המחברים מודים תמיכה של איגוד הלב האמריקני (0735136N), דנה קרן, הקרן הלאומית למדע (CBET/0737731), וקרן קולטר.

1. הדמיה ההתקנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מצלמה עם עדשת זום מאקרו צריך להיות מותקן על הבמה אנכי או מיקרוסקופ כירורגי (במקום עדשת זום מאקרו, מטרה מיקרוסקופ ואת העדשה או מערכת פשוטה עם שתי עדשות יכול לשמש בהתאם ההגדלה הרצוי). </li><li style=";text-align:right;direction:rtl"> הורד את התוכנה המתאימה מאתר האינטרנט שלנו לשלוט על המצלמה ( <a href="http://bach.bme.utexas.edu/mediawiki/index.php/Software">http://bach.bme.utexas.edu/mediawiki/index.php/Software</a> ). </li><li style=";text-align:right;direction:rtl"> תוכנת המצלמה יש להשתמש כדי לאשר חפץ הוא להתמקד בגובה הרצוי </li><li style=";text-align:right;direction:rtl"> דיודת לייזר עם ערכת collimation אמור להיות להגדיר כך מסתעף אור לייזר מאירה את האובייקט. </li><li style=";text-align:right;direction:rtl"> הפעל את / כיבוי כל אור הסביבה כדי לאשר את אור הלייזר הוא שווה להאיר את כל שדה הראייה של המצלמה. </li><li style=";text-align:right;direction:rtl"> בדוגמה זו, אור אדום לייזר שימש כי זה היה יותר קל להדגים כיצד לבנות את המערכת, אבל אור לייזר אינפרא אדום יכול בקלות לשמש והיה התועלת בנוסף לחדור עמוק יותר לרקמות. כמו כן, עם מסננים מתאימים מול המצלמה כדי לחסום את האור הנראה, אור אינפרא אדום יכול לשמש עם אורות חדר. </li></ol> 2. כירורגי הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זהו ניתוח לא הישרדות, כי הניסוי יכול להיעשות באופן כרוני באמצעות חלון חדר במחקר הישרדות. </li><li style=";text-align:right;direction:rtl"> להרדים את החיה ולמקם אותו למסגרת stereotactic. </li><li style=";text-align:right;direction:rtl"> הסר את העור ואת הרקמה הסובבת את הגולגולת. </li><li style=";text-align:right;direction:rtl"> בעזרת מקדח שיניים, דק את הגולגולת על האזור במוח הרצוי שקיפות נזהר לשטוף את המשטח עם תמיסת מלח לעתים קרובות על מנת למנוע נזק למוח. </li><li style=";text-align:right;direction:rtl"> השתמש בטון שיניים כדי ליצור היטב סביב האזור הרצוי ולאחר מכן למקם טיפת שמן מינרלי או ג&#39;ל סיליקון לתוך הבאר כדי לשפר את הראות. </li><li style=";text-align:right;direction:rtl"> לחלופין, הגולגולת ניתן להסיר את חלון החדר יכול להיווצר כאן. </li></ol> 3. איסוף נתונים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש בתוכנת המצלמה לרכוש תמונות גם לחשב רבב ערכי הניגודיות. </li><li style=";text-align:right;direction:rtl"> מניחים את החיה לתוך שדה הראייה של המצלמה ולהתאים את גובה המצלמה או מוקד העדשה עד תמונות ברורות של vasculature נראים. </li><li style=";text-align:right;direction:rtl"> ודא שיש מספיק אור לייזר הוא מגיע ללא מצלמה להרוות אותו. שימוש בהיסטוגרמה של התמונה, להתאים את כוח לייזר כדי להבטיח את הרוב של פיקסלים במצלמה שמחים קיבולת כמחצית שלהם. </li><li style=";text-align:right;direction:rtl"> בחר את מספר התמונות שאתה רוצה לרכוש כמה ממוצעים לעשות לפני תחילת הניסוי. </li><li style=";text-align:right;direction:rtl"> לאחר שהניסוי יתחיל, שינויים בזרימת הדם יכול בקלות להיות במעקב על ידי בחירת אזורים של עניין או על ידי יצירת דימוי של זרימת הדם יחסית. </li></ol> 4. נציג תוצאות איור 1 מציג דוגמה תמונה טיפוסית רבב גלם תמונה בניגוד המרה רבב כי צריך להיות שנוצר בעת שימוש בתוכנה כדי לבדוק את זרימת הדם במוח. עבור חזותי שינויים בזרימת הדם, קל יותר לקבל את התוכנה ליצירת מפות היחסי של זרימת הדם. תרשים 2 מציג סדרה טיפוסית של תמונות זרימת הדם יחסית במהלך עלייה חולפת בזרימת הדם העוברים בשדה הראייה. הצבע האדום מייצג עלייה בזרימת הדם, בעוד הצבע הכחול מראה על ירידה. הצבע הירוק מציין כי אין שינוי בזרימת הדם יחסית הבסיס נתון. <img src="/files/ftp_upload/2004/2004fig1.jpg" alt="איור 1" /> באיור 1. דוגמה גלם רבב תמונה (משמאל) לעומת רבב תמונה (מימין). <img src="/files/ftp_upload/2004/2004fig2.jpg" alt="איור 2" /> איור 2. דוגמה מספר תמונות יחסית תזרים דם בנקודות זמן שונות במהלך עלייה חולפת בזרימת הדם בעקבות ירידה בזרימת הדם.

<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>

אין ניגודי אינטרסים הכריז.

בסרטון זה אנו הוכיחו כמה קל לבנות ולהשתמש לייזר רבב הדמיה הניגוד (LSCI) מערכת להסתכל על השינויים בזרימת הדם. LSCI פותחה בשנת 1980 כדרך ליצירת מפות של זרימת הדם ברשתית 1. אמנם עדיין משמשת התמונה זלוף הרשתית ועור, זה הפך להיות מאוד פופולרי כטכניקה לזרום תמונת הדם במוח 2. זהו בעיקר בשל הרזולוציה במרחב ובזמן מעולה בתנאי ואת הפשטות של המכשור. LSCI נעשה שימוש כדי לחקור את השינויים בזרימת הדם עקב הפעלת תפקודית 3,4, דיכאון הפצת קליפת המוח 5, 6,7 שבץ. כמו כן, יש את היתרון של שילוב בקלות עם טכניקות אחרות כמו ההחזרה הדמיה 8, dyes9 רגיש מתח, או בדיקות חמצן 10,11 כך פיזיולוגיים מרובות ניתן הדמיה בו זמנית.

הטכניקה היא צדדי מספיק כדי לשמש עם מגוון רחב של ציוד. הדברים היחידים הדרושים כדי לבצע את הניסוי הן מצלמה עם עדשה תואמת, דיודת לייזר מכל סוג שהוא עם בקר, ואת התוכנה המסופקת. טבלה של הציוד הספציפי נעשה שימוש בסרטון נכלל לעיל. רשימה מלאה של חלקים נוספים שניתן להשתמש בהם בניסוי הזה נמצא באתר האינטרנט שלנו, <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

לייזר רבב הדמיה ניגודיות (LSCI) היא טכניקה פשוטה אך רבת עוצמה המשמש הדמיה שדה מלא של זרימת הדם. טכניקה מנתח תנודות דפוס רבב דינמי לזהות תנועה של חלקיקים דומים מנתחת כיצד לייזר דופלר משמרות תדר כדי לקבוע מהירות החלקיקים. כי זה יכול לשמש כדי לפקח על התנועה של כדוריות דם אדומות, LSCI הפך לכלי פופולרי למדידת זרימת הדם ברקמות כגון הרשתית, העור, המוח. זה הפך להיות שימושי במיוחד במדעי המוח שבו זרימת הדם משתנה במהלך אירועים פיזיולוגיים כמו שבץ ההפעלה פונקציונלי, ואת שלילת קוטביות הפצת ניתן לכמת. LSCI גם אטרקטיבי מכיוון שהוא מספק רזולוציה במרחב ובזמן מעולה, תוך שימוש במכשור יקר זה יכול בקלות להיות משולבת עם שיטות הדמיה אחרות. כאן אנו מראים כיצד לבנות תוכנית ההתקנה LSCI ולהפגין את יכולתה לפקח על זרימת הדם שינויים במוח במהלך ניסוי בבעלי חיים.

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

וידאו זה מדגים כיצד לבנות לייזר רבב הדמיה ניגודיות (LSCI) מערכת שיכולה בקלות לשמש כדי לפקח על זרימת הדם.

כיצד לבנות לייזר רבב הדמיה ניגודיות (LSCI) מערכת לפקח זרימת הדם

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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

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

23.5K Views.  University of Texas at Austin. וידאו זה מדגים כיצד לבנות לייזר רבב הדמיה ניגודיות (LSCI) מערכת שיכולה בקלות לשמש כדי לפקח על זרימת הדם.

Video: 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.

הדם במוח חמצון מדידה על בסיס מרווה חמצן תלויי של זרחני

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

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.

איך ליצור ולהשתמש היריבות המשקפת

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.

הזרקת דם עצמיים על דגם דימום intracerebral ספונטנית עכברים

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).

בהדמית vivo של יושרה סיבי עצב הראייה על ידי contrast-enhanced MRI בעכברים

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.

שימוש בcaspase ריבוב Assay לקביעת אפופטוזיס בדגם נייד ההיפותלמוס

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.

הדמיה חיה של<em&gt; דרוזופילה</em&gt; הזחל Neuroblasts

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.

Microglia בתור biosensor סרוגייט לקביעת Nanoparticle neurotoxicity

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

How to Find Effects of Stimulus Processing on Event Related Brain Potentials of Close Others when Hyperscanning Partners

The partners of each pair must be able to pass the McGill Friendship Questionnaire without communicating. Each partner is then seated in front of a screen in one of two adjacent rooms. These rooms are separated by a glass window through which participants communicate to maintain feelings of togetherness while being fitted with the EEG cap. After checking for adequate EEG signals, the glass is covered by a curtain to prevent visual communication. Then, partners must be silent but are instructed to try to feel in the presence of their partner during the entire experiment. Just before it starts, participants are told that each of them will be presented with one image at a time and that these images will occur at the same time for both of them on their own screen. They are also instructed that, for each trial, the simultaneous images will always be different. However, unbeknownst to them, trials are randomized: only half of them are consistent with this instruction and actually include two different images. These trials form the DSC, that is, the different-stimuli condition. The other half of the trials are inconsistent with the instruction. They include two identical images and form the ISC (identical-stimuli condition). After the experiment, participants are sorted into two groups: those who reported that they felt in the presence of their partner during the majority of the trials and those who reported they did not. The impact of the stimulus processing of the partner is found by subtracting the mean voltages of the ERPs of the ISC (inconsistent with the instructions) from the ERPs of the DSC (consistent with the instructions) in at least two time windows (TWs): firstly, in the 75 to 150 ms TW, where the absolute values of these subtractions are greater, especially at right frontal sites, in those who felt in the presence of their partner than in those who did not; secondly, in the LPP time window (i.e., from 650 to 950 ms post onset), where ERPs are significantly less positive in the DSC than in the ISC in those in whom the raw results of the early (75-150ms) subtractions are negative.

This protocol describes key steps involved in assessing the sensitivity of the brain of one person to the stimulus processing of a close other by selecting pairs of partners, recording their electroencephalogram (EEG) simultaneously and computing their event-related brain potentials (ERPs).

כיצד למצוא את ההשפעות של גירוי עיבוד על אירועים הקשורים המוח פוטנציאל של קרובים אחרים כאשר Hyperscanning שותפים

The partners of each pair must be able to pass the McGill Friendship Questionnaire without communicating. Each partner is then seated in front of a screen ...

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.

חיים הדמיה ואחריו תא בודד מעקב צג Cell Biology, ההתקדמות שושלת היוחסין של אוכלוסיות עצבית מרובים

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.