Cranial Suture Regeneration Mitigates Skull and Neurocognitive Defects in Craniosynostosis.

Craniosynostosis results from premature fusion of the cranial suture(s), which contain mesenchymal stem cells (MSCs) that are crucial for calvarial expansion in coordination with brain growth. Infants with craniosynostosis have skull dysmorphology, increased intracranial pressure, and complications such as neurocognitive impairment that compromise quality of life. Animal models recapitulating these phenotypes are lacking, hampering development of urgently needed innovative therapies. Here, we show that Twist1+/- mice with craniosynostosis have increased intracranial pressure and neurocognitive behavioral abnormalities, recapitulating features of human Saethre-Chotzen syndrome. Using a biodegradable material combined with MSCs, we successfully regenerated a functional cranial suture that corrects skull deformity, normalizes intracranial pressure, and rescues neurocognitive behavior deficits. The regenerated suture creates a niche into which endogenous MSCs migrated, sustaining calvarial bone homeostasis and repair. MSC-based cranial suture regeneration offers a paradigm shift in treatment to reverse skull and neurocognitive abnormalities in this devastating disease.

Prenatal disruption of blood-brain barrier formation via cyclooxygenase activation leads to lifelong brain inflammation.

Gestational maternal immune activation (MIA) in mice induces persistent brain microglial activation and a range of neuropathologies in the adult offspring. Although long-term phenotypes are well documented, how MIA in utero leads to persistent brain inflammation is not well understood. Here, we found that offspring of mothers treated with polyriboinosinic­polyribocytidylic acid [poly(I:C)] to induce MIA at gestational day 13 exhibit blood­brain barrier (BBB) dysfunction throughout life. Live MRI in utero revealed fetal BBB hyperpermeability 2 d after MIA. Decreased pericyte­endothelium coupling in cerebral blood vessels and increased microglial activation were found in fetal and 1- and 6-mo-old offspring brains. The long-lasting disruptions result from abnormal prenatal BBB formation, driven by increased proliferation of cyclooxygenase-2 (COX2; Ptgs2)-expressing microglia in fetal brain parenchyma and perivascular spaces. Targeted deletion of the Ptgs2 gene in fetal myeloid cells or treatment with the inhibitor celecoxib 24 h after immune activation prevented microglial proliferation and disruption of BBB formation and function, showing that prenatal COX2 activation is a causal pathway of MIA effects. Thus, gestational MIA disrupts fetal BBB formation, inducing persistent BBB dysfunction, which promotes microglial overactivation and behavioral alterations across the offspring life span. Taken together, the data suggest that gestational MIA disruption of BBB formation could be an etiological contributor to neuropsychiatric disorders.

Longitudinal manganese-enhanced magnetic resonance imaging of neural projections and activity.

Manganese-enhanced magnetic resonance imaging (MEMRI) holds exceptional promise for preclinical studies of brain-wide physiology in awake-behaving animals. The objectives of this review are to update the current information regarding MEMRI and to inform new investigators as to its potential. Mn(II) is a powerful contrast agent for two main reasons: (1) high signal intensity at low doses; and (2) biological interactions, such as projection tracing and neural activity mapping via entry into electrically active neurons in the living brain. High-spin Mn(II) reduces the relaxation time of water protons: at Mn(II) concentrations typically encountered in MEMRI, robust hyperintensity is obtained without adverse effects. By selectively entering neurons through voltage-gated calcium channels, Mn(II) highlights active neurons. Safe doses may be repeated over weeks to allow for longitudinal imaging of brain-wide dynamics in the same individual across time. When delivered by stereotactic intracerebral injection, Mn(II) enters active neurons at the injection site and then travels inside axons for long distances, tracing neuronal projection anatomy. Rates of axonal transport within the brain were measured for the first time in "time-lapse" MEMRI. When delivered systemically, Mn(II) enters active neurons throughout the brain via voltage-sensitive calcium channels and clears slowly. Thus behavior can be monitored during Mn(II) uptake and hyperintense signals due to Mn(II) uptake captured retrospectively, allowing pairing of behavior with neural activity maps for the first time. Here we review critical information gained from MEMRI projection mapping about human neuropsychological disorders. We then discuss results from neural activity mapping from systemic Mn(II) imaged longitudinally that have illuminated development of the tonotopic map in the inferior colliculus as well as brain-wide responses to acute threat and how it evolves over time. MEMRI posed specific challenges for image data analysis that have recently been transcended. We predict a bright future for longitudinal MEMRI in pursuit of solutions to the brain-behavior mystery.

Evolution of brain-wide activity in the awake behaving mouse after acute fear by longitudinal manganese-enhanced MRI.

Life threatening fear after a single exposure evolves in a subset of vulnerable individuals to anxiety, which may persist for their lifetime. Yet neither the whole brain's response to innate acute fear nor how brain activity evolves over time is known. Sustained neuronal activity may be a factor in the development of a persistent fear response. We couple two experimental protocols to provoke acute fear leading to prolonged fear: Predator stress (PS), a naturalistic approach to induce fear in rodents; and Serotonin transporter knockout mouse (SERT-KO) that responds to PS with sustained defensive behavior. Behavior was monitored before, during and at short and long times after PS in wild type (WT) and SERT-KO mice. Both genotypes responded to PS with defensive behavior. SERT-KO retained defensive behavior for 23 days, while WT mice returned to baseline exploratory behavior by 9 days. Thus, differences in neural activity between WT and SERT-KO 9 days after PS identifies neural correlates of persistent defensive behavior, in mice. We used longitudinal manganese-enhanced magnetic resonance imaging (MEMRI) to identify brain-wide neural activity associated with different behaviors. Mn2+ accumulation in active neurons occurs in awake, behaving mice and is retrospectively imaged. Following the same two cohorts of mice, WT and SERT-KO, longitudinally allowed unbiased quantitative comparisons of brain-wide activity by statistical parametric mapping (SPM). During natural behavior in WT, only low levels of activity-induced Mn2+-accumulation were detected, while much more accumulation appeared immediately after PS in both WT and SERT-KO, and evolved at 9 days to a new activity pattern (p < 0.0001, uncorr., T = 5.4). Patterns of accumulation differed between genotypes, with more regions of the brain and larger volumes within regions involved in SERT-KO than WT. A new computational segmentation analysis, using our InVivo Atlas based on a manganese-enhanced MR image of a living mouse, revealed dynamic changes in the volume of significantly enhanced voxels within each segment that differed between genotypes across 45 of 87 segmented regions. At Day 9 after PS, the striatum and ventral pallidum were active in both genotypes but more so in the SERT-KO. SERT-KO also displayed sustained or increased volume of Mn2+ accumulations between Post-Fear and Day 9 in eight segments where activity was decreased or silenced in WT. C-fos staining, an alternative neural activity marker, of brains from the same mice fixed at conclusion of imaging sessions confirmed that MEMRI detected active neurons. Intensity measurements in 12 regions of interest (ROIs) supported the SPM results. Between group comparisons by SPM and of ROI measurements identified specific regions differing between time points and genotypes. We report brain-wide activity in response to a single exposure of acute fear, and, for the first time, its evolution to new activity patterns over time in individuals vulnerable to persistent fear. Our results show multiple regions with dynamic changes in neural activity and that the balance of activity between segments is disordered in the SERT-KO. Thus, longitudinal MEMRI represents a powerful approach to discover how brain-wide activity evolves from the natural state either after an experience or during a disease process.

Conditional Creation and Rescue of Nipbl-Deficiency in Mice Reveals Multiple Determinants of Risk for Congenital Heart Defects.

Elucidating the causes of congenital heart defects is made difficult by the complex morphogenesis of the mammalian heart, which takes place early in development, involves contributions from multiple germ layers, and is controlled by many genes. Here, we use a conditional/invertible genetic strategy to identify the cell lineage(s) responsible for the development of heart defects in a Nipbl-deficient mouse model of Cornelia de Lange Syndrome, in which global yet subtle transcriptional dysregulation leads to development of atrial septal defects (ASDs) at high frequency. Using an approach that allows for recombinase-mediated creation or rescue of Nipbl deficiency in different lineages, we uncover complex interactions between the cardiac mesoderm, endoderm, and the rest of the embryo, whereby the risk conferred by genetic abnormality in any one lineage is modified, in a surprisingly non-additive way, by the status of others. We argue that these results are best understood in the context of a model in which the risk of heart defects is associated with the adequacy of early progenitor cell populations relative to the sizes of the structures they must eventually form.

Vascular dysfunction-The disregarded partner of Alzheimer's disease.

Increasing evidence recognizes Alzheimer's disease (AD) as a multifactorial and heterogeneous disease with multiple contributors to its pathophysiology, including vascular dysfunction. The recently updated AD Research Framework put forth by the National Institute on Aging-Alzheimer's Association describes a biomarker-based pathologic definition of AD focused on amyloid, tau, and neuronal injury. In response to this article, here we first discussed evidence that vascular dysfunction is an important early event in AD pathophysiology. Next, we examined various imaging sequences that could be easily implemented to evaluate different types of vascular dysfunction associated with, and/or contributing to, AD pathophysiology, including changes in blood-brain barrier integrity and cerebral blood flow. Vascular imaging biomarkers of small vessel disease of the brain, which is responsible for >50% of dementia worldwide, including AD, are already established, well characterized, and easy to recognize. We suggest that these vascular biomarkers should be incorporated into the AD Research Framework to gain a better understanding of AD pathophysiology and aid in treatment efforts.

Manganese-enhanced magnetic resonance imaging reveals increased DOI-induced brain activity in a mouse model of schizophrenia.

Maternal infection during pregnancy increases the risk for schizophrenia in offspring. In rodent models, maternal immune activation (MIA) yields offspring with schizophrenia-like behaviors. None of these behaviors are, however, specific to schizophrenia. The presence of hallucinations is a key diagnostic symptom of schizophrenia. In mice, this symptom can be defined as brain activation in the absence of external stimuli, which can be mimicked by administration of hallucinogens. We find that, compared with controls, adult MIA offspring display an increased stereotypical behavioral response to the hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI), an agonist for serotonin receptor 2A (5-HT2AR). This may be explained by increased levels of 5-HT2AR and downstream signaling molecules in unstimulated MIA prefrontal cortex (PFC). Using manganese-enhanced magnetic resonance imaging to identify neuronal activation elicited by DOI administration, we find that, compared with controls, MIA offspring exhibit a greater manganese (Mn(2+)) accumulation in several brain areas, including the PFC, thalamus, and striatum. The parafascicular thalamic nucleus, which plays the role in the pathogenesis of hallucinations, is activated by DOI in MIA offspring only. Additionally, compared with controls, MIA offspring demonstrate higher DOI-induced expression of early growth response protein 1, cyclooxygenase-2, and brain-derived neurotrophic factor in the PFC. Chronic treatment with the 5-HT2AR antagonist ketanserin reduces DOI-induced head twitching in MIA offspring. Thus, the MIA mouse model can be successfully used to investigate activity induced by DOI in awake, behaving mice. Moreover, manganese-enhanced magnetic resonance imaging is a useful, noninvasive method for accurately measuring this type of activity.

Optimal acquisition and modeling parameters for accurate assessment of low Ktrans blood-brain barrier permeability using dynamic contrast-enhanced MRI.

PURPOSE: To determine optimal parameters for acquisition and processing of dynamic contrast-enhanced MRI (DCE-MRI) to detect small changes in near normal low blood-brain barrier (BBB) permeability. METHODS: Using a contrast-to-noise ratio metric (K-CNR) for Ktrans precision and accuracy, the effects of kinetic model selection, scan duration, temporal resolution, signal drift, and length of baseline on the estimation of low permeability values was evaluated with simulations. RESULTS: The Patlak model was shown to give the highest K-CNR at low Ktrans . The Ktrans transition point, above which other models yielded superior results, was highly dependent on scan duration and tissue extravascular extracellular volume fraction (ve ). The highest K-CNR for low Ktrans was obtained when Patlak model analysis was combined with long scan times (10-30 min), modest temporal resolution (<60 s/image), and long baseline scans (1-4 min). Signal drift as low as 3% was shown to affect the accuracy of Ktrans estimation with Patlak analysis. CONCLUSION: DCE acquisition and modeling parameters are interdependent and should be optimized together for the tissue being imaged. Appropriately optimized protocols can detect even the subtlest changes in BBB integrity and may be used to probe the earliest changes in neurodegenerative diseases such as Alzheimer's disease and multiple sclerosis.

ROCKETSHIP: a flexible and modular software tool for the planning, processing and analysis of dynamic MRI studies.

BACKGROUND: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is a promising technique to characterize pathology and evaluate treatment response. However, analysis of DCE-MRI data is complex and benefits from concurrent analysis of multiple kinetic models and parameters. Few software tools are currently available that specifically focuses on DCE-MRI analysis with multiple kinetic models. Here, we developed ROCKETSHIP, an open-source, flexible and modular software for DCE-MRI analysis. ROCKETSHIP incorporates analyses with multiple kinetic models, including data-driven nested model analysis. RESULTS: ROCKETSHIP was implemented using the MATLAB programming language. Robustness of the software to provide reliable fits using multiple kinetic models is demonstrated using simulated data. Simulations also demonstrate the utility of the data-driven nested model analysis. Applicability of ROCKETSHIP for both preclinical and clinical studies is shown using DCE-MRI studies of the human brain and a murine tumor model. CONCLUSION: A DCE-MRI software suite was implemented and tested using simulations. Its applicability to both preclinical and clinical datasets is shown. ROCKETSHIP was designed to be easily accessible for the beginner, but flexible enough for changes or additions to be made by the advanced user as well. The availability of a flexible analysis tool will aid future studies using DCE-MRI. A public release of ROCKETSHIP is available at https://github.com/petmri/ROCKETSHIP .

Multimodality PET/MRI agents targeted to activated macrophages.

The recent emergence of multimodality imaging, particularly the combination of PET and MRI, has led to excitement over the prospect of improving detection of disease. Iron oxide nanoparticles have become a popular platform for the fabrication of PET/MRI probes owing to their advantages of high MRI detection sensitivity, biocompatibility, and biodegradability. In this article, we report the synthesis of dextran-coated iron oxide nanoparticles (DIO) labeled with the positron emitter (64)Cu to generate a PET/MRI probe, and modified with maleic anhydride to increase the negative surface charge. The modified nanoparticulate PET/MRI probe (MDIO-(64)Cu-DOTA) bears repetitive anionic charges on the surface that facilitate recognition by scavenger receptor type A (SR-A), a ligand receptor found on activated macrophages but not on normal vessel walls. MDIO-(64)Cu-DOTA has an average iron oxide core size of 7-8 nm, an average hydrodynamic diameter of 62.7 nm, an r1 relaxivity of 16.8 mM(-1) s(-1), and an r 2 relaxivity of 83.9 mM(-1) s(-1) (37 °C, 1.4 T). Cell studies confirmed that the probe was nontoxic and was specifically taken up by macrophages via SR-A. In comparison with the nonmodified analog, the accumulation of MDIO in macrophages was substantially improved. These characteristics demonstrate the promise of MDIO-(64)Cu-DOTA for identification of vulnerable atherosclerotic plaques via the targeting of macrophages.

MRI Detection of Lymph Node Metastasis through Molecular Targeting of C-C Chemokine Receptor Type 2 and Monocyte Hitchhiking.

Biopsy is the clinical standard for diagnosing lymph node (LN) metastasis, but it is invasive and poses significant risk to patient health. Magnetic resonance imaging (MRI) has been utilized as a noninvasive alternative but is limited by low sensitivity, with only ∼35% of LN metastases detected, as clinical contrast agents cannot discriminate between healthy and metastatic LNs due to nonspecific accumulation. Nanoparticles targeted to the C-C chemokine receptor 2 (CCR2), a biomarker highly expressed in metastatic LNs, have the potential to guide the delivery of contrast agents, improving the sensitivity of MRI. Additionally, cancer cells in metastatic LNs produce monocyte chemotactic protein 1 (MCP1), which binds to CCR2+ inflammatory monocytes and stimulates their migration. Thus, the molecular targeting of CCR2 may enable nanoparticle hitchhiking onto monocytes, providing an additional mechanism for metastatic LN targeting and early detection. Hence, we developed micelles incorporating gadolinium (Gd) and peptides derived from the CCR2-binding motif of MCP1 (MCP1-Gd) and evaluated the potential of MCP1-Gd to detect LN metastasis. When incubated with migrating monocytes in vitro, MCP1-Gd transport across lymphatic endothelium increased 2-fold relative to nontargeting controls. After administration into mouse models with initial LN metastasis and recurrent LN metastasis, MCP1-Gd detected metastatic LNs by increasing MRI signal by 30-50% relative to healthy LNs. Furthermore, LN targeting was dependent on monocyte hitchhiking, as monocyte depletion decreased accumulation by >70%. Herein, we present a nanoparticle contrast agent for MRI detection of LN metastasis mediated by CCR2-targeting and demonstrate the potential of monocyte hitchhiking for enhanced nanoparticle delivery.

KCNJ2 inhibition mitigates mechanical injury in a human brain organoid model of traumatic brain injury.

Traumatic brain injury (TBI) strongly correlates with neurodegenerative disease. However, it remains unclear which neurodegenerative mechanisms are intrinsic to the brain and which strategies most potently mitigate these processes. We developed a high-intensity ultrasound platform to inflict mechanical injury to induced pluripotent stem cell (iPSC)-derived cortical organoids. Mechanically injured organoids elicit classic hallmarks of TBI, including neuronal death, tau phosphorylation, and TDP-43 nuclear egress. We found that deep-layer neurons were particularly vulnerable to injury and that TDP-43 proteinopathy promotes cell death. Injured organoids derived from C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) patients displayed exacerbated TDP-43 dysfunction. Using genome-wide CRISPR interference screening, we identified a mechanosensory channel, KCNJ2, whose inhibition potently mitigated neurodegenerative processes in vitro and in vivo, including in C9ORF72 ALS/FTD organoids. Thus, targeting KCNJ2 may reduce acute neuronal death after brain injury, and we present a scalable, genetically flexible cerebral organoid model that may enable the identification of additional modifiers of mechanical stress.

Reconfiguration of brain-wide neural activity after early life adversity.

Early life adversity (ELA) predisposes individuals to both physical and mental disorders lifelong. How ELA affects brain function leading to this vulnerability is under intense investigation. Research has begun to shed light on ELA effects on localized brain regions within defined circuits. However, investigations into brain-wide neural activity that includes multiple localized regions, determines relationships of activity between regions and identifies shifts of activity in response to experiential conditions is necessary. Here, we performed longitudinal manganese-enhanced magnetic resonance imaging (MEMRI) to image the brain in normally reared or ELA-exposed adults. Images were captured in the freely moving home cage condition, and short- and long-term after naturalistic threat. Images were analyzed with new computational methods, including automated segmentation and fractional activation or difference volumes. We found that neural activity was increased after ELA compared to normal rearing in multiple brain regions, some of which are involved in defensive and/or reward circuitry. Widely distributed patterns of neural activity, "brain states", and their dynamics after threat were altered with ELA. Upon acute threat, ELA-mice retained heightened neural activity within many of these regions, and new hyperactive responses emerged in monoaminergic centers of the mid- and hindbrain. Nine days after acute threat, heightened neural activity remained within locus coeruleus and increased within posterior amygdala, ventral hippocampus, and dorso- and ventromedial hypothalamus, while reduced activity emerged within medial prefrontal cortical regions (prelimbic, infralimbic, anterior cingulate). These results reveal that functional imbalances arise between multiple brain-systems which are dependent upon context and cumulative experiences after ELA.

Harnessing axonal transport to map reward circuitry: Differing brain-wide projections from medial prefrontal cortical domains.

Neurons project long axons that contact other distant neurons. Neurons in the medial prefrontal cortex project into the limbic system to regulate responses to reward or threat. Diminished neural activity in prefrontal cortex is associated with loss of executive function leading to drug use, yet the specific circuitry that mediate these effects is unknown. Different regions within the medial prefrontal cortex may project to differing limbic system nuclei. Here, we exploited the cell biology of intracellular membrane trafficking, fast axonal transport, to map projections from two adjacent medial prefrontal cortical regions. We used Mn(II), a calcium analog, to trace medial prefrontal cortical projections in the living animal by magnetic resonance imaging (MRI). Mn(II), a contrast agent for MRI, enters neurons through voltage-activated calcium channels and relies on kinesin-1 and amyloid-precursor protein to transport out axons to distal destinations. Aqueous MnCl2 together with fluorescent dextran (3--5 nL) was stereotactically injected precisely into two adjacent regions of the medial prefrontal cortex: anterior cingulate area (ACA) or infralimbic/prelimbic (IL/PL) region. Projections were traced, first live by manganese-enhanced MRI (MEMRI) at four time points in 3D, and then after fixation by microscopy. Data-driven unbiased voxel-wise statistical maps of aligned normalized MR images after either ACA or IL/PL injections revealed statistically significant progression of Mn(II) over time into deeper brain regions: dorsal striatum, globus pallidus, amygdala, hypothalamus, substantia nigra, dorsal raphe and locus coeruleus. Quantitative comparisons of these distal accumulations at 24 h revealed dramatic differences between ACA and IL/PL injection groups throughout the limbic system, and most particularly in subdomains of the hypothalamus. ACA projections targeted dorsomedial nucleus of the hypothalamus, posterior part of the periventricular region and mammillary body nuclei as well as periaqueductal gray, while IL/PL projections accumulated in anterior hypothalamic areas and lateral hypothalamic nuclei as well as amygdala. As hypothalamic subsegments relay CNS activity to the body, our results suggest new concepts about mind-body relationships and specific roles of distinct yet adjacent medial prefrontal cortical segments. Our MR imaging strategy, when applied to follow other cell biological processes in the living organism, will undoubtedly lead to an expanded perspective on how minute details of cellular processes influence whole body health and wellbeing.

The C9ORF72 repeat expansion alters neurodevelopment.

Genetic mutations that cause adult-onset neurodegenerative diseases are often expressed during embryonic stages, but it is unclear whether they alter neurodevelopment and how this might influence disease onset. Here, we show that the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), a repeat expansion in C9ORF72, restricts neural stem cell proliferation and reduces cortical and thalamic size in utero. Surprisingly, a repeat expansion-derived dipeptide repeat protein (DPR) not known to reduce neuronal viability plays a key role in impairing neurodevelopment. Pharmacologically mimicking the effects of the repeat expansion on neurodevelopment increases susceptibility of C9ORF72 mice to motor defects. Thus, the C9ORF72 repeat expansion stunts development of the brain regions prominently affected in C9ORF72 FTD/ALS patients.

Synthesis and Preclinical Evaluation of 22-[18F]Fluorodocosahexaenoic Acid as a Positron Emission Tomography Probe for Monitoring Brain Docosahexaenoic Acid Uptake Kinetics.

Docosahexaenoic acid [22:6(n-3), DHA], a polyunsaturated fatty acid, has an important role in regulating neuronal functions and in normal brain development. Dysregulated brain DHA uptake and metabolism are found in individuals carrying the APOE4 allele, which increases the genetic risk for Alzheimer's disease (AD), and are implicated in the progression of several neurodegenerative disorders. However, there are limited tools to assess brain DHA kinetics in vivo that can be translated to humans. Here, we report the synthesis of an ω-radiofluorinated PET probe of DHA, 22-[18F]fluorodocosahexaenoic acid (22-[18F]FDHA), for imaging the uptake of DHA into the brain. Using the nonradiolabeled 22-FDHA, we confirmed that fluorination of DHA at the ω-position does not significantly alter the anti-inflammatory effect of DHA in microglial cells. Through dynamic PET-MR studies using mice, we observed the accumulation of 22-[18F]FDHA in the brain over time and estimated DHA's incorporation coefficient (K*) using an image-derived input function. Finally, DHA brain K* was validated using intravenous administration of 15 mg/kg arecoline, a natural product known to increase the DHA K* in rodents. 22-[18F]FDHA is a promising PET probe that can reveal altered lipid metabolism in APOE4 carriers, AD, and other neurologic disorders. This new probe, once translated into humans, would enable noninvasive and longitudinal studies of brain DHA dynamics by guiding both pharmacological and nonpharmacological interventions for neurodegenerative diseases.

Deficits in axonal transport in hippocampal-based circuitry and the visual pathway in APP knock-out animals witnessed by manganese enhanced MRI.

Mounting evidence implicates axonal transport defects, typified by the presence of axonal varicosities with aberrant accumulations of cargo, as an early event in Alzheimer's disease (AD) pathogenesis. Work identifying amyloid precursor protein (APP) as a vesicular motor receptor for anterograde axonal transport further implicates axonal transport in AD. Manganese-enhanced MRI (MEMRI) detects axonal transport dynamics in preclinical studies. Here we pursue an understanding of the role of APP in axonal transport in the central nervous system by applying MEMRI to hippocampal circuitry and to the visual pathway in living mice homozygous for either wild type or a deletion in the APP gene (n=12 for each genotype). Following intra-ocular or stereotaxic hippocampal injection, we performed time-lapse MRI to detect Mn(2+) transport. Three dimensional whole brain datasets were compared on a voxel-wise basis using within-group pair-wise analysis. Quantification of transport to structures connected to injection sites via axonal fiber tracts was also performed. Histology confirmed consistent placement of hippocampal injections and no observable difference in glial-response to the injections. APP-/- mice had significantly reduced transport from the hippocampus to the septal nuclei and amygdala after 7h and reduced transport to the contralateral hippocampus after 25 h; axonal transport deficits in the APP-/- animals were also identified in the visual pathway. These data support a system-wide role for APP in axonal transport within the central nervous system and demonstrate the power of MEMRI for assessing neuronal circuitry involved in memory and learning.

Estrogen treatment prevents gray matter atrophy in experimental autoimmune encephalomyelitis.

Gray matter atrophy is an important correlate to clinical disability in multiple sclerosis (MS), and many treatment trials include atrophy as an outcome measure. Atrophy has been shown to occur in experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS. The clinical severity of EAE is reduced in estrogen-reated mice, but it remains unknown whether estrogen treatment can reduce gray matter atrophy in EAE. In this study, mice with EAE were treated with either estrogen receptor (ER)-α ligand or ER-ß ligand, and diffusion tensor images (DTI) were collected and neuropathology was performed. DTI showed atrophy in the cerebellar gray matter of vehicle-treated EAE mice compared with healthy controls but not in ER-α or ER-ß ligand-treated EAE mice. Neuropathology demonstrated that Purkinje cell numbers were decreased in vehicle-treated EAE mice, whereas neither ER ligand-treated EAE groups showed a decrease. This is the first report of a neuroprotective therapy in EAE that unambiguously prevents gray matter atrophy while sparing a major neuronal cell type. Fractional anisotropy (FA) in the cerebellar white matter was decreased in vehicle- and ER-ß ligand-treated but not in ER-α ligand-treated EAE mice. Inflammatory cell infiltration was increased in vehicle- and ER-ß ligand-treated but not in ER-α ligand-treated EAE mice. Myelin staining was decreased in vehicle-treated EAE mice and was spared in both ER ligand-treated groups. This is consistent with decreased FA as a potential biomarker for inflammation rather than myelination or axonal damage in the cerebellum in EAE.

Quantitative pharmacologic MRI in mice.

Pharmacologic MRI (phMRI) uses functional MRI techniques to provide a noninvasive in vivo measurement of the hemodynamic effects of drugs. The cerebral blood volume change (ΔCBV) serves as a surrogate for neuronal activity via neurovascular coupling mechanisms. By assessing the location and time course of brain activity in mouse mutant studies, phMRI can provide valuable insights into how different behavioral phenotypes are expressed in deferring brain activity response to drug challenge. In this report, we evaluate the utility of three different intravascular ultrasmall superparamagnetic iron oxide (USPIO) contrast agents for phMRI using a gradient-echo technique, with temporal resolution of one min at high magnetic field. The tissue half-life of the USPIOs was studied using a nonlinear detrending model. The three USPIOs are candidates for CBV weighted phMRI experiments, with r(2)/r(1) ratios ≥ 20 and apparent half-lives ≥ 1.5 h at the described doses. An echo-time of about 10 ms or longer results in a functional contrast to noise ratio (fCNR) > 75 after USPIO injection, with negligible decrease between 1.5-2 h. phMRI experiments were conducted at 7 T using cocaine as a psychotropic substance and acetazolamide, a global vasodilator, as a positive control. Cocaine acts as a dopamine-serotonin-norepinephrine reuptake inhibitor, increasing extracellular concentrations of these neurotransmitters, and thus increasing dopaminergic, serotonergic and noradrenergic neurotransmission. phMRI results showed that CBV was reduced in the normal mouse brain after cocaine challenge, with the largest effects in the nucleus accumbens, whereas after acetazolamide, blood volume was increased in both cerebral and extracerebral tissue.

Diffusion MRI Connections in the Octopus Brain.

Using high angle resolution diffusion magnetic resonance imaging (HARDI) with fiber tractography analysis we map out a meso-scale connectome of the Octopus bimaculoides brain. The brain of this cephalopod has a qualitatively different organization than that of vertebrates, yet it exhibits complex behavior, an elaborate sensory system and high cognitive abilities. Over the last 60 years wide ranging and detailed studies of octopus brain anatomy have been undertaken, including classical histological sectioning/staining, electron microscopy and neuronal tract tracing with injected dyes. These studies have elucidated many neuronal connections within and among anatomical structures. Diffusion MRI based tractography utilizes a qualitatively different method of tracing connections within the brain and offers facile three-dimensional images of anatomy and connections that can be quantitatively analyzed. Twenty-five separate lobes of the brain were segmented in the 3D MR images of each of three samples, including all five sub-structures in the vertical lobe. These parcellations were used to assay fiber tracings between lobes. The connectivity matrix constructed from diffusion MRI data was largely in agreement with that assembled from earlier studies. The one major difference was that connections between the vertical lobe and more basal supra-esophageal structures present in the literature were not found by MRI. In all, 92 connections between the 25 different lobes were noted by diffusion MRI: 53 between supra-esophageal lobes and 26 between the optic lobes and other structures. These represent the beginnings of a mesoscale connectome of the octopus brain.