Les auteurs reconnaissent le soutien de l&#39;American Heart Association (0735136N), Dana Foundation, la National Science Foundation (CBET/0737731), et la Fondation Coulter.

1. Imagerie Setup <ol><li> Un appareil photo avec un zoom macro doit être monté sur une scène verticale ou microscope chirurgical (au lieu d&#39;un zoom macro, un objectif de microscope et de l&#39;objectif ou un simple système à deux lentilles peuvent être utilisés selon l&#39;agrandissement désiré). </li><li> Télécharger un logiciel approprié à partir de notre site Web pour contrôler la caméra ( <a href="http://bach.bme.utexas.edu/mediawiki/index.php/Software">http://bach.bme.utexas.edu/mediawiki/index.php/Software</a> ). </li><li> Le logiciel de la caméra doit être utilisée pour confirmer un objet est la mise au point à la hauteur désirée </li><li> Une diode laser avec un kit de collimation doit alors être mis en place afin que la lumière laser divergent éclaire l&#39;objet. </li><li> Baissez / désactiver toutes de la lumière ambiante afin de confirmer que la lumière laser est uniformément éclairant le champ de vision de la caméra. </li><li> Dans cet exemple, le rouge la lumière laser a été utilisé parce qu&#39;il était plus facile de démontrer la façon de construire le système, mais une lumière laser infrarouge pourrait tout aussi bien être utilisée et aurait l&#39;avantage supplémentaire de pénétrer plus profondément dans les tissus. Aussi, avec des filtres appropriés en face de la caméra pour bloquer la lumière visible, l&#39;infrarouge peut être utilisée avec les lumières de la pièce de suite. </li></ol> 2. Préparation chirurgicale <ol><li> Il s&#39;agit d&#39;une chirurgie non-survie, bien que l&#39;expérience pourrait être fait de façon chronique dans une étude de survie en utilisant une fenêtre de chambre. </li><li> Anesthésier les animaux et le placer dans un cadre stéréotaxique. </li><li> Retirez la peau et des tissus entourant le crâne. </li><li> L&#39;utilisation d&#39;une fraise dentaire, mince du crâne sur la région du cerveau à la transparence voulue en prenant soin de rincer la surface avec une solution saline fréquemment afin d&#39;éviter d&#39;endommager le cerveau. </li><li> Utiliser du ciment dentaire à créer un puits dans la région souhaitée, puis déposer une goutte d&#39;huile minérale ou de gel de silicone dans le puits pour améliorer la visibilité. </li><li> Alternativement, le crâne pourrait être retirée et une fenêtre de la chambre pourrait être créé ici. </li></ol> 3. Collecte des données <ol><li> Utilisez le logiciel de la caméra pour acquérir des images et aussi de calculer des valeurs de contraste de speckle. </li><li> Placez l&#39;animal dans le champ de vision de la caméra et ajuster la hauteur de la caméra ou de se concentrer objectif jusqu&#39;à ce que des images claires de la vascularisation sont vus. </li><li> Assurez-vous que la lumière laser est suffisante pour atteindre l&#39;appareil photo sans le saturer. Utilisation de l&#39;histogramme de l&#39;image, ajuster la puissance du laser pour assurer la majorité des pixels de la caméra sont excités à environ la moitié de leur capacité. </li><li> Sélectionnez le nombre d&#39;images que vous souhaitez acquérir et combien en moyenne à faire avant de commencer l&#39;expérience. </li><li> Une fois que l&#39;expérience commence, les modifications du flux sanguin peut être facilement contrôlé en sélectionnant des régions d&#39;intérêt ou en générant une image du flux sanguin relatif. </li></ol> 4. Les résultats représentatifs La figure 1 montre un exemple typique d&#39;une crue de speckle image et un contraste de l&#39;image convertie chatoiement qui devraient être générés lors de l&#39;utilisation du logiciel pour examiner le flux sanguin dans le cerveau. Pour visualiser les changements du flux sanguin, il est plus facile d&#39;avoir le logiciel de générer des cartes relatives de la circulation sanguine. La figure 2 montre une série d&#39;images typiques de la circulation sanguine par rapport au cours d&#39;une augmentation transitoire du débit sanguin qui circule à travers le champ de vision. La couleur rouge représente une augmentation du débit sanguin tandis que la couleur bleue montre une diminution. La couleur verte indique qu&#39;il n&#39;y a pas de changement dans le flux sanguin par rapport à une référence donnée. <img src="/files/ftp_upload/2004/2004fig1.jpg" alt="Figure 1" /> Figure 1. Exemple d&#39;une image de speckle premières (à gauche) et le chatoiement contraste de l&#39;image (à droite). <img src="/files/ftp_upload/2004/2004fig2.jpg" alt="Figure 2" /> Figure 2. Exemple de plusieurs images relatives circulation sanguine à différents moments au cours d&#39;une augmentation transitoire du débit sanguin suivie par une diminution du débit sanguin.

<ol>
	<li>Briers, J. D., Fercher, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+blood-flow+visualization+by+means+of+laser+speckle+photography.">Retinal blood-flow visualization by means of laser speckle photography.</a> Invest. Ophthalmol. Vis. Sci. 22, 255-259 (1982).</li><li>Boas, D. A., Dunn, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+speckle+contrast+imaging+in+biomedical+optics.">Laser speckle contrast imaging in biomedical optics.</a> J. Biomed. Opt. 15, 011109-011109 (2010).</li><li>Dunn, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+imaging+of+total+cerebral+hemoglobin+concentration,+oxygenation,+and+blood+flow+during+functional+activation.">Simultaneous imaging of total cerebral hemoglobin concentration, oxygenation, and blood flow during functional activation.</a> Opt Lett. 28, 28-30 (2003).</li><li>Devor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+the+cortical+hemodynamic+response+to+cortical+and+thalamic+neuronal+activity.">Coupling of the cortical hemodynamic response to cortical and thalamic neuronal activity.</a> Proc Natl Acad Sci U S A. 102, 3822-3827 (2005).</li><li>Ayata, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pronounced+hypoperfusion+during+spreading+depression+in+mouse+cortex.">Pronounced hypoperfusion during spreading depression in mouse cortex.</a> J Cereb Blood Flow Metab. 24, 1172-1182 (2004).</li><li>Jones, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+multispectral+reflectance+imaging+and+laser+speckle+flowmetry+of+cerebral+blood+flow+and+oxygen+metabolism+in+focal+cerebral+ischemia.">Simultaneous multispectral reflectance imaging and laser speckle flowmetry of cerebral blood flow and oxygen metabolism in focal cerebral ischemia.</a> J. Biomed. Opt. 13, (2008).</li><li>Dunn, A. K., Bolay, H., Moskowitz, M. A., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+cerebral+blood+flow+using+laser+speckle.">Dynamic imaging of cerebral blood flow using laser speckle.</a> J Cereb Blood Flow Metab. 21, 195-201 (2001).</li><li>Dunn, A. K., Devor, A., Dale, A. M., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+extent+of+oxygen+metabolism+and+hemodynamic+changes+during+functional+activation+of+the+rat+somatosensory+cortex.">Spatial extent of oxygen metabolism and hemodynamic changes during functional activation of the rat somatosensory cortex.</a> Neuroimage. 27, 279-290 (2005).</li><li>Farkas, E., Bari, F., Obrenovitch, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-modal+imaging+of+anoxic+depolarization+and+hemodynamic+changes+induced+by+cardiac+arrest+in+the+rat+cerebral+cortex.">Multi-modal imaging of anoxic depolarization and hemodynamic changes induced by cardiac arrest in the rat cerebral cortex.</a> Neuroimage. 51, 734-742 (2010).</li><li>Sakadzic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+imaging+of+cerebral+partial+pressure+of+oxygen+and+blood+flow+during+functional+activation+and+cortical+spreading+depression.">Simultaneous imaging of cerebral partial pressure of oxygen and blood flow during functional activation and cortical spreading depression.</a> Appl. Opt. 48, (2009).</li><li>Ponticorvo, A., Dunn, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+imaging+of+oxygen+tension+and+blood+flow+in+animals+using+a+digital+micromirror+device.">Simultaneous imaging of oxygen tension and blood flow in animals using a digital micromirror device.</a> Opt Express. 18, 8160-8170 (2010).</li></ol>

Dans cette vidéo, nous avons démontré combien il est facile à construire et à utiliser un laser speckle imagerie de contraste (LSCI) système pour étudier les changements du flux sanguin. LSCI a été développé dans les années 1980 comme un moyen de générer des cartes de débit sanguin dans la rétine 1. Alors qu&#39;il était encore utilisé pour l&#39;image de la rétine et la perfusion cutanée, il est devenu extrêmement populaire comme une technique à l&#39;image du flux sanguin dans le cerve...

La technique est assez polyvalent pour être utilisé avec une large gamme d&#39;équipements. Les seules choses nécessaires pour réaliser l&#39;expérience sont d&#39;une caméra compatible avec une lentille, une diode laser de tout type avec un contrôleur, et le logiciel fourni. Un tableau des équipements spécifiques utilisés dans la vidéo est inclus ci-dessus. Une liste complète des pièces supplémentaires qui peuvent être utilisés dans cette expérience se trouve sur notre site, <a href="http://bach.bme.utexas.edu/mediawiki/index.php/Hardware">http://bach.bme.utexas.edu/mediawiki/index.php/Hardware</a>

how to build a laser speckle contrast imaging (lsci) system to monitor blood flow

Speckle du laser d&#39;imagerie de contraste (LSCI) est une technique simple mais puissant qui est utilisé pour plein Imagerie du flux sanguin. La technique de l&#39;analyse des fluctuations dans une dynamique speckle pour détecter le mouvement des particules similaires à la façon dont les analyses laser Doppler décalages de fréquence pour déterminer la vitesse des particules. Parce qu&#39;il peut être utilisé pour surveiller le mouvement des globules rouges, LSCI est devenu un outil populaire pour mesurer le débit sanguin dans les tissus tels que la rétine, la peau et du cerveau. Il est devenu particulièrement utile dans les neurosciences, où les changements du flux sanguin lors d&#39;événements physiologiques comme l&#39;activation fonctionnelle, d&#39;AVC et la dépolarisation d&#39;épandage peut être quantifiée. LSCI est également intéressante car elle offre une excellente résolution spatiale et temporelle en utilisant l&#39;instrumentation peu coûteuse qui peut facilement être combiné avec d&#39;autres modalités d&#39;imagerie. Ici, nous montrons comment construire une installation LSCI et démontrer sa capacité à surveiller les changements du flux sanguin dans le cerveau au cours d&#39;une expérimentation animale.

Watch this Scientific Journal Video about How to Build a Laser Speckle Contrast Imaging LSCI System to Monitor Blood Flow at JoVE.com

How to Build a Laser Speckle Contrast Imaging LSCI System to Monitor Blood Flow

Cette vidéo montre comment construire un laser speckle imagerie de contraste (LSCI) système qui peut facilement être utilisé pour surveiller la circulation sanguine.

Comment construire un laser speckle imagerie de contraste (LSCI) Système pour surveiller la circulation sanguine

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow.  The technique analyzes ...

how-to-build-laser-speckle-contrast-imaging-lsci-system-to-monitor

Research

JoVE Journal

Neuroscience

How to Build a Laser Speckle Contrast Imaging (LSCI) System to Monitor Blood Flow

23.4K vues.  University of Texas at Austin. Cette vidéo montre comment construire un laser speckle imagerie de contraste (LSCI) système qui peut facilement être utilisé pour surveiller la circulation sanguine.

Vidéo: How to Build a Laser Speckle Contrast Imaging LSCI System to Monitor Blood Flow

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher 
spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis 
and treatment of neurovascular diseases such as stroke, Alzheimer's disease, and head injury.
Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. 
Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.
Click here to view the related article <a href="http://www.jove.com/Details.php?ID=1731">Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.</a>

We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

Mesure cérébrale oxygénation sanguine à base d&#39;oxygène-dépendant trempe de phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

Recherche

Neurosciences

How to Create and Use Binocular Rivalry

Each of our eyes normally sees a slightly different image of the world around us. The brain can combine these two images into a single coherent representation. However, when the eyes are presented with images that are sufficiently different from each other, an interesting thing happens: Rather than fusing the two images into a combined conscious percept, what transpires is a pattern of perceptual alternations where one image dominates awareness while the other is suppressed; dominance alternates between the two images, typically every few seconds. This perceptual phenomenon is known as binocular rivalry. Binocular rivalry is considered useful for studying perceptual selection and awareness in both human and animal models, because unchanging visual input to each eye leads to alternations in visual awareness and perception. To create a binocular rivalry stimulus, all that is necessary is to present each eye with a different image at the same perceived location. There are several ways of doing this, but newcomers to the field are often unsure which method would best suit their specific needs. The purpose of this article is to describe a number of inexpensive and straightforward ways to create and use binocular rivalry. We detail methods that do not require expensive specialized equipment and describe each method's advantages and disadvantages. The methods described include the use of red-blue goggles, mirror stereoscopes and prism goggles.

Binocular rivalry occurs when the eyes are presented with different images at the same location: one image dominates while the other is suppressed, and dominance alternates periodically. Rivalry is useful for investigating perceptual selection and visual awareness. Here we describe several easy methods for creating and using binocular rivalry stimuli.

Comment créer et utiliser la rivalité binoculaire

Each of our eyes normally sees a slightly different image of the world around us. The brain can combine these two images into a single coherent ...

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model. While ICH
accounts for 10-15% of all strokes, there remains no specific
effective therapy. The autologous blood injection model in mice
involves the stereotaxic injection of arterial blood into the basal
ganglia mimicking a spontaneous hypertensive hemorrhage in man. The
response to hemorrhage can then be studied in vivo and the
neurobehavioral deficits quantified, allowing for description of the
ensuing pathology and the testing of potential therapeutic agents. The
procedure described in this protocol uses the double injection
technique to minimize risk of blood reflux up the needle track, no
anticoagulants in the pumping system, and eliminates all dead space
and expandable tubing in the system.

The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.

Injection de sang autologue au modèle spontané Hémorragie intracérébrale chez la souris

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

Dans l&#39;imagerie in vivo de Optic Nerve Fiber intégrité par IRM de contraste amélioré chez la souris

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

Utilisation d&#39;un multiplexage Caspase test destiné à déterminer l&#39;apoptose dans une cellule du modèle hypothalamique

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Imagerie en direct de<em&gt; Drosophila</em&gt; larves Neuroblastes

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

Microglies comme un biocapteur Surrogate pour déterminer Nanoparticules neurotoxicité

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Vivre l’imagerie suivie d’une cellule unique de suivi pour surveiller la biologie cellulaire et la Progression de la lignée de plusieurs Populations de neurones

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

How to Obtain Reliable Visual Event-related Potentials in Newborns

The present study discusses the characteristics of visual event-related potentials (VEPs) and outlines methodological steps for obtaining reliable measurements in newborns. Obtaining high-quality, reliable VEPs is crucial for the early detection of abnormal development of the central nervous system in at-risk newborns, and for implementing successful early interventions. Recommendations are based on a previous study which showed that when post-conceptional age, polysomnography-identified sleep stages, and light-emitting diodes (LEDs) googles as the luminous source are controlled, no more than 4 repetitions of VEP averages are required to obtain replicable recordings, variability decreases, and reliable VEPs can be obtained. By controlling for these sources of variability and using statistical analyses, we were able to clearly and reliably identify the amplitude and latency of three main components (NII, PII and NIII) present in 100% of newborns (n = 20) during active sleep. Recording VEPs during awake states, quiet sleep and transitional sleep is not recommended because VEP morphology may differ significantly from one average to the next, leading to the risk of misleading clinical prognoses. Moreover, it is easier to obtain VEPs during active sleep because this state can be clearly and reliably identified at this stage of development, sleep cycles are short enough to allow measurements to be taken in a reasonable time, and the method does not require new o expensive equipment.

Several important points for obtaining high-quality reliable visual evoked potentials (VEPs) in newborns while minimizing variability and the risk of misleading prognoses are presented.

Comment obtenir des potentiels fiables liés aux événements visuels chez les nouveau-nés

The present study discusses the characteristics of visual event-related potentials (VEPs) and outlines methodological steps for obtaining reliable ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

Un système de neuronavigation stéréotaxique et d’ultrasons focalisés guidé par image à haut débit pour l’ouverture de la barrière hémato-encéphalique chez les rongeurs

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...