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.

Noradrenergic dysfunction accelerates LPS-elicited inflammation-related ascending sequential neurodegeneration and deficits in non-motor/motor functions.

The loss of central norepinephrine (NE) released by neurons of the locus coeruleus (LC) occurs with aging, and is thought to be an important factor in producing the many of the nonmotor symptoms and exacerbating the degenerative process in animal models of Parkinson's disease (PD). We hypothesize that selectively depleting noradrenergic LC neurons prior to the induction of chronic neuroinflammation may not only accelerate the rate of progressive neurodegeneration throughout the brain, but may exacerbate nonmotor and motor behavioral phenotypes that recapitulate symptoms of PD. For this reason, we used a "two-hit" mouse model whereby brain NE were initially depleted by DSP-4 one week prior to exposing mice to LPS. We found that pretreatment with DSP-4 potentiated LPS-induced sequential neurodegeneration in SNpc, hippocampus, and motor cortex, but not in VTA and caudate/putamen. Mechanistic study revealed that DSP-4 enhanced LPS-induced microglial activation and subsequently elevated neuronal oxidative stress in affected brain regions in a time-dependent pattern. To further characterize the effects of DSP-4 on non-motor and motor symptoms in the LPS model, physiological and behavioral tests were performed at different time points following injection. Consistent with the enhanced neurodegeneration, DSP-4 accelerated the progressive deficits of non-motor symptoms including hyposmia, constipation, anxiety, sociability, exaggerated startle response and impaired learning. Furthermore, notable decreases of motor functions, including decreased rotarod activity, grip strength, and gait disturbance, were observed in treated mice. In summary, our studies provided not only an accelerated "two-hit" PD model that recapitulates the features of sequential neuron loss and the progression of motor/non-motor symptoms of PD, but also revealed the critical role of early LC noradrenergic neuron damage in the pathogenesis of PD-like symptoms.

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.

Post-treatment with an ultra-low dose of NADPH oxidase inhibitor diphenyleneiodonium attenuates disease progression in multiple Parkinson's disease models.

Nicotinamide adenine dinucleotide phosphate oxidase, a key superoxide-producing enzyme, plays a critical role in microglia-mediated chronic neuroinflammation and subsequent progressive dopaminergic neurodegeneration in Parkinson's disease. Although nicotinamide adenine dinucleotide phosphate oxidase-targeting anti-inflammatory therapy for Parkinson's disease has been proposed, its application in translational research remains limited. The aim of this study was to obtain preclinical evidence supporting this therapeutic strategy by testing the efficacy of an ultra-low dose of the nicotinamide adenine dinucleotide phosphate oxidase inhibitor diphenyleneiodonium in both endotoxin (lipopolysaccharide)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice using post-treatment regimens. Our data revealed that post-treatment with diphenyleneiodonium significantly attenuated progressive dopaminergic degeneration and improved rotarod activity. Remarkably, post-treatment with diphenyleneiodonium 10 months after lipopolysaccharide injection when mice had 30% loss of nigral dopaminergic neurons, showed high efficacy in protecting the remaining neuronal population and restoring motor function. Diphenyleneiodonium-elicited neuroprotection was associated with the inhibition of microglial activation, a reduction in the expression of proinflammatory factors and an attenuation of α-synuclein aggregation. A pathophysiological evaluation of diphenyleneiodonium-treated mice, including assessment of body weight, organs health, and neuronal counts, revealed no overt signs of toxicity. In summary, infusion of ultra-low dose diphenyleneiodonium potently reduced microglia-mediated chronic neuroinflammation by selectively inhibiting nicotinamide adenine dinucleotide phosphate oxidase and halted the progression of neurodegeneration in mouse models of Parkinson's disease. The robust neuroprotective effects and lack of apparent toxic side effects suggest that diphenyleneiodonium at ultra-low dose may be a promising candidate for future clinical trials in Parkinson's disease patients.

Substance P exacerbates dopaminergic neurodegeneration through neurokinin-1 receptor-independent activation of microglial NADPH oxidase.

Although dysregulated substance P (SP) has been implicated in the pathophysiology of Parkinson's disease (PD), how SP affects the survival of dopaminergic neurons remains unclear. Here, we found that mice lacking endogenous SP (TAC1(-/-)), but not those deficient in the SP receptor (neurokinin-1 receptor, NK1R), were more resistant to lipopolysaccharide (LPS)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigral dopaminergic neurodegeneration than wild-type controls, suggesting a NK1R-independent toxic action of SP. In vitro dose-response studies revealed that exogenous SP enhanced LPS- and 1-methyl-4-phenylpyridinium (MPP(+))-induced dopaminergic neurodegeneration in a bimodal manner, peaking at submicromolar and subpicomolar concentrations, but was substantially less effective at intermediate concentrations. Mechanistically, the actions of submicromolar levels of SP were NK1R-dependent, whereas subpicomolar SP-elicited actions required microglial NADPH oxidase (NOX2), the key superoxide-producing enzyme, but not NK1R. Subpicomolar concentrations of SP activated NOX2 by binding to the catalytic subunit gp91(phox) and inducing membrane translocation of the cytosolic subunits p47(phox) and p67(phox). The importance of NOX2 was further corroborated by showing that inhibition or disruption of NOX2 blocked subpicomolar SP-exacerbated neurotoxicity. Together, our findings revealed a critical role of microglial NOX2 in mediating the neuroinflammatory and dopaminergic neurodegenerative effects of SP, which may provide new insights into the pathogenesis of PD.

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.

A novel role of microglial NADPH oxidase in mediating extra-synaptic function of norepinephrine in regulating brain immune homeostasis.

Although the peripheral anti-inflammatory effect of norepinephrine (NE) is well documented, the mechanism by which this neurotransmitter functions as an anti-inflammatory/neuroprotective agent in the central nervous system (CNS) is unclear. This article aimed to determine the anti-inflammatory/neuroprotective effects and underlying mechanisms of NE in inflammation-based dopaminergic neurotoxicity models. In mice, NE-depleting toxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) was injected at 6 months of lipopolysaccharide (LPS)-induced neuroinflammation. It was found that NE depletion enhanced LPS-induced dopaminergic neuron loss in the substantia nigra. This piece of in vivo data prompted us to conduct a series of studies in an effort to elucidate the mechanism as to how NE affects dopamine neuron survival by using primary midbrain neuron/glia cultures. Results showed that submicromolar concentrations of NE dose-dependently protected dopaminergic neurons from LPS-induced neurotoxicity by inhibiting microglia activation and subsequent release of pro-inflammatory factors. However, NE-elicited neuroprotection was not totally abolished in cultures from ß2-adrenergic receptor (ß2-AR)-deficient mice, suggesting that novel pathways other than ß2-AR are involved. To this end, It was found that submicromolar NE dose-dependently inhibited NADPH oxidase (NOX2)-generated superoxide, which contributes to the anti-inflammatory and neuroprotective effects of NE. This novel mechanism was indeed adrenergic receptors independent since both (+) and (-) optic isomers of NE displayed the same potency. We further demonstrated that NE inhibited LPS-induced NOX2 activation by blocking the translocation of its cytosolic subunit to plasma membranes. In summary, we revealed a potential physiological role of NE in maintaining brain immune homeostasis and protecting neurons via a novel mechanism.

Substance P enhances microglial density in the substantia nigra through neurokinin-1 receptor/NADPH oxidase-mediated chemotaxis in mice.

The distribution of microglia varies greatly throughout the brain. The substantia nigra (SN) contains the highest density of microglia among different brain regions. However, the mechanism underlying this uneven distribution remains unclear. Substance P (SP) is a potent proinflammatory neuropeptide with high concentrations in the SN. We recently demonstrated that SP can regulate nigral microglial activity. In the present study, we further investigated the involvement of SP in modulating nigral microglial density in postnatal developing mice. Nigral microglial density was quantified in wild-type (WT) and SP-deficient mice from postnatal day 1 (P1) to P30. SP was detected at high levels in the SN as early as P1 and microglial density did not peak until around P30 in WT mice. SP-deficient mice (TAC1(-/-)) had a significant reduction in nigral microglial density. No differences in the ability of microglia to proliferate were observed between TAC1(-/-) and WT mice, suggesting that SP may alter microglial density through chemotaxic recruitment. SP was confirmed to dose-dependently attract microglia using a trans-well culture system. Mechanistic studies revealed that both the SP receptor neurokinin-1 receptor (NK1R) and the superoxide-producing enzyme NADPH oxidase (NOX2) were necessary for SP-mediated chemotaxis in microglia. Furthermore, genetic ablation and pharmacological inhibition of NK1R or NOX2 attenuated SP-induced microglial migration. Finally, protein kinase Cδ (PKCδ) was recognized to couple SP/NK1R-mediated NOX2 activation. Altogether, we found that SP partly accounts for the increased density of microglia in the SN through chemotaxic recruitment via a novel NK1R-NOX2 axis-mediated pathway.

Subpicomolar diphenyleneiodonium inhibits microglial NADPH oxidase with high specificity and shows great potential as a therapeutic agent for neurodegenerative diseases.

Activation of microglial NADPH oxidase (NOX2) plays a critical role in mediating neuroinflammation, which is closely linked with the pathogenesis of a variety of neurodegenerative diseases, including Parkinson's disease (PD). The inhibition of NOX2-generated superoxide has become an effective strategy for developing disease-modifying therapies for PD. However, the lack of specific and potent NOX2 inhibitors has hampered the progress of this approach. Diphenyleneiodonium (DPI) is a widely used, long-acting NOX2 inhibitor. However, due to its non-specificity for NOX2 and high cytotoxicity at standard doses (µM), DPI has been precluded from human studies. In this study, using ultra-low doses of DPI, we aimed to: (1) investigate whether these problems could be circumvented and (2) determine whether ultra-low doses of DPI were able to preserve its utility as a potent NOX2 inhibitor. We found that DPI at subpicomolar concentrations (10(-14) and 10(-13) M) displays no toxicity in primary midbrain neuron-glia cultures. More importantly, we observed that subpicomolar DPI inhibited phorbol myristate acetate (PMA)-induced activation of NOX2. The same concentrations of DPI did not inhibit the activities of a series of flavoprotein-containing enzymes. Furthermore, potent neuroprotective efficacy was demonstrated in a post-treatment study. When subpicomolar DPI was added to neuron-glia cultures pretreated with lipopolysaccharide, 1-methyl-4-phenylpyridinium or rotenone, it potently protected the dopaminergic neurons. In summary, DPI's unique combination of high specificity toward NOX2, low cytotoxicity and potent neuroprotective efficacy in post-treatment regimens suggests that subpicomolar DPI may be an ideal candidate for further animal studies and potential clinical trials.

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.

Mitochondriotropic lanthanide nanorods: implications for multimodal imaging.

Two-photon active mitochondriotropic lanthanide nanorods for high resolution fluorescence imaging. The presence of Gd in the nanorods also enabled us to utilize this material as a T1-T2 dual-mode contrast reagent for recording magnetic resonance images of the mouse brain.

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.