in apoptotic cell density was observed in the GD9-exposed offspring, while an increase in the density of neurons and glia with ultrastructural features reflective of increased neuroinflammation and oxidative stress was observed in GD17-exposed offspring, particularly in females. Overall, our findings integrate imaging techniques across different scales to identify differential impact of MIA timing on the earliest stages of neurodevelopment.

TDP-43-M323K causes abnormal brain development and progressive cognitive and motor deficits associated with mislocalised and increased levels of TDP-43.

TDP-43 pathology is found in several neurodegenerative disorders, collectively referred to as "TDP-43 proteinopathies". Aggregates of TDP-43 are present in the brains and spinal cords of >97% of amyotrophic lateral sclerosis (ALS), and in brains of ∼50% of frontotemporal dementia (FTD) patients. While mutations in the TDP-43 gene (TARDBP) are usually associated with ALS, many clinical reports have linked these mutations to cognitive impairments and/or FTD, but also to other neurodegenerative disorders including Parkinsonism (PD) or progressive supranuclear palsy (PSP). TDP-43 is a ubiquitously expressed, highly conserved RNA-binding protein that is involved in many cellular processes, mainly RNA metabolism. To investigate systemic pathological mechanisms in TDP-43 proteinopathies, aiming to capture the pleiotropic effects of TDP-43 mutations, we have further characterised a mouse model carrying a point mutation (M323K) within the endogenous Tardbp gene. Homozygous mutant mice developed cognitive and behavioural deficits as early as 3 months of age. This was coupled with significant brain structural abnormalities, mainly in the cortex, hippocampus, and white matter fibres, together with progressive cortical interneuron degeneration and neuroinflammation. At the motor level, progressive phenotypes appeared around 6 months of age. Thus, cognitive phenotypes appeared to be of a developmental origin with a mild associated progressive neurodegeneration, while the motor and neuromuscular phenotypes seemed neurodegenerative, underlined by a progressive loss of upper and lower motor neurons as well as distal denervation. This is accompanied by progressive elevated TDP-43 protein and mRNA levels in cortex and spinal cord of homozygous mutant mice from 3 months of age, together with increased cytoplasmic TDP-43 mislocalisation in cortex, hippocampus, hypothalamus, and spinal cord at 12 months of age. In conclusion, we find that Tardbp M323K homozygous mutant mice model many aspects of human TDP-43 proteinopathies, evidencing a dual role for TDP-43 in brain morphogenesis as well as in the maintenance of the motor system, making them an ideal in vivo model system to study the complex biology of TDP-43.

Mouse models of immune dysfunction: their neuroanatomical differences reflect their anxiety-behavioural phenotype.

Extensive evidence supports the role of the immune system in modulating brain function and behaviour. However, past studies have revealed striking heterogeneity in behavioural phenotypes produced from immune system dysfunction. Using magnetic resonance imaging, we studied the neuroanatomical differences among 11 distinct genetically modified mouse lines (n = 371), each deficient in a different element of the immune system. We found a significant and heterogeneous effect of immune dysfunction on the brains of both male and female mice. However, by imaging the whole brain and using Bayesian hierarchical modelling, we were able to identify patterns within the heterogeneous phenotype. Certain structures-such as the corpus callosum, midbrain, and thalamus-were more likely to be affected by immune dysfunction. A notable brain-behaviour relationship was identified with neuroanatomy endophenotypes across mouse models clustering according to anxiety-like behaviour phenotypes reported in literature, such as altered volume in brains regions associated with promoting fear response (e.g., the lateral septum and cerebellum). Interestingly, genes with preferential spatial expression in the most commonly affected regions are also associated with multiple sclerosis and other immune-mediated diseases. In total, our data suggest that the immune system modulates anxiety behaviour through well-established brain networks.

P63 expression plays a role in developmental rate, embryo size, and local morphogenesis.

BACKGROUND: The p63 gene is integral to the development of many body parts including limb, palate, teeth, and urogenital tract. Loss of p63 expression may alter developmental rate, which is crucial to normal morphogenesis. To validate a novel, unbiased embryo phenotyping software tool, we tested whether delayed development contributes to the pathological phenotype of a p63 mouse mutant (p63-/- ). We quantified dysmorphology in p63-/- embryos and tested for universal growth delay relative to wild-type (WT) embryos. Fixed embryos (n = 6; p63-/- ) aged day (E) 15.5 were micro-CT scanned and quantitatively analyzed using a digital WT atlas that defined volumetric differences between p63-/- and WT embryos. RESULTS: p63-/- embryos showed a growth delay of approximately 22 hr (0.9 days). Among the E15.5 mutants, overall size was closest to WT E14.6 mice but shape was closest to WT E14.0. The atlas clearly identified in p63-/- embryos malformations of epithelial derivatives including limbs, tail, urogenital structures, brain, face, and tooth. CONCLUSIONS: The software atlas technique described the p63-/- phenotype as a combination of developmental delay (i.e., heterochrony) and malformation (i.e., pathological shape; failed organogenesis). This study identifies for the first time global and local roles for p63 in prenatal growth and development. Developmental Dynamics 247:779-787, 2018. © 2018 Wiley Periodicals, Inc.

Impact of X/Y genes and sex hormones on mouse neuroanatomy.

Biological sex influences brain anatomy across many species. Sex differences in brain anatomy have classically been attributed to differences in sex chromosome complement (XX versus XY) and/or in levels of gonadal sex steroids released from ovaries and testes. Using the four core genotype (4CG) mouse model in which gonadal sex and sex chromosome complement are decoupled, we previously found that sex hormones and chromosomes influence the volume of distinct brain regions. However, recent studies suggest there may be more complex interactions between hormones and chromosomes, and that circulating steroids can compensate for and/or mask underlying chromosomal effects. Moreover, the impact of pre vs post-pubertal sex hormone exposure on this sex hormone/sex chromosome interplay is not well understood. Thus, we used whole brain high-resolution ex-vivo MRI of intact and pre-pubertally gonadectomized 4CG mice to investigate two questions: 1) Do circulating steroids mask sex differences in brain anatomy driven by sex chromosome complement? And 2) What is the contribution of pre- versus post-pubertal hormones to sex-hormone-dependent differences in brain anatomy? We found evidence of both cooperative and compensatory interactions between sex chromosomes and sex hormones in several brain regions, but the interaction effects were of low magnitude. Additionally, most brain regions affected by sex hormones were sensitive to both pre- and post-pubertal hormones. This data provides further insight into the biological origins of sex differences in brain anatomy.

4D atlas of the mouse embryo for precise morphological staging.

After more than a century of research, the mouse remains the gold-standard model system, for it recapitulates human development and disease and is quickly and highly tractable to genetic manipulations. Fundamental to the power and success of using a mouse model is the ability to stage embryonic mouse development accurately. Past staging systems were limited by the technologies of the day, such that only surface features, visible with a light microscope, could be recognized and used to define stages. With the advent of high-throughput 3D imaging tools that capture embryo morphology in microscopic detail, we now present the first 4D atlas staging system for mouse embryonic development using optical projection tomography and image registration methods. By tracking 3D trajectories of every anatomical point in the mouse embryo from E11.5 to E14.0, we established the first 4D atlas compiled from ex vivo 3D mouse embryo reference images. The resulting 4D atlas comprises 51 interpolated 3D images in this gestational range, resulting in a temporal resolution of 72 min. From this 4D atlas, any mouse embryo image can be subsequently compared and staged at the global, voxel and/or structural level. Assigning an embryonic stage to each point in anatomy allows for unprecedented quantitative analysis of developmental asynchrony among different anatomical structures in the same mouse embryo. This comprehensive developmental data set offers developmental biologists a new, powerful staging system that can identify and compare differences in developmental timing in wild-type embryos and shows promise for localizing deviations in mutant development.

Magnetic resonance thermometry of flowing blood.

Blood temperature is a key determinant of tissue temperature and can be altered under normal physiological states, such as exercise, in diseases such as stroke or iatrogenically in therapies which modulate tissue temperature, such as therapeutic hypothermia. Currently available methods for the measurement of arterial and venous temperatures are invasive and, for small animal models, are impractical. Here, we present a methodology for the measurement of intravascular and tissue temperature by magnetic resonance imaging (MRI) using the lanthanide agent TmDOTMA- (DOTMA, tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; Tm, thulium). The approach makes use of phase-sensitive imaging measurements, combined with spectrally selective excitation, to monitor the temperature-dependent shift in the resonance of proton nuclei associated with water and with methyl groups of TmDOTMA- . Measurements were first made in a flow phantom modelling diastolic blood flow in the mouse aorta or inferior vena cava (IVC) and imaged using 7-T preclinical MRI with a custom-built surface coil. Flowing and static fluid temperatures agreed to within 0.12°C for these experiments. Proof-of-concept experiments were also performed on three healthy adult mice, demonstrating temperature measurements in the aorta, IVC and kidney following a bolus injection of contrast agent. A small (0.7-1°C), but statistically significant, higher kidney temperature compared with the aorta (p = 0.002-0.007) and IVC (p = 0.003-0.03) was shown in all animals. These findings demonstrate the feasibility of the technique for in vivo applications and illustrate how the technique could be used to explore the relationship between blood and tissue temperature for a wide range of applications.

Hippocampal volumes differ across the mouse estrous cycle, can change within 24 hours, and associate with cognitive strategies.

Recent human and rodent brain imaging studies have shown that the shape of the brain can be changed by experience. These mesoscopic alterations in neuroanatomy are hypothesized to be driven by changes at the level of neuronal processes. To examine whether the shape of the brain changes rapidly, we used MRI to examine changes in the volume of the hippocampus across the 4-6 day estrous cycle in the female mouse. It is well known that changing steroid levels across the cycle influence dendritic spine maturation and alter synapse density in the hippocampus; our results show that the estrous cycle is associated with approximately 2-3% changes in hippocampal volume as seen by high-resolution ex-vivo MRI. Changes in hippocampal volume are, moreover, associated with a switch between hippocampal and striatal based navigation strategies in solving the dual choice T-maze in the same mice. A second experiment, using in-vivo MRI, suggests that these changes in hippocampal volume can occur over a 24 hour period. In summary, we show that the brain is highly plastic at a mesoscopic level of resolution detectable by MRI, that volumetric increases and decreases in hippocampal volume follow previously established patterns of changes in neuropil, and that these changes in volume predict changes in cognition.

Mutation in the FUS nuclear localisation signal domain causes neurodevelopmental and systemic metabolic alterations.

Variants in the ubiquitously expressed DNA/RNA-binding protein FUS cause aggressive juvenile forms of amyotrophic lateral sclerosis (ALS). Most FUS mutation studies have focused on motor neuron degeneration; little is known about wider systemic or developmental effects. We studied pleiotropic phenotypes in a physiological knock-in mouse model carrying the pathogenic FUSDelta14 mutation in homozygosity. RNA sequencing of multiple organs aimed to identify pathways altered by the mutant protein in the systemic transcriptome, including metabolic tissues, given the link between ALS-frontotemporal dementia and altered metabolism. Few genes were commonly altered across all tissues, and most genes and pathways affected were generally tissue specific. Phenotypic assessment of mice revealed systemic metabolic alterations related to the pathway changes identified. Magnetic resonance imaging brain scans and histological characterisation revealed that homozygous FUSDelta14 brains were smaller than heterozygous and wild-type brains and displayed significant morphological alterations, including a thinner cortex, reduced neuronal number and increased gliosis, which correlated with early cognitive impairment and fatal seizures. These findings show that the disease aetiology of FUS variants can include both neurodevelopmental and systemic alterations.

Preparation of fixed mouse brains for MRI.

In fixed mouse brain magnetic resonance images, a high prevalence of fixation artifacts have been observed. Of more than 1700 images of fixed brains acquired at our laboratory, fixation artifacts were present in approximately 30%. In this study, two of these artifacts are described and their causes are identified. A hyperintense rim around the brain is observed when using perfusates reconstituted from powder and delivered at a high flow rate. It is proposed that these perfusion conditions cause blockage of the capillary beds and an increase in pressure that ruptures the vessels, resulting in a blister of liquid below the dura mater. Secondly, gray-white matter contrast inversion is observed when too short a fixation time or too low a concentration of fixative is used, resulting in inadequate fixation. The deleterious consequences of these artifacts for quantitative data analysis are discussed, and precautions for their prevention are provided.

A mouse model of Down syndrome trisomic for all human chromosome 21 syntenic regions.

Down syndrome (DS) is caused by the presence of an extra copy of human chromosome 21 (Hsa21) and is the most common genetic cause for developmental cognitive disability. The regions on Hsa21 are syntenically conserved with three regions located on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. In this report, we describe a new mouse model for DS that carries duplications spanning the entire Hsa21 syntenic regions on all three mouse chromosomes. This mouse mutant exhibits DS-related neurological defects, including impaired cognitive behaviors, reduced hippocampal long-term potentiation and hydrocephalus. These results suggest that when all the mouse orthologs of the Hsa21 genes are triplicated, an abnormal cognitively relevant phenotype is the final outcome of the elevated expressions of these orthologs as well as all the possible functional interactions among themselves and/or with other mouse genes. Because of its desirable genotype and phenotype, this mutant may have the potential to serve as one of the reference models for further understanding the developmental cognitive disability associated with DS and may also be used for developing novel therapeutic interventions for this clinical manifestation of the disorder.

Structural brain abnormalities in endothelial nitric oxide synthase-deficient mice revealed by high-resolution magnetic resonance imaging.

INTRODUCTION: Endothelial nitric oxide synthase (eNOS) produces nitric oxide, which is essential for a variety of physiological functions in the brain. Previous work has demonstrated the detrimental effects of eNOS deficiency on brain function in male eNOS knockout (eNOS KO) mice. However, the effect of eNOS deficiency on brain structure and any association between these effects and sex is unknown. METHODS: This study used three-dimensional high-resolution ex vivo magnetic resonance imaging and behavioral tests of anxiety and cognitive performance to investigate structure-function relationships in the brain of female and male eNOS KO mice in young adulthood. RESULTS: While there were no differences in anxiety-like behavior or locomotion, there was a sex-specific deficit in contextual fear memory retention in male, but not in female, eNOS mice compared to wild-type controls. Moreover, we found that eNOS deficiency induced changes in multiple brain regions that are involved in learning and fear memory including the hippocampus, amygdala, hypothalamus, and areas of the cortex. Several of these MRI-detectable neuroanatomical changes were dependent on sex. CONCLUSION: The observation that eNOS deficiency impacts brain structure at an early age demonstrates the importance of eNOS for healthy brain development.

A functional cerebral endothelium is necessary to protect against cognitive decline.

A vascular insult occurring early in disease onset may initiate cognitive decline leading to dementia, while pharmacological and lifestyle interventions can prevent this progression. Mice with a selective, tamoxifen-inducible deletion of NF-κB essential modulator (Nemo) in brain endothelial cells were studied as a model of vascular cognitive impairment. Groups included NemoFl controls and three NemobeKO groups: One untreated, and two treated with simvastatin or exercise. Social preference and nesting were impaired in NemobeKO mice and were not countered by treatments. Cerebrovascular function was compromised in NemobeKO groups regardless of treatment, with decreased changes in sensory-evoked cerebral blood flow and total hemoglobin levels, and impaired endothelium-dependent vasodilation. NemobeKO mice had increased string vessel pathology, blood-brain barrier disruption, neuroinflammation, and reduced cortical somatostatin-containing interneurons. These alterations were reversed when endothelial function was recovered. Findings strongly suggest that damage to the cerebral endothelium can trigger pathologies associated with dementia and its functional integrity should be an effective target in future therapeutic efforts.

Age-dependent gray matter demyelination is associated with leptomeningeal neutrophil accumulation.

People living with multiple sclerosis (MS) experience episodic CNS white matter lesions instigated by autoreactive T cells. With age, patients with MS show evidence of gray matter demyelination and experience devastating nonremitting symptomology. What drives progression is unclear and studying this has been hampered by the lack of suitable animal models. Here, we show that passive experimental autoimmune encephalomyelitis (EAE) induced by an adoptive transfer of young Th17 cells induced a nonremitting clinical phenotype that was associated with persistent leptomeningeal inflammation and cortical pathology in old, but not young, SJL/J mice. Although the quantity and quality of T cells did not differ in the brains of old versus young EAE mice, an increase in neutrophils and a decrease in B cells were observed in the brains of old mice. Neutrophils were also found in the leptomeninges of a subset of progressive MS patient brains that showed evidence of leptomeningeal inflammation and subpial cortical demyelination. Taken together, our data show that while Th17 cells initiate CNS inflammation, subsequent clinical symptoms and gray matter pathology are dictated by age and associated with other immune cells, such as neutrophils.

Exposure to maternal high-fat diet induces extensive changes in the brain of adult offspring.

Maternal environmental exposures, such as high-fat diets, diabetes and obesity, can induce long-term effects in offspring. These effects include increased risk of neurodevelopmental disorders (NDDs) including autism spectrum disorder (ASD), depression and anxiety. The mechanisms underlying these late-life neurologic effects are unknown. In this article, we measured changes in the offspring brain and determined which brain regions are sensitive to maternal metabolic milieu and therefore may mediate NDD risk. We showed that mice exposed to a maternal high-fat diet display extensive brain changes in adulthood despite being switched to a low-fat diet at weaning. Brain regions impacted by early-life diet include the extended amygdalar system, which plays an important role in reward-seeking behaviour. Genes preferentially expressed in these regions have functions related to feeding behaviour, while also being implicated in human NDDs, such as autism. Our data demonstrated that exposure to maternal high-fat diet in early-life leads to brain alterations that persist into adulthood, even after dietary modifications.

Subtle alterations in neonatal neurodevelopment following early or late exposure to prenatal maternal immune activation in mice.

Prenatal exposure to maternal immune activation (MIA) is a risk factor for a variety of neurodevelopmental and psychiatric disorders. The timing of MIA-exposure has been shown to affect adolescent and adult offspring neurodevelopment, however, less is known about these effects in the neonatal period. To better understand the impact of MIA-exposure on neonatal brain development in a mouse model, we assess neonate communicative abilities with the ultrasonic vocalization task, followed by high-resolution ex vivo magnetic resonance imaging (MRI) on the neonatal (postnatal day 8) mouse brain. Early exposed offspring displayed decreased communicative ability, while brain anatomy appeared largely unaffected, apart from some subtle alterations. By integrating MRI and behavioural assays to investigate the effects of MIA-exposure on neonatal neurodevelopment we show that offspring neuroanatomy and behaviour are only subtly affected by both early and late exposure. This suggests that the deficits often observed in later stages of life may be dormant, not yet developed in the neonatal period, or not as easily detectable using a cross-sectional approach.

Peroxisome Proliferator-Activated Receptor-δ Deficiency in Microglia Results in Exacerbated Axonal Injury and Tissue Loss in Experimental Autoimmune Encephalomyelitis.

Peroxisome proliferator-activated receptor (PPAR)-δ is a nuclear receptor that functions to maintain metabolic homeostasis, regulate cell growth, and limit the development of excessive inflammation during immune responses. Previously, we reported that PPAR-δ-deficient mice develop a more severe clinical course of experimental autoimmune encephalomyelitis (EAE); however, it was difficult to delineate the role that microglia played in this disease phenotype since PPAR-δ-deficient mice exhibited a number of immune defects that enhanced CNS inflammation upstream of microglia activation. Here, we specifically investigated the role of PPAR-δ in microglia during EAE by using mice where excision of a floxed Ppard allele was driven by expression of a tamoxifen (TAM)-inducible CX3C chemokine receptor 1 promoter-Cre recombinase transgene (Cx3cr1CreERT2: Ppardfl/fl). We observed that by 30 days of TAM treatment, Cx3cr1CreERT2: Ppardfl/fl mice exhibited Cre-mediated deletion primarily in microglia and this was accompanied by efficient knockdown of Ppard expression in these cells. Upon induction of EAE, TAM-treated Cx3cr1CreERT2: Ppardfl/fl mice presented with an exacerbated course of disease compared to TAM-treated Ppardfl/fl controls. Histopathological and magnetic resonance (MR) studies on the spinal cord and brains of EAE mice revealed increased Iba-1 immunoreactivity, axonal injury and CNS tissue loss in the TAM-treated Cx3cr1CreERT2: Ppardfl/fl group compared to controls. In early EAE, a time when clinical scores and the infiltration of CD45+ leukocytes was equivalent between Cx3cr1CreERT2: Ppardfl/fl and Ppardfl/fl mice, Ppard-deficient microglia exhibited a more reactive phenotype as evidenced by a shorter maximum process length and lower expression of genes associated with a homeostatic microglia gene signature. In addition, Ppard-deficient microglia exhibited increased expression of genes associated with reactive oxygen species generation, phagocytosis and lipid clearance, M2-activation, and promotion of inflammation. Our results therefore suggest that PPAR-δ has an important role in microglia in limiting bystander tissue damage during neuroinflammation.

Cerebral asymmetries in 12-week-old C57Bl/6J mice measured by magnetic resonance imaging.

Asymmetries of multiple components of the rodent cerebrum have been described at various levels of organization. Yet, despite its ubiquitous nature, many confusing and sometimes contradictory reports regarding structural asymmetries in the rodent brain have been published. There is a need, therefore, for a whole-brain imaging analysis technique for asymmetry studies that is both accurate, reproducible and robust. To this end, a comprehensive three-dimensional examination of differences in brain structure in an inbred mouse strain was undertaken. The goal of this study was thus to use high-resolution magnetic resonance imaging to assess structural asymmetries in the adult C57Bl/6J mouse brain. Fixed brain T2-weighted images of 20 male C57Bl/6J mice were acquired on a 7T scanner at 32 microm isotropic resolution. We used voxel-based analyses to examine structural asymmetries throughout the whole mouse brain. The striatum, medial-posterior regions of the thalamus, and motor, sensorimotor, and visual cortex were found to be asymmetrical. The most significant asymmetry was found in the hippocampus and, specifically, the dentate gyrus. In each case, the left region was larger than the right. No other regions of the mouse brain showed structural asymmetry. The results in the dentate gyrus were confirmed using stereology, revealing a correlation of r=0.61 between magnetic resonance and stereological measures. Hippocampal, along with cortical asymmetry, has been discussed repeatedly in the literature, yet a clear pattern of directionality, until this point, has not been described. The findings of asymmetry in the striatum and absence of asymmetry in the rest of the brain are novel and show the advantage of using the whole-brain three-dimensional techniques developed herein for assessing asymmetry.