We thank the Scientific Computing and Imaging Institute of the University of Utah for use of FluoRender software (R01GM09815). This work was supported by grants from the Glaucoma Research Foundation, Melsa M. and Frank Theodore Barr Foundation and the US National Institute of Health, (R01EY020878 and R01EY023621) to M.L.V., and (R01EY017182 and R01EY017950) to B.K.A.

The authors have nothing to disclose.

رصد حي لعدد الخلايا دبقية وتفعيل الصرفي أثناء مرض الاعصاب يتطلب استخدام أساليب التصوير غير الغازية التي تسمح التصور التفصيلي من الميزات الخلية. بعد التصوير، والخلايا الدبقية يجب أن تكون معزولة (مجزأة) للتحليل المظهرية عن طريق استخدام الخطوات عتبة متعددة لتقييم حجم ...

الخلايا الدبقية الصغيرة هي خلايا neuroimmune الموجودة حصرا في الجهاز العصبي المركزي (CNS) منذ التطور الجنيني المبكرة وطوال فترة البلوغ. مجهزة ذخيرة معقدة من المستقبلات، وينظم النشاط دبقية وعدم التجانس الإقليمي من خلال تفاعلها ثنائي الاتجاه مع الخلايا العصبية المجاورة، الدبقية، حاجز الدم في الدماغ والخلايا التسلل neuroinflammatory 1،2. تساهم ظائف دبقية القاعدية لصيانة وإصلاح الفسيولوجية، كما عينة أراضيها لاضطرابات في التوازن 3،4. أثناء إصابة الجهاز العصبي المركزي أو المرض، والخلايا الدبقية الصغيرة هي أول المستجيبين للإشارات العصبية التي تؤدي ثم انتقالها إلى النمط الظاهري على رد الفعل، يسمى &quot;تنشيط الخلايا الدبقية الصغيرة 2،5-7. تنشيط الخلايا الدبقية الصغيرة ينطوي على دورة معقدة من الجينات والبروتينات التعبير، التي تقترن إلى سوما الخلية وعملية تغيير الحجم ويعيد 6-9. تنشيط الخلايا الدبقية الصغيرة، فضلا عن إعادة توزيع الخلايا والمجموعات، يمكن رافق الزيادة الإجمالية المحلية في عدد الخلايا (كثرة الدبيقيات تسميته). هذا يمكن أن تنجم عن تكاثر الخلايا وتجديد الذات، مع أو بدون توظيف حيدات الدم المستمدة 3،4،7،10-14. في مجموعة واسعة من وأمراض الجهاز العصبي المركزي المزمنة التي تعتمد على العمر وأصيب كثرة الدبيقيات وتفعيل الخلايا الدبقية الصغيرة مرض مواز التقدم 15-19. كيف أثر الدبقية الصغيرة التنكس العصبي لا تزال غير واضحة، وذلك أساسا لأنها تلعب الدورين اعصاب والضارة التي قد يكون لها مساهمات متنوعة لظهور المرض وتطور. وقد رصدت الدراسات التصويرية الحية تهدف إلى فهم مرض مزمن CNS السلوك دبقية في الجهاز العصبي المركزي المتضرر من النماذج الحيوانية والبشر، وأثبتت أن التعديلات دبقية هي بداية اكتشافها في مراحل المرض المبكرة 15-17،19،20. وبالتالي، فمن الأهمية بمكان لوضع نهج لكشف ورصد تفعيل دبقية في الجسم الحي. DETE غير الغازيةتأسست ction التغيرات الإقليمية في تنشيط الخلايا الدبقية الصغيرة في الدماغ باعتبارها مهمة في مؤشر الجسم الحي من تطور المرض الاعصاب، وذلك باستخدام التصوير الجزيئي أو تلألؤ بيولوجي وانبعاث البوزيترون التصوير المقطعي أو التصوير بالرنين المغناطيسي 18،21،22. هذه الطرق التصوير الجزيئي والنووي الكمية للغاية وغير الغازية كشف دباق لقرار الإقليمي. بدلا من ذلك، واثنين من الفوتون التصوير متحد البؤر في CX3CR1 GFP / + الفئران قد سمح مراقبة الخلايا الدبقية الصغيرة في الدماغ لقرار الخلوية 3،4،9،20،23-28. ومع ذلك، فإن هذا النهج يحد على المدى الطويل والمراقبة المتكررة من التعديلات دبقية المزمنة، وبالنظر إلى المخاطر المحتملة للإزعاج سلوكهم حتى مينيملي الإجراءات تصوير الدماغ 29. بدلا من ذلك، الشبكية تقدم الظروف المثلى لمباشرة في الجسم الحي التصور ورصد المتكررة من الخلايا الدبقية الصغيرة في حياتهم سليمة المتخصصة CNS طوال الشيخوخة، بعد الإصابة الحادة، ويحتمل أن تكون خلال الأمراض العصبية المزمنة. وهكذا، فقد أثبتت الدراسات الحديثة جدوى عالية الدقة التصوير من الخلايا الدبقية الصغيرة في شبكية العين معربا عن GFP من خلال تكييف تنظير العين مسح متحد البؤر الليزر (cSLO) إلى الصورة الحية CX3CR1 GFP / + الفئران. وقد استخدم هذا لتتبع التغيرات الأسبوعية في GFP + الخلايا الأرقام في الفئران الفردي لمدة تصل إلى 10 أسابيع بعد الإصابة التي يسببها ارتفاع ضغط الدم الحاد أو العين 30-36. فإننا قمنا بمد هذا النهج لأداء التصوير على المدى الطويل على مدى عدة أشهر، والكمية تعقب التغييرات في تنشيط الخلايا الدبقية الصغيرة استنادا إلى حجم سوما باستخدام تحليل المورفومترية. وقد عرفت حجم Somal كمقياس المفيد تفعيل الخلايا الدبقية الصغيرة في دراسات التصوير الحية باستخدام اثنين من الفوتون المجهري متحد البؤر في شرائح القشرية لأداء في مجال التصوير المجراة من CX3CR1-GFP + الخلايا الدبقية الصغيرة 9. كما أظهرت هذه الدراسات وغيرها العلاقة بين حجم somal ومستويات البروتين Iba1 التعبير، ذوي الخوذات البيضاءالتراث الثقافي غير المادي يزيد أيضا مع تفعيل 9،37. وبالتالي، يمكن تحديد تنشيط الخلايا الدبقية الصغيرة في الفئران الحية، ورصد أعدادها وتوزيعها على مر الزمن خلال CNS الصحة والمرض. يصف هذا البروتوكول طرق cSLO الحصول على الصور الحية وتحليل لرصد أعداد الخلايا الدبقية، توزيع وتفعيل الصرفي أثناء الشبكية خلايا العقدة (RGC) انحطاط (الشكل 1). وبالتالي، تستخدم هذه الدراسة: 1) نموذج الفأر من الزرق ورثت (DBA / 2J) أن يخضع تعتمد على سن العصب البصري والشبكية والتنكس العصبي يظهر التباين الملحوظ في تطور المرض ما بين 5 و 10 شهرا من العمر 38،39، 2) شهريا cSLO في الجسم الحي التصوير لرؤية طويلة الأجل لGFP + الخلايا في شبكية العين والعصب البصري امياليني من متخالف Cx3cr1 GFP / + DBA / 2J الفئران الذين تتراوح أعمارهم بين 3-5 أشهر، 3) تحليل التصوير الحي من قبل تجزئة والعتبة لعزل somata الخلية والتدبير المنطقة الأشعة تحت الحمراء. يتم تطبيق هذه الاستراتيجيات لتقييم حركية الشبكية دول تفعيل دبقية خلال المراحل المبكرة من الزرق المزمن.

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			<td height="21" style="height:21px;width:617px;">Name</td>
			<td style="width:444px;">Company</td>
			<td style="width:208px;">Catalog Number</td>
			<td style="width:741px;">Comments</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:617px;">DBA/2J and 
			CX3CR1-GFP/+ mice</td>
			<td>The Jackson Laboratory, Bar Harbor, ME</td>
			<td>000671 and 00582</td>
			<td>Mice are breed in-house, introducing new breeders every 3 to 4 generations to prevent genetic drift.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">301/2G needle and 1 ml tuberculin slip-tip syringe</td>
			<td>BD, East Rutherford, NJ</td>
			<td>305106 and 309659</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:617px;">2,2,2-tribromoethanol, tert-Amyl alcohol 99% and phosphate buffer saline tablets</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>T48402, 152463 and P4417</td>
			<td>Avertin solution (pH 7.3, sterile filtered) must be freshly made solution or stored at 4&#186;C for up to 1 week.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heat therapy T/Pump</td>
			<td>Gaymar Industries, Orchard Park, NY</td>
			<td>TP-650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cotton-tipped applicators</td>
			<td>Fisher Scientific, Pittsburgh, PA</td>
			<td>23-400-100</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Tropicamide 1%&#160;</td>
			<td>Bausch &#38; Lomb, Rochester, NY</td>
			<td>NDC 24208-585-64</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PMMA contact lenses, 1.70-base curve, 3.2 mm total diameter, 0.40 mm-thick center</td>
			<td>Cantor &#38; Nissel Ltd., Northamptonshire, UK</td>
			<td>G003709</td>
			<td>Lenses are rinsed with sterile PBS and stored in polypropylene boxes.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HRA/Spectralis confocal scanning laser ophthalmoscope and Eye Explorer software</td>
			<td>Heidelberg Engineering GmbH, Carlsbad, CA</td>
			<td>Version 1.7.1.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PowerPoint</td>
			<td>Microsoft, Redmond, WA</td>
			<td>Version 14.4.3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adobe Photoshop</td>
			<td>Adobe, San Jose, CA</td>
			<td>Version CS3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FluoRender</td>
			<td>Scientific Computing and Imaging Institute, University of Utah</td>
			<td>Version 2.13</td>
			<td>Freeware. http://www.sci.utah.edu/software/13-software/127-fluorender.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NIS-Elements C</td>
			<td>Nikon, Melville, NY</td>
			<td>Version 4.30.01</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo dynamics of retinal microglial activation during neurodegeneration: confocal ophthalmoscopic imaging and cell morphometry in mouse glaucoma

<html> الخلايا الدبقية الصغيرة، وهي الخلايا neuroimmune CNS المقيمة، وتحويل التشكل وحجمها استجابة لأضرار CNS، والتحول إلى حالة تنشيط وظائف متميزة وملامح التعبير الجيني. أدوار تفعيل دبقية في مجالات الصحة والاصابة والمرض لا تزال غير مفهومة تماما بسبب التنظيم الديناميكي والمعقد في استجابة للتغيرات في المكروية بهم. وبالتالي، فمن الأهمية بمكان أن مراقبة غير جراحية وتحليل التغيرات في تفعيل دبقية مع مرور الوقت في الكائن الحي سليمة. في الجسم الحي تأخرت الدراسات تفعيل دبقية التي كتبها القيود التقنية لتتبع سلوك دبقية دون تغيير البيئة CNS. وقد كان هذا تحديا من نوع خاص خلال التنكس العصبي المزمن، حيث يجب أن تعقب التغييرات على المدى الطويل. شبكية العين، وهو جهاز العصبي المركزي قابلة للتصوير العيش غير الغازية، ويوفر نظام قوي لتصور وتميز ديناميات تنشيط الخلايا الدبقية الصغيرة خلال الاضطرابات المزمنة. الحمار = &quot;jove_content&quot;&gt; يعرض هذا البروتوكول الطرق على المدى الطويل، والتصوير في الجسم الحي من الخلايا الدبقية الصغيرة في شبكية العين، وذلك باستخدام تنظير العين متحد البؤر (cSLO) وCX3CR1 GFP الفئران / + المراسل، لتصور الخلايا الدبقية الصغيرة لقرار الخلوية. أيضا، نحن تصف أساليب لقياس التغيرات الشهرية في تنشيط الخلايا وكثافة في مجموعات فرعية الخلايا الكبيرة (200-300 الخلايا في شبكية العين). نحن نؤكد استخدام منطقة somal كمقياس مفيدة لتتبع مباشر لتفعيل دبقية في شبكية العين من خلال تطبيق آلية تحليل المورفومترية القائم على عتبة في الجسم الحي الصور. نحن نستخدم هذه الحصول على الصور الحية ويحلل استراتيجيات لرصد التغيرات الديناميكية في تفعيل دبقية وكثرة الدبيقيات خلال المراحل المبكرة من التنكس العصبي في شبكية العين في نموذج الفأر من الزرق المزمن. وينبغي أن يكون هذا النهج مفيدا لمعرفة مدى مساهمة الخلايا الدبقية الصغيرة إلى انخفاض العصبية ومحور عصبي في اضطرابات الجهاز العصبي المركزي المزمنة التي تؤثر على شبكية العين والعصب البصري.

استخدمت لدينا مؤخرا في الدراسات المجراة هذه الحصول على الصور وتحليل أساليب العيش لتصور وتتبع حركية وأنماط ONH والتغيرات دبقية صغيرة في شبكية العين خلال المراحل المبكرة من الزرق المزمن وعلاقتها أواخر التنكس العصبي 59. نحن هنا توضيح cSLO بروتوكول الحصول ع?...

Watch this Scientific Journal Video about In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma at JoVE.co

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

تنشيط الخلايا الدبقية الصغيرة وكثرة الدبيقيات استجابات الرئيسية لالتنكس العصبي المزمن. هنا، فإننا نقدم طرق لفي الجسم الحي، والتصور على المدى الطويل CX3CR1-GFP + الخلايا الدبقية الشبكية بواسطة تنظير العين متحد البؤر، وعتبة والمورفولوجية تحليل لتحديد وقياس تفعيلها. نحن نراقب التغيرات دبقية خلال المراحل المبكرة من الزرق المرتبطة بالتقدم في السن.

في فيفو ديناميات الشبكية دبقية التنشيط خلال تنكس عصبي: متحد البؤر Ophthalmoscopic التصوير والقياس الشكلي خلية في الماوس الزرق

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

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In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

10.5K Views.  University of Utah.  تنشيط الخلايا الدبقية الصغيرة وكثرة الدبيقيات استجابات الرئيسية لالتنكس العصبي المزمن. هنا، فإننا نقدم طرق لفي الجسم الحي، والتصور على المدى الطويل CX3CR1-GFP + الخلايا الدبقية الشبكية بواسطة تنظير العين متحد البؤر، وعتبة والمورفولوجية ...

Video: In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

متعدد الفوتون التصوير من غزو خلايا الورم في الفأر نموذج مثلي الفم سرطان الخلايا الحرشفية

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

في ضوء بارد التصوير حيوية الجسم الحي من نقص الأكسجة ورم من الدماغ الانبثاث سرطان الثدي في نموذج الفأر

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

في الجسم الحي التصوير من ورم الأوعية الدموية باستخدام الإسفار متحد البؤر Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

في الجسم الحي 19 F التصوير بالرنين المغناطيسي لتتبع الخليوي

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

تسلسلي<em&gt; في الجسم الحي</em&gt; التصوير من الخلايا الجذعية المكونة للعظم / السلف خلال اصلاح الكسر

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

القياس عن بعد داخل الرحم لقياس ماوس مقلص الضغط<em&gt; في فيفو</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

روز البنغال Photothrombosis بواسطة التصوير الضوئي متحد البؤر<em&gt; في فيفو</em&gt;: نموذج لسفينة واحدة السكتة الدماغية

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

الإجراءات في يحفز الزرق<em&gt; في فيفو</em&gt; نموذج الفأر وجبل لالجامع إعداد الشبكية

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

نضوب خلايا الفأر من Xenografts ورم الإنسان يحسن بشكل ملحوظ تحليل المصب من الخلايا المستهدفة

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

صبغة الفلورسنت وصفها من كرات الدم الحمراء والكريات البيض لدراسة ديناميات التدفق في الماوس دوران الشبكية

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...