الكتاب يقر بدعم من جمعية القلب الاميركية (0735136N) ، ومؤسسة دانا ، المؤسسة الوطنية للعلوم (CBET/0737731) ، ومؤسسة كولتر.

1. الإعداد والتصوير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وينبغي أن شنت الكاميرا مع عدسة التكبير الماكرو إلى مرحلة الرأسي أو الميكروسكوب الجراحي (بدلا من عدسة التكبير الكلي ، وهو الهدف عدسة المجهر وبسيطة أو نظام العدسة يمكنهما أن تستخدم اعتمادا على التكبير المطلوب). </li><li style=";text-align:right;direction:rtl"> تحميل البرمجيات المناسبة من موقعنا على الانترنت للسيطرة على الكاميرا ( <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"> وينبغي استخدام برنامج الكاميرا للتأكد من كائن هو في التركيز على الارتفاع المطلوب </li><li style=";text-align:right;direction:rtl"> ثم ينبغي ليزر ديود مع مجموعة إيزاء تقام متباعدة بحيث يضيء ضوء الليزر الكائن. </li><li style=";text-align:right;direction:rtl"> رفض / إيقاف كافة الإضاءة المحيطة للتأكد من أن ضوء الليزر هو إلقاء الضوء بالتساوي الحقل بأكمله نظر الكاميرا. </li><li style=";text-align:right;direction:rtl"> في هذا المثال ، تم استخدام ضوء الليزر الأحمر لأنه كان من الأسهل لشرح كيفية بناء النظام ، ولكن الأشعة تحت الحمراء ضوء الليزر يمكن بسهولة مثلما يتم استخدام وسيكون له فائدة على اختراق أعمق وبالإضافة إلى ذلك في الأنسجة. أيضا ، مع الفلاتر المناسبة أمام الكاميرا لحجب الضوء المرئي ، يمكن أن تستخدم الأشعة تحت الحمراء مع أضواء على الغرفة. </li></ol> 2. تحضير جراحي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> هذه هي عملية جراحية غير البقاء ، رغم أنه يمكن القيام به التجربة مزمن في دراسة البقاء على قيد الحياة باستخدام نافذة الغرفة. </li><li style=";text-align:right;direction:rtl"> تخدير الحيوانات ووضعها في إطار المجسم. </li><li style=";text-align:right;direction:rtl"> إزالة الجلد والأنسجة المحيطة الجمجمة. </li><li style=";text-align:right;direction:rtl"> باستخدام حفر الأسنان والجمجمة رقيقة فوق المنطقة المطلوب الدماغ على الشفافية والحرص على مسح السطح مع المياه المالحة في كثير من الأحيان من أجل تجنب إلحاق الضرر في الدماغ. </li><li style=";text-align:right;direction:rtl"> استخدام الاسمنت الأسنان لإنشاء بئر في جميع أنحاء المنطقة المرغوبة ومن ثم وضع قطرة من الزيت المعدني أو هلام السيليكون في البئر لتحسين الرؤية. </li><li style=";text-align:right;direction:rtl"> بدلا من ذلك ، يمكن إزالة الجمجمة ويمكن إنشاء نافذة الغرفة هنا. </li></ol> 3. جمع البيانات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام برنامج الكاميرا للحصول على الصور وأيضا حساب القيم رقطة التباين. </li><li style=";text-align:right;direction:rtl"> وضع الحيوانات في مجال الرؤية للكاميرا وضبط ارتفاع تركيز عدسة الكاميرا أو حتى ينظر إليها صورا واضحة من الأوعية الدموية. </li><li style=";text-align:right;direction:rtl"> تأكد من أن ما يكفي من ضوء الليزر هو الوصول إلى الكاميرا دون أن تشبع. باستخدام الرسم البياني للصورة ، ضبط قوة الليزر لضمان متحمسون غالبية بكسل الكاميرا لقدرتها حوالي النصف. </li><li style=";text-align:right;direction:rtl"> حدد عدد الصور التي ترغب في الحصول على ومدى المتوسط ​​يجب القيام به قبل بداية التجربة. </li><li style=";text-align:right;direction:rtl"> بمجرد أن تبدأ التجربة ، يمكن بسهولة تغيير تدفق الدم يمكن رصدها من خلال تحديد مناطق المصالح أو عن طريق توليد صورة من تدفق الدم النسبي. </li></ol> 4. ممثل النتائج ويظهر الشكل 1 مثالا لصورة نموذجية رقطة الخام وصورة النقيض تحويل رقطة التي ينبغي إنشاؤها عند استخدام البرنامج لدراسة تدفق الدم في الدماغ. لتصور التغيرات في تدفق الدم ، فمن الأسهل الحصول على برنامج توليد خرائط النسبية لتدفق الدم. ويبين الشكل 2 سلسلة الصور النمطية للالنسبية تدفق الدم خلال زيادة عابرة في تدفق الدم التي تنتقل عبر مجال الرؤية. اللون الأحمر يمثل زيادة في تدفق الدم في حين أن اللون الأزرق يدل على انخفاض. اللون الأخضر يشير إلى أنه لا يوجد أي تغيير في نسبة تدفق الدم إلى خط أساس معين. <img src="/files/ftp_upload/2004/2004fig1.jpg" alt="الشكل 1" /> الشكل 1. مثال لطخة الخام الصورة (يسار) ورقطة صورة النقيض (يمين). <img src="/files/ftp_upload/2004/2004fig2.jpg" alt="الشكل 2" /> الشكل 2. مثال قريب من عدة لوحات من تدفق الدم في نقاط زمنية مختلفة خلال زيادة عابرة في تدفق الدم يتبعه انخفاض في تدفق الدم.

<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>

الإعلان عن أي تضارب في المصالح.

في هذا الفيديو أظهرنا كم هو سهل لبناء واستخدام ليزر رقطة النقيض من التصوير (LSCI) نظام للنظر في التغيرات في تدفق الدم. وقد وضعت في 1980s LSCI كوسيلة لتوليد خرائط تدفق الدم في شبكية العين 1. في حين لا تزال تستخدم لصورة شبكية العين والجلد التروية ، فقد أصبح من شعبية للغاية ...

هذه التقنية هي مرنة بما فيه الكفاية ليتم استخدامها مع مجموعة واسعة من المعدات. الأشياء الوحيدة اللازمة لتنفيذ هذه التجربة هي كاميرا متوافقة مع العدسة ، ليزر ديود من أي نوع مع وحدة تحكم ، والبرامج المقدمة. يتم تضمين جدول محدد للمعدات المستخدمة في الفيديو أعلاه. وتوجد قائمة كاملة من أجزاء إضافية التي يمكن استخدامها في هذه التجربة على موقعنا ، <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

الليزر الرقطة التصوير التباين (LSCI) هي تقنية بسيطة لكنها قوية يستخدم لحقل التصوير الكامل لتدفق الدم. تقنية تحليل تقلبات في نمط رقطة الحيوية للكشف عن حركة جزيئات مشابهة لطريقة الليزر دوبلر يحلل التحولات التردد في تحديد سرعة الجسيمات. لأنه يمكن استخدامها لرصد حركة خلايا الدم الحمراء ، وأصبحت أداة LSCI شعبية لقياس تدفق الدم في الأنسجة مثل شبكية العين والجلد والدماغ. أصبح مفيدا بشكل خاص في علم الأعصاب ، حيث يمكن قياس التغيرات تدفق الدم خلال الأحداث الفسيولوجية مثل التنشيط وظيفية ، والسكتة الدماغية ، والاستقطاب الانتشار. LSCI جذابة أيضا لأنه يوفر ممتازة القرار المكانية والزمانية أثناء استخدام الأجهزة الرخيصة التي يمكن بسهولة دمجها مع غيرها من طرائق التصوير. هنا نبين كيفية بناء الإعداد LSCI وتثبت قدرتها على رصد التغيرات في تدفق الدم في الدماغ خلال تجربة الحيوانية.

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

هذا الفيديو يوضح كيفية بناء ليزر الرقطة التباين التصوير (LSCI) النظام التي يمكن استخدامها بسهولة لمراقبة تدفق الدم.

كيفية بناء ليزر الرقطة التباين التصوير (LSCI) نظام لمراقبة تدفق الدم

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 de Cartagena UDEC

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. هذا الفيديو يوضح كيفية بناء ليزر الرقطة التباين التصوير (LSCI) النظام التي يمكن استخدامها بسهولة لمراقبة تدفق الدم.

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://jove.unicartagenaproxy.elogim.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.

الأكسجة الدماغية قياس الأوكسجين في الدم واستنادا إلى الرواية التي تعتمد على التفسفر

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.

كيفية إنشاء واستخدام التنافس بينوكلر

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.

حقن الدم ذاتي النزيف الدماغي الداخلي النموذجي لالعفوي في الفئران

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).

في الجسم الحي التصوير من الألياف البصرية النزاهة العصب عن طريق التصوير بالرنين المغناطيسي تعزيز التباين في الفئران

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.

استخدام كاسباس المتعدد الفحص لتحديد موت الخلايا المبرمج في المهاد الخليوي نموذج

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.

التصوير المباشر ل<em&gt; ذبابة الفاكهة</em&gt; اليرقات Neuroblasts

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

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

التصوير الفيديو وخرائط الزمانية المكانية لتحليل الجهاز الهضمي على الحركة في الفئران

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

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.

الخلايا الدبقية الصغيرة بمثابة جهاز الاستشعار البيولوجي الأب البديل لتحديد الجسيمات النانوية العصبية

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.

يعيش تصوير متبوعاً بخلية واحدة تتبع لرصد خلية علم الأحياء وتطور نسب السكان العصبية متعددة

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.

كيفية بناء ليزر الرقطة التباين التصوير (LSCI) نظام لمراقبة تدفق الدم

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