One-pot synthesis of carboxymethyl-dextran coated iron oxide nanoparticles (CION) for preclinical fMRI and MRA applications.

Superparamagnetic iron-oxide nanoparticles are robust contrast agents for magnetic resonance imaging (MRI) used for sensitive structural and functional mapping of the cerebral blood volume (CBV) when administered intravenously. To date, many CBV-MRI studies are conducted with Feraheme, manufactured for the clinical treatment of iron-deficiency. Unfortunately, Feraheme is currently not available outside the United States due to commercial and regulatory constraints, making CBV-MRI methods either inaccessible or very costly to achieve. To address this barrier, we developed a simple, one-pot recipe to synthesize Carboxymethyl-dextran coated Iron Oxide Nanoparticles, namely, "CION", suitable for preclinical CBV-MRI applications. Here we disseminate a step-by-step instruction of our one-pot synthesis protocol, which allows CION to be produced in laboratories with minimal cost. We also characterized different CION-conjugations by manipulating polymer to metal stoichiometric ratio in terms of their size, surface chemistry, and chemical composition, and shifts in MR relaxivity and pharmacokinetics. We performed several proof-of-concept experiments in vivo, demonstrating the utility of CION for functional and structural MRI applications, including hypercapnic CO2 challenge, visual stimulation, targeted optogenetic stimulation, and microangiography. We also present evidence that CION can serve as a cross-modality research platform by showing concurrent in vivo optical and MRI measurement of CBV using fluorescent-labeled CION. The simplicity and cost-effectiveness of our one-pot synthesis method should allow researchers to reproduce CION and tailor the relaxivity and pharmacokinetics according to their imaging needs. It is our hope that this work makes CBV-MRI more openly available and affordable for a variety of research applications.

Meditation and Mindfulness in Pregnancy and Postpartum: A Review of the Evidence.

Given their growing popularity, mindfulness practices including meditation are actively being studied in clinical trials to assess their efficacy at improving health outcomes during pregnancy and the postpartum period. We conducted a literature review to compile these studies and assessed their findings. There is sufficient evidence to support the practice of mindfulness practices in pregnancy to reduce anxiety, depression, and stress during pregnancy, which may continue to have beneficial effects through the postpartum period. There is limited evidence on the benefits of mindfulness and meditation for other aspects of pregnancy. However, due to the low-risk nature of these techniques, all women should be encouraged to engage in mindfulness practices during pregnancy.

Mind-Body Techniques in Pregnancy and Postpartum.

Maternal stress can perturb physiology and psychiatric health leading to adverse outcomes. This review investigates the effectiveness of several mind-body therapies-namely biofeedback, progressive muscle relaxation, guided imagery, tai chi, and yoga-as interventions in reducing maternal stress and other pregnancy-related conditions. Through randomized trials, these techniques have shown promising benefits for reducing pain, high blood pressure, stress, anxiety, depressive symptoms, labor pain and outcomes, and postpartum mood disturbances. As these interventions are easy to implement, low cost, and safe to perform in pregnancy, they should be considered as alternative, nonpharmaceutical interventions to use during pregnancy and postpartum care.

Locus coeruleus neurons are most sensitive to chronic neuroinflammation-induced neurodegeneration.

Parkinson's disease (PD) develops over decades through spatiotemporal stages that ascend from the brainstem to the forebrain. The mechanism behind this caudo-rostral neurodegeneration remains largely undefined. In unraveling this phenomenon, we recently developed a lipopolysaccharide (LPS)-elicited chronic neuroinflammatory mouse model that displays sequential losses of neurons in brainstem, substantia nigra, hippocampus and cortex. In this study, we aimed to investigate the mechanisms of caudo-rostral neurodegeneration and focused our efforts on the earliest neurodegeneration of vulnerable noradrenergic locus coeruleus (NE-LC) neurons in the brainstem. We found that compared with neurons in other brain regions, NE-LC neurons in untreated mice displayed high levels of mitochondrial oxidative stress that was severely exacerbated in the presence of LPS-elicited chronic neuroinflammation. In agreement, NE-LC neurons in LPS-treated mice displayed early reduction of complex IV expression and mitochondrial swelling and loss of cristae. Mechanistically, the activation of the superoxide-generating enzyme NADPH oxidase (NOX2) on NE-LC neurons was essential for their heightened vulnerability during chronic neuroinflammation. LPS induced early and high expressions of NOX2 in NE-LC neurons. Genetic or pharmacological inactivation of NOX2 markedly reduced mitochondrial oxidative stress and dysfunction in LPS-treated mice. Furthermore, inhibition of NOX2 significantly ameliorated LPS-induced NE-LC neurodegeneration. More importantly, post-treatment with NOX2 inhibitor diphenyleneiodonium when NE-LC neurodegeneration had already begun, still showed high efficacy in protecting NE-LC neurons from degeneration in LPS-treated mice. This study strongly supports that chronic neuroinflammation and NOX2 expression among vulnerable neuronal populations contribute to caudo-rostral degeneration in PD.

Genetic identification of a population of noradrenergic neurons implicated in attenuation of stress-related responses.

Noradrenergic signaling plays a well-established role in promoting the stress response. Here we identify a subpopulation of noradrenergic neurons, defined by developmental expression of Hoxb1, that has a unique role in modulating stress-related behavior. Using an intersectional chemogenetic strategy, in combination with behavioral and physiological analyses, we show that activation of Hoxb1-noradrenergic (Hoxb1-NE) neurons decreases anxiety-like behavior and promotes an active coping strategy in response to acute stressors. In addition, we use cerebral blood volume-weighted functional magnetic resonance imaging to show that chemoactivation of Hoxb1-NE neurons results in reduced activity in stress-related brain regions, including the bed nucleus of the stria terminalis, amygdala, and locus coeruleus. Thus, the actions of Hoxb1-NE neurons are distinct from the well-documented functions of the locus coeruleus in promoting the stress response, demonstrating that the noradrenergic system contains multiple functionally distinct subpopulations.

Aspm sustains postnatal cerebellar neurogenesis and medulloblastoma growth in mice.

Alterations in genes that regulate brain size may contribute to both microcephaly and brain tumor formation. Here, we report that Aspm, a gene that is mutated in familial microcephaly, regulates postnatal neurogenesis in the cerebellum and supports the growth of medulloblastoma, the most common malignant pediatric brain tumor. Cerebellar granule neuron progenitors (CGNPs) express Aspm when maintained in a proliferative state by sonic hedgehog (Shh) signaling, and Aspm is expressed in Shh-driven medulloblastoma in mice. Genetic deletion of Aspm reduces cerebellar growth, while paradoxically increasing the mitotic rate of CGNPs. Aspm-deficient CGNPs show impaired mitotic progression, altered patterns of division orientation and differentiation, and increased DNA damage, which causes progenitor attrition through apoptosis. Deletion of Aspm in mice with Smo-induced medulloblastoma reduces tumor growth and increases DNA damage. Co-deletion of Aspm and either of the apoptosis regulators Bax or Trp53 (also known as p53) rescues the survival of neural progenitors and reduces the growth restriction imposed by Aspm deletion. Our data show that Aspm functions to regulate mitosis and to mitigate DNA damage during CGNP cell division, causes microcephaly through progenitor apoptosis when mutated, and sustains tumor growth in medulloblastoma.

Role of Genetic Variation in Collateral Circulation in the Evolution of Acute Stroke: A Multimodal Magnetic Resonance Imaging Study.

BACKGROUND AND PURPOSE: No studies have determined the effect of differences in pial collateral extent (number and diameter), independent of differences in environmental factors and unknown genetic factors, on severity of stroke. We examined ischemic tissue evolution during acute stroke, as measured by magnetic resonance imaging and histology, by comparing 2 congenic mouse strains with otherwise identical genetic backgrounds but with different alleles of the Determinant of collateral extent-1 (Dce1) genetic locus. We also optimized magnetic resonance perfusion and diffusion-deficit thresholds by using histological measures of ischemic tissue. METHODS: Perfusion, diffusion, and T2-weighted magnetic resonance imaging were performed on collateral-poor (congenic-Bc) and collateral-rich (congenic-B6) mice at 1, 5, and 24 hours after permanent middle cerebral artery occlusion. Magnetic resonance imaging-derived penumbra and ischemic core volumes were confirmed by histology in a subset of mice at 5 and 24 hours after permanent middle cerebral artery occlusion. RESULTS: Although perfusion-deficit volumes were similar between strains 1 hour after permanent middle cerebral artery occlusion, diffusion-deficit volumes were 32% smaller in collateral-rich mice. At 5 hours, collateral-rich mice had markedly restored perfusion patterns showing reduced perfusion-deficit volumes, smaller infarct volumes, and smaller perfusion-diffusion mismatch volumes compared with the collateral-poor mice (P<0.05). At 24 hours, collateral-rich mice had 45% smaller T2-weighted lesion volumes (P<0.005) than collateral-poor mice, with no difference in perfusion-diffusion mismatch volumes because of penumbral death occurring 5 to 24 hours after permanent middle cerebral artery occlusion in collateral-poor mice. CONCLUSIONS: Variation in collateral extent significantly alters infarct volume expansion, transiently affects perfusion and diffusion magnetic resonance imaging signatures, and impacts salvage of ischemic penumbra after stroke onset.

Microglial regulation of immunological and neuroprotective functions of astroglia.

Microglia and astroglia play critical roles in the development, function, and survival of neurons in the CNS. However, under inflammatory conditions the role of astrogliosis in the inflammatory process and its effects on neurons remains unclear. Here, we used several types of cell cultures treated with the bacterial inflammogen LPS to address these questions. We found that the presence of astroglia reduced inflammation-driven neurotoxicity, suggesting that astrogliosis is principally neuroprotective. Neutralization of supernatant glial cell line-derived neurotrophic factor (GDNF) released from astroglia significantly reduced this neuroprotective effect during inflammation. To determine the immunological role of astroglia, we optimized a highly-enriched astroglial culture protocol and demonstrated that LPS failed to induce the synthesis and release of TNF-α and iNOS/NO. Instead we found significant enhancement of TNF-α and iNOS expression in highly-enriched astroglial cultures required the presence of 0.5-1% microglia, respectively. Thus suggesting that microglial-astroglial interactions are required for LPS to induce the expression of pro-inflammatory factors and GDNF from astroglia. Specifically, we found that microglia-derived TNF-α plays a pivotal role as a paracrine signal to regulate the neuroprotective functions of astrogliosis. Taken together, these findings suggest that astroglia may not possess the ability to directly recognize the innate immune stimuli LPS, but rather depend on crosstalk with microglia to elicit release of neurotrophic factors as a counterbalance to support neuronal survival from the collateral damage generated by activated microglia during neuroinflammation.

Dynamic perfusion and diffusion MRI of cortical spreading depolarization in photothrombotic ischemia.

Cortical spreading depolarization (CSD) is known to exacerbate ischemic damage, as the number of CSDs correlates with the final infarct volumes and suppressing CSDs improves functional outcomes. To investigate the role of CSD in ischemic damage, we developed a novel rat model of photothrombotic ischemia using a miniature implantable optic fiber that allows lesion induction inside the magnetic resonance imaging (MRI) scanner. We were able to precisely control the location and the size of the ischemic lesion, and continuously monitor dynamic perfusion and diffusion MRI signal changes at high temporal resolution before, during and after the onset of focal ischemia. Our model showed that apparent diffusion coefficient (ADC) and cerebral blood flow (CBF) in the ischemic core dropped immediately after lesion onset by 20±6 and 41±23%, respectively, and continually declined over the next 5h. Meanwhile, CSDs were observed in all animals (n=36) and displayed either a transient decrease of ADC by 17±3% or an increase of CBF by 104±15%. All CSDs were initiated from the rim of the ischemic core, propagated outward, and confined to the ipsilesional cortex. Additionally, we demonstrated that by controlling the size of perfusion-diffusion mismatch (which approximates the penumbra) in our model, the number of CSDs correlated with the mismatch area rather than the final infarct volume. This study introduces a novel platform to study CSDs in real-time with high reproducibility using MRI.

Spectral fiber photometry derives hemoglobin concentration changes for accurate measurement of fluorescent sensor activity.

Fiber photometry is an emerging technique for recording fluorescent sensor activity in the brain. However, significant hemoglobin absorption artifacts in fiber photometry data may be misinterpreted as sensor activity changes. Because hemoglobin exists widely in the brain, and its concentration varies temporally, such artifacts could impede the accuracy of photometry recordings. Here we present use of spectral photometry and computational methods to quantify photon absorption effects by using activity-independent fluorescence signals, which can be used to derive oxy- and deoxy-hemoglobin concentration changes. Although these changes are often temporally delayed compared with the fast-responding fluorescence spikes, we found that erroneous interpretation may occur when examining pharmacology-induced sustained changes and that sometimes hemoglobin absorption could flip the GCaMP signal polarity. We provide hemoglobin-based correction methods to restore fluorescence signals and compare our results with other commonly used approaches. We also demonstrated the utility of spectral fiber photometry for delineating regional differences in hemodynamic response functions.

Chemogenetic stimulation of tonic locus coeruleus activity strengthens the default mode network.

The default mode network (DMN) of the brain is functionally associated with a wide range of behaviors. In this study, we used functional magnetic resonance imaging (fMRI), positron emission tomography (PET), and spectral fiber photometry to investigate the selective neuromodulatory effect of norepinephrine (NE)-releasing noradrenergic neurons in the locus coeruleus (LC) on the mouse DMN. Chemogenetic-induced tonic LC activity decreased cerebral blood volume (CBV) and glucose uptake and increased synchronous low-frequency fMRI activity within the frontal cortices of the DMN. Fiber photometry results corroborated these findings, showing that LC-NE activation induced NE release, enhanced calcium-weighted neuronal spiking, and reduced CBV in the anterior cingulate cortex. These data suggest that LC-NE alters conventional coupling between neuronal activity and CBV in the frontal DMN. We also demonstrated that chemogenetic activation of LC-NE neurons strengthened functional connectivity within the frontal DMN, and this effect was causally mediated by reduced modulatory inputs from retrosplenial and hippocampal regions to the association cortices of the DMN.

Loss of Brain Norepinephrine Elicits Neuroinflammation-Mediated Oxidative Injury and Selective Caudo-Rostral Neurodegeneration.

Environmental toxicant exposure has been strongly implicated in the pathogenesis of Parkinson's disease (PD). Clinical manifestations of non-motor and motor symptoms in PD stem from decades of progressive neurodegeneration selectively afflicting discrete neuronal populations along a caudo-rostral axis. However, recapitulating this spatiotemporal neurodegenerative pattern in rodents has been unsuccessful. The purpose of this study was to generate such animal PD models and delineate mechanism underlying the ascending neurodegeneration. Neuroinflammation, oxidative stress, and neuronal death in mice brains were measured at different times following a single systemic injection of lipopolysaccharide (LPS). We demonstrate that LPS produced an ascending neurodegeneration that temporally afflicted neurons initially in the locus coeruleus (LC), followed by substantia nigra, and lastly the primary motor cortex and hippocampus. To test the hypothesis that LPS-elicited early loss of noradrenergic LC neurons may underlie this ascending pattern, we used a neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to deplete brain norepinephrine. DSP-4 injection resulted in a time-dependent ascending degenerative pattern similar to that generated by the LPS model. Mechanistic studies revealed that increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-2 (NOX2)-dependent superoxide/reactive oxygen species (ROS) production plays a key role in both LPS- and DSP-4-elicited neurotoxicity. We found that toxin-elicited chronic neuroinflammation, oxidative neuronal injuries, and neurodegeneration were greatly suppressed in mice deficient in NOX2 gene or treated with NOX2-specific inhibitor. Our studies document the first rodent PD model recapturing the ascending neurodegenerative pattern of PD patients and provide convincing evidence that the loss of brain norepinephrine is critical in initiating and maintaining chronic neuroinflammation and the discrete neurodegeneration in PD.

A novel method for assessing sex-specific and genotype-specific response to injury in astrocyte culture.

Female astrocytes sustain less cell death from oxygen-glucose deprivation (OGD) than male astrocytes. Arimidex, an aromatase inhibitor, abolishes these sex differences. To verify sex-dependent differences in P450 aromatase function in astrocyte cell death following OGD, we developed a novel method that uses sex-specific and genotype-specific single pup primary astrocyte cultures from wild-type (WT) and aromatase-knockout (ArKO) mice. After determining sex by external and internal examination as well as PCR and genotype by PCR amplification of tail cDNA, we established cultures from 1-3-day-old male and female WT and ArKO mice pups and grew them to confluence in estrogen-free media. Cell death was measured by lactate dehydrogenase (LDH) assay. Our study shows that, while WT female astrocytes are more resistant to OGD than WT male cells, sex differences disappear in ArKO cells. Cell death is significantly increased in ArKO compared to WT in female astrocytes but not male cells. Therefore, P450 aromatase appears to be essential in endogenous neuroprotection in females, and this finding may have clinical implications. This innovative technique may also be applied to other in vitro studies of sex-related functional differences.

Physiological Concentration of Prostaglandin E2 Exerts Anti-inflammatory Effects by Inhibiting Microglial Production of Superoxide Through a Novel Pathway.

This study investigated the physiological regulation of brain immune homeostasis in rat primary neuron-glial cultures by sub-nanomolar concentrations of prostaglandin E2 (PGE2). We demonstrated that 0.01 to 10 nM PGE2 protected dopaminergic neurons against LPS-induced neurotoxicity through a reduction of microglial release of pro-inflammatory factors in a dose-dependent manner. Mechanistically, neuroprotective effects elicited by PGE2 were mediated by the inhibition of microglial NOX2, a major superoxide-producing enzyme. This conclusion was supported by (1) the close relationship between inhibition of superoxide and PGE2-induced neuroprotective effects; (2) the mediation of PGE2-induced reduction of superoxide and neuroprotection via direct inhibition of the catalytic subunit of NOX2, gp91phox, rather than through the inhibition of conventional prostaglandin E2 receptors; and (3) abolishment of the neuroprotective effect of PGE2 in NOX2-deficient cultures. In summary, this study revealed a potential physiological role of PGE2 in maintaining brain immune homeostasis and protecting neurons via an EP receptor-independent mechanism.

Efficacy of supplemented buffered peptone water for the isolation of Campylobacter jejuni and C. coli from broiler retail products.

Broiler retail samples (n=113) were analyzed to determine (i) the effectiveness of buffered peptone water (BPW) supplemented with blood and antibiotics for the isolation of Campylobacter jejuni and C. coli, (ii) if a 1:4 enrichment ratio performs similarly as a 1:9 ratio, and (iii) if BPW is similar to Bolton broth for enumeration of Campylobacter spp. in retail broiler meat using the most probably number (MPN) procedure. Chi-square comparison showed that BPW performed similarly as Bolton broth (P< or =0.05) for Campylobacter isolation in breast tenders, boneless breasts, split breasts and skin samples. However, BPW showed a lower detection rate (P> or =0.05) for thighs and boneless thighs. When the results were combined, BPW performed similarly as Bolton broth for the isolation of Campylobacter spp. (P< or =0.05). BPW at an enrichment ratio of 1:4 was statistically similar to Bolton broth or BPW at a ratio of 1:9. No differences were observed between the MPN data from Bolton broth and the MPN data from BPW (P< or =0.50). A multiplex PCR assay revealed that ca. 48% of the isolates obtained from Bolton broth and 59% of the isolates obtained with BPW were C. coli. Both Bolton broth and BPW allowed for the growth of C. jejuni and C. coli from the same sample. Remarkably, a large genomic variability was observed by PFGE analysis of the isolates collected from the same sample with Bolton broth or BPW, which confirms that more than one genotype can successfully multiply during enrichment and be recoverable on agar plates. These findings suggest that BPW could be used as an enrichment medium for isolation of Campylobacter from retail broiler samples. The implications of the high number of C. coli isolates found in this study is discussed.

Critical role of the Mac1/NOX2 pathway in mediating reactive microgliosis-generated chronic neuroinflammation and progressive neurodegeneration.

As average life expectancy rises throughout the world, neurodegenerative diseases have emerged as one of the greatest global public heath challenges in modern times. Substantial efforts have been made in researching neurodegenerative diseases over the last few decades, yet their predominantly sporadic nature has made uncovering their etiologies challenging. Mounting evidence has suggested that factors like damage-associated molecular patterns (DAMPs) released by stressed and dying neurons are likely involved in disease pathology and in stimulating chronic activation of microglia that contributes to neuronal oxidative stress and degeneration. This review focuses on how the microglial integrin receptor Mac1 and its downstream effector NADPH oxidase (NOX2) contribute to maintaining chronic neuroinflammation and are crucial in inflammation-driven neurotoxicity in neurodegenerative diseases. Our hope is to provide new insights on novel targets and therapies that could slow or even halt neurodegeneration.

Preparation of rodent primary cultures for neuron-glia, mixed glia, enriched microglia, and reconstituted cultures with microglia.

Microglia, neurons, and macroglia (astrocytes and oligodendrocytes) are the major cell types in the central nervous system. In the past decades, primary microglia-enriched cultures have been widely used to study the biological functions of microglia in vitro. In order to study the interactions between microglia and other brain cells, neuron-glia, neuron-microglia, and mixed glia cultures were developed. The aim of this chapter is to provide basic and adaptable protocols for the preparation of these microglia-containing primary cultures from rodent. Meanwhile, we also want to provide a collection of tips from our collective experiences doing primary brain cell cultures.