Synaptogenesis by Cholinergic Stimulation of Astrocytes.

Astrocytes release numerous factors known to contribute to the process of synaptogenesis, yet knowledge about the signals that control their release is limited. We hypothesized that neuron-derived signals stimulate astrocytes, which respond to neurons through the modulation of astrocyte-released synaptogenic factors. Here we investigate the effect of cholinergic stimulation of astrocytes on synaptogenesis in co-cultured neurons. Using a culture system where primary rat astrocytes and primary rat neurons are first grown separately allowed us to independently manipulate astrocyte cholinergic signaling. Subsequent co-culture of pre-stimulated astrocytes with naïve neurons enabled us to assess how prior stimulation of astrocyte acetylcholine receptors uniquely modulates neuronal synapse formation. Pre-treatment of astrocytes with the acetylcholine receptor agonist carbachol increased the expression of synaptic proteins, the number of pre- and postsynaptic puncta, and the number of functional synapses in hippocampal neurons after 24 h in co-culture. Astrocyte secretion of the synaptogenic protein thrombospondin-1 increased after cholinergic stimulation and inhibition of the receptor for thrombospondins prevented the increase in neuronal synaptic structures. Thus, we identified a novel mechanism of neuron-astrocyte-neuron communication, where neuronal release of acetylcholine stimulates astrocytes to release synaptogenic proteins leading to increased synaptogenesis in neurons. This study provides new insights into the role of neurotransmitter receptors in developing astrocytes and into our understanding of the modulation of astrocyte-induced synaptogenesis.

Synaptogenesis by Cholinergic Stimulation of Astrocytes.

Astrocytes release numerous factors known to contribute to the process of synaptogenesis, yet knowledge about the signals that control their release is limited. We hypothesized that neuron-derived signals stimulate astrocytes, which respond by signaling back to neurons through the modulation of astrocyte-released synaptogenic factors. Here we investigate the effect of cholinergic stimulation of astrocytes on synaptogenesis in co-cultured neurons. Using a culture system where primary rat astrocytes and primary rat neurons are first grown separately allowed us to independently manipulate astrocyte cholinergic signaling. Subsequent co-culture of pre-stimulated astrocytes with naïve neurons enabled us to assess how prior stimulation of astrocyte acetylcholine receptors uniquely modulates neuronal synapse formation. Pre-treatment of astrocytes with the acetylcholine receptor agonist carbachol increased the expression of synaptic proteins, the number of pre- and postsynaptic puncta, and the number of functional synapses in hippocampal neurons after 24 hours in co-culture. Astrocyte secretion of the synaptogenic protein thrombospondin-1 increased after cholinergic stimulation and the inhibition of the target receptor for thrombospondins prevented the observed increase in neuronal synaptic structures. Thus, we identified a novel mechanism of neuron-astrocyte-neuron communication, i.e. , neuronal release of acetylcholine stimulates astrocytes to release synaptogenic proteins leading to increased synaptogenesis in neurons. This study provides new insights into the role of neurotransmitter receptors in developing astrocytes and into our understanding of the modulation of astrocyte-induced synaptogenesis.

Pathogenic T cell cytokines in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system that is believed to have an autoimmune etiology. As MS is the most common nontraumatic disease that causes disability in young adults, extensive research has been devoted to identifying therapeutic targets. In this review, we discuss the current understanding derived from studies of patients with MS and animal models of how specific cytokines produced by autoreactive CD4 T cells contribute to the pathogenesis of MS. Defining the roles of these cytokines will lead to a better understanding of the potential of cytokine-based therapies for patients with MS.

Myelin-specific CD8+ T cells exacerbate brain inflammation in CNS autoimmunity.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the CNS. Although CD4+ T cells are implicated in MS pathogenesis and have been the main focus of MS research using the animal model experimental autoimmune encephalomyelitis (EAE), substantial evidence from patients with MS points to a role for CD8+ T cells in disease pathogenesis. We previously showed that an MHC class I-restricted epitope of myelin basic protein (MBP) is presented in the CNS during CD4+ T cell-initiated EAE. Here, we investigated whether naive MBP-specific CD8+ T cells recruited to the CNS during CD4+ T cell-initiated EAE engaged in determinant spreading and influenced disease. We found that the MBP-specific CD8+ T cells exacerbated brain but not spinal cord inflammation. We show that a higher frequency of monocytes and monocyte-derived cells presented the MHC class I-restricted MBP ligand in the brain compared with the spinal cord. Infiltration of MBP-specific CD8+ T cells enhanced ROS production in the brain only in these cell types and only when the MBP-specific CD8+ T cells expressed Fas ligand (FasL). These results suggest that myelin-specific CD8+ T cells may contribute to disease pathogenesis via a FasL-dependent mechanism that preferentially promotes lesion formation in the brain.

Co-Culture of Neurons and Microglia.

Microglia, the resident immune cells of the brain, have been implicated in numerous neurodegenerative and neurodevelopmental diseases. Activation of microglia by a variety of stimuli induces the release of factors, including pro- and anti-inflammatory cytokines and reactive oxygen species, that contribute to modulating neuro-inflammation and oxidative stress, two crucial processes linked to disorders of the central nervous system. The in vitro techniques described here will provide a set of protocols for the isolation and plating of primary cerebellar granule neurons, primary cortical microglia from a mixed glia culture, and methods for co-culturing both cell types. These methods allow the study of how microglia and the factors they release in this shared environment mediate the effects of toxicants on neuronal function and survival. The protocols presented here allow for flexibility in experimental design, the study of numerous toxicological endpoints, and the opportunity to explore neuroprotective strategies. © 2017 by John Wiley & Sons, Inc.

Reporte de nuestra experiencia en la identificación del ganglio centinela en cáncer de mama: comparación entre la técnica de azul patente y la técnica combinada (azul patente + tecnecio 99) / Diagnostic accuracy of sentinel lymph node biopsy (sln) in the management of breast cancer at Hospital Privado Universitario de Córdoba: comparison of the detection rate between blue dye (patent blue) technique and combined technique (blue dye + radioactive coloid Tc99)

Objetivo Determinar la capacidad diagnóstica de la técnica del ganglio centinela (gc) en cáncer de mama temprano en el Hospital Privado Universitario de Córdoba. Comparar la tasa de detección entre la técnica de azul patente y la técnica combinada (azul patente + Tecnecio 99). Material y método Se estudiaron 640 pacientes con cáncer de mama en las que se realizó la biopsia del gc entre los años 2008 y 2016. Fueron divididas en: Grupo 1 (565 pacientes), en el que se utilizó solo azul patente como técnica para su identificación; y Grupo 2 (75 pacientes), en el que se empleó la técnica combinada. El estudio del gc se llevó a cabo con impronta citológica para el examen intraoperatorio y con hematoxilina y eosina (h/e) para el examen diferido. Se concretó la linfadenectomía axilar (la) en aquellos casos donde el gc fue positivo (por congelación o diferido) o bien en los que no pudo ser identificado. Resultados La tasa de detección global del gc fue del 94% (587), siendo del 91% (514) cuando se utilizó solo azul patente y de 97% (73) con la técnica combinada. El 18,6% (109) del total de los gc fueron positivos. La sensibilidad de la impronta citológica del gc fue del 69,7%. Conclusiones La biopsia del gc es el método de elección para la estadificación axilar en el cáncer mama inicial, ya que ha demostrado ser seguro, eficaz y asociarse a una baja morbilidad. Los porcentajes de identificación del gc ascendieron del 91% al 97% con la incorporación del Tc 99. Al comparar con series internacionales y nacionales, no hubo diferencias significativas en cuanto a la sensibilidad y tasa de falsos negativos del método intraoperatorio.

Objectives To determine the diagnostic accuracy of sentinel lymph node biopsy (sln) in the management of breast cancer at Hospital Privado Universitario de Córdoba. To compare the detection rate between blue dye (patent blue) technique and combined technique (blue dye + radioactive coloid Tc99). Materials and method 640 breast cancer patients treated with sln were studied between 2008 and 2016. They were divided in 2: Group 1, where snl was identified using blue dye; and Group 2, where a combined technique was used. The imprint cytology was used for intraoperative examination, and the evaluation with hematoxylin and eosin (h&e) was used for the deferred examination. An axillary lymph node dissection (alnd) was performed in positive sln cases and when the sln wasn´t founded. Results Overall detection rate of sln was 94% (587), while only 91% (514) when using patent blue, and 97% (73) when using blue dye and radioactive tracer combined. The 18,6% (109) of all sln were positive. The overall sensitivity of imprint cytology of sln was 69,7%. Conclusions sln biopsy is an accurate and safe technique to assess the axillary stage in our population. The identification percentage rose from 91% to 97% with the introduction of Tc 99. It was no difference in sensitivity and false negative rate between our results and national and international data.

Neurotoxicity of traffic-related air pollution.

The central nervous system is emerging as an important target for adverse health effects of air pollution, where it may contribute to neurodevelopmental and neurodegenerative disorders. Air pollution comprises several components, including particulate matter (PM) and ultrafine particulate matter (UFPM), gases, organic compounds, and metals. An important source of ambient PM and UFPM is represented by traffic-related air pollution, primarily diesel exhaust (DE). Human epidemiological studies and controlled animal studies have shown that exposure to air pollution, and to traffic-related air pollution or DE in particular, may lead to neurotoxicity. In particular, air pollution is emerging as a possible etiological factor in neurodevelopmental (e.g. autism spectrum disorders) and neurodegenerative (e.g. Alzheimer's disease) disorders. The most prominent effects caused by air pollution in both humans and animals are oxidative stress and neuro-inflammation. Studies in mice acutely exposed to DE (250-300µg/m3 for 6h) have shown microglia activation, increased lipid peroxidation, and neuro-inflammation in various brain regions, particularly the hippocampus and the olfactory bulb. An impairment of adult neurogenesis was also found. In most cases, the effects of DE were more pronounced in male mice, possibly because of lower antioxidant abilities due to lower expression of paraoxonase 2.

Microglia mediate diesel exhaust particle-induced cerebellar neuronal toxicity through neuroinflammatory mechanisms.

In addition to the well-established effects of air pollution on the cardiovascular and respiratory systems, emerging evidence has implicated it in inducing negative effects on the central nervous system. Diesel exhaust particulate matter (DEP), a major component of air pollution, is a complex mixture of numerous toxicants. Limited studies have shown that DEP-induced dopaminergic neuron dysfunction is mediated by microglia, the resident immune cells of the brain. Here we show that mouse microglia similarly mediate primary cerebellar granule neuron (CGN) death in vitro. While DEP (0, 25, 50, 100µg/2cm2) had no effect on CGN viability after 24h of treatment, in the presence of primary cortical microglia neuronal cell death increased by 2-3-fold after co-treatment with DEP, suggesting that microglia are important contributors to DEP-induced CGN neurotoxicity. DEP (50µg/2cm2) treatment of primary microglia for 24h resulted in morphological changes indicative of microglia activation, suggesting that DEP may induce the release of cytotoxic factors. Microglia-conditioned medium after 24h treatment with DEP, was also toxic to CGNs. DEP caused a significant increase in reactive oxygen species in microglia, however, antioxidants failed to protect neurons from DEP/microglia-induced toxicity. DEP increased mRNA levels of the pro-inflammatory cytokines IL-6 and IL1-ß, and the release of IL-6. The antibiotic minocycline (50µM) and the peroxisome proliferator-activated receptor-γ agonist pioglitazone (50µM) attenuated DEP-induced CGN death in the co-culture system. Microglia and CGNs from male mice appeared to be somewhat more susceptible to DEP neurotoxicity than cells from female mice possibly because of lower paraoxonase-2 expression. Together, these results suggest that microglia-induced neuroinflammation may play a critical role in modulating the effect of DEP on neuronal viability. .

Mechanisms of Neuroprotection by Quercetin: Counteracting Oxidative Stress and More.

Increasing interest has recently focused on determining whether several natural compounds, collectively referred to as nutraceuticals, may exert neuroprotective actions in the developing, adult, and aging nervous system. Quercetin, a polyphenol widely present in nature, has received the most attention in this regard. Several studies in vitro, in experimental animals and in humans, have provided supportive evidence for neuroprotective effects of quercetin, either against neurotoxic chemicals or in various models of neuronal injury and neurodegenerative diseases. The exact mechanisms of such protective effects remain elusive, though many hypotheses have been formulated. In addition to a possible direct antioxidant effect, quercetin may also act by stimulating cellular defenses against oxidative stress. Two such pathways include the induction of Nrf2-ARE and induction of the antioxidant/anti-inflammatory enzyme paraoxonase 2 (PON2). In addition, quercetin has been shown to activate sirtuins (SIRT1), to induce autophagy, and to act as a phytoestrogen, all mechanisms by which quercetin may provide its neuroprotection.

Role of glutamate receptors in tetrabrominated diphenyl ether (BDE-47) neurotoxicity in mouse cerebellar granule neurons.

The polybrominated diphenyl ether (PBDE) flame retardants are developmental neurotoxicants, as evidenced by numerous in vitro, animal and human studies. PBDEs can alter the homeostasis of thyroid hormone and directly interact with brain cells. Induction of oxidative stress, leading to DNA damage and apoptotic cell death is a prominent mechanism of PBDE neurotoxicity, though other mechanisms have also been suggested. In the present study we investigated the potential role played by glutamate receptors in the in vitro neurotoxicity of the tetrabromodiphenyl ether BDE-47, one of the most abundant PBDE congeners. Toxicity of BDE-47 in mouse cerebellar neurons was diminished by antagonists of glutamate ionotropic receptors, but not by antagonists of glutamate metabotropic receptors. Antagonists of NMDA and AMPA/Kainate receptors also inhibited BDE-47-induced oxidative stress and increases in intracellular calcium. The calcium chelator BAPTA-AM also inhibited BDE-47 cytotoxicity and oxidative stress. BDE-47 caused a rapid increase of extracellular glutamate levels, which was not antagonized by any of the compounds tested. The results suggest that BDE-47, by still unknown mechanisms, increases extracellular glutamate which in turn activates ionotropic glutamate receptors leading to increased calcium levels, oxidative stress, and ultimately cell death.

The brominated flame retardant BDE-47 causes oxidative stress and apoptotic cell death in vitro and in vivo in mice.

Polybrominated diphenyl ethers (PBDEs), used for decades as flame retardants, have become widespread environmental contaminants. Exposure is believed to occur primarily through diet and dust, and infants and toddlers have the highest body burden, raising concern for potential developmental neurotoxicity. The exact mechanisms of PBDE neurotoxicity have not been elucidated, but two relevant modes of action relate to impairment of thyroid hormone homeostasis and to direct effects on brain cells causing alterations in signal transduction, oxidative stress and apoptotic cell death. The present study shows that BDE-47 (2,2',4,4'-tetrabromodiphenyl ether) induces oxidative stress and ensuing apoptotic cell death in mouse cerebellar granule neurons in vitro. Similarly, in vivo administration of BDE-47, according to an exposure protocol shown to induce behavioral and biochemical alterations (10mg/kg, per os on post-natal day 10), induces oxidative stress and apoptosis, without altering serum levels of thyroid hormones. The effects of BDE-47 both in vitro and in vivo were more pronounced in a mouse model lacking the modifier subunit of glutamate cysteine ligase (GCLM) which results in reduced anti-oxidant capability due to low levels of GSH. Concentrations of BDE-47 in brain were in the mid-nanomolar range. These findings indicate that effects observed with BDE-47 in vitro are also present after in vivo administration, suggesting that in addition to potential endocrine effects, which were not seen here, direct interactions with brain cells should be considered as a potential mechanism of BDE-47 neurotoxicity.

Synaptic structure quantification in cultured neurons.

Behavioral problems (e.g., learning and memory) following developmental exposure to toxicants suggests that dysregulation of the process of synapse formation and function may occur. The ability to assess these changes is thus of value. This unit describes a method to investigate toxicant-induced changes to synaptic structure formation in primary hippocampal neurons using immunocytochemical labeling of the pre- and post-synaptic markers synaptophysin and PSD-95, confocal imaging, and three-dimensional object analysis. Protocols for the long-term culturing of primary hippocampal neurons and of primary cortical astrocytes, as well as their co-culture, are included. While the described methods focus on how astrocytes influence synapse formation and how toxicants may interfere in this process, modifications to the experimental plan can easily be implemented. This would allow for the investigation of the effects of toxicants after treating neurons alone, or both astrocytes and neurons in co-culture. With the common endpoint of synapse structure formation, differences between varying treatment paradigms can expand the understanding of the influence of particular toxicants on these diverse cell types and provide insight into potential mechanisms of effect and the contributions of each to synapse formation.

Neurotoxicants are in the air: convergence of human, animal, and in vitro studies on the effects of air pollution on the brain.

In addition to increased morbidity and mortality caused by respiratory and cardiovascular diseases, air pollution may also negatively affect the brain and contribute to central nervous system diseases. Air pollution is a mixture comprised of several components, of which ultrafine particulate matter (UFPM; <100 nm) is of much concern, as these particles can enter the circulation and distribute to most organs, including the brain. A major constituent of ambient UFPM is represented by traffic-related air pollution, mostly ascribed to diesel exhaust (DE). Human epidemiological studies and controlled animal studies have shown that exposure to air pollution may lead to neurotoxicity. In addition to a variety of behavioral abnormalities, two prominent effects caused by air pollution are oxidative stress and neuroinflammation, which are seen in both humans and animals and are confirmed by in vitro studies. Among factors which can affect neurotoxic outcomes, age is considered the most relevant. Human and animal studies suggest that air pollution (and DE) may cause developmental neurotoxicity and may contribute to the etiology of neurodevelopmental disorders, including autistic spectrum disorders. In addition, air pollution exposure has been associated with increased expression of markers of neurodegenerative disease pathologies.

Quantification of synaptic structure formation in cocultures of astrocytes and hippocampal neurons.

The ability to quantify changes of synaptic structure, whether associated with the formation of synapse in early development or the degeneration of synapses in adult life in an in vitro culture system, is important for understanding the underlying mechanisms. Astrocytes play a vital role in neuronal development and functioning, including synapse formation and stabilization. The method described in this chapter allows for the determination of the modulation by astrocytes of synaptic structure formation in hippocampal neurons. Using a sandwich coculture system, highly pure, hippocampal neurons are grown in culture for 14 days on glass coverslips, after which they are inverted, without contact, over separately cultured astrocytes or pretreated astrocytes for 24 h. Neuronal immunocytochemical staining of the presynaptic marker, synaptophysin, and the postsynaptic marker, PSD-95, is used to assess the localization of synaptic proteins into pre and postsynaptic structures. Deconvolved, confocal images are used to determine a mean puncta intensity threshold for use in rendering the surface of the synaptic structures using three-dimensional object analysis software. Once rendered in three-dimensional space, automatic quantification of the number of pre- and postsynaptic specializations and the number of those structures that overlap is obtained, allowing the ability to compare how different treatments may modulate the formation of synapses. Because synapses not only consist of distinct pre- and postsynaptic specializations, but are also defined by their apposition, the determination and study of synapse formation can only benefit by methods that use all of the available data to assess the actual structure.