This work was funded by National Health and Medical Research Project Grant APP1021582 and a Hunter Medical Research Institute grant sponsored by Sparke Helmore/NBN Triathlon and the Estate of the late Leslie Kenneth McFarlane.

يتم تنفيذ جميع الأعمال الحيوان في هذا البروتوكول مع الالتزام الصارم من جامعة نيوكاسل جنة الأخلاقيات الحيوانية وافق الإجراءات. 1. إعداد الآلات، وسائل الإعلام الثقافة وصحون <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل دافئ وسائل الإعلام 199 (TC199) أو Dulbecco لتعديل النسر متوسطة (DMEM) وسائل الاعلام إلى 37 ° C. قبل الأوكسجين والمتوسطة التي ظهرت على السطح مع 95٪ O 2/5٪ CO 2. تأكد من وسيلة لديه درجة الحموضة النهائية من 7.3. </li><li style=";text-align:right;direction:rtl"> إعداد خياطة بقطع اثنين من 5 أقسام سم لكل كيس. حلقة الغرز إلى عقدة مغلقة. </li></ol> 2. تشريح وإعداد الجهاز الهضمي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> سحب الصلبة الغذاء 12 ساعة قبل القتل الرحيم. إذا رغبت في ذلك، وضع الحيوانات على ملاحق جل المواد الغذائية خلال هذا الوقت. </li><li style=";text-align:right;direction:rtl"> الموت ببطء الفئران عن طريق جرعة زائدة من الصوديوم بنتوباربيتون ([200 مغ / كغ]، حقن داخل الصفاق) تليها خلع عنق الرحم وفقا لأخلاقيات بروتو المؤسسيالعواميد ورذاذ الايثانول 70٪ على البطن والصدر. </li><li style=";text-align:right;direction:rtl"> باستخدام مقص، وجعل شق أفقي في منتصف البطن وفضح البريتوني. </li><li style=";text-align:right;direction:rtl"> انتقل إلى فصل وإزالة الجهاز الهضمي عن طريق خفض الأمعاء الدقيقة العلوي من المعدة في العضلة العاصرة البوابية وقطع الأمعاء الغليظة على حافة الشرج. استخدام ملقط لإزالة بلطف مساريق. ضع الأمعاء في قبل تحسنت والمتوسطة الاوكسيجين. </li><li style=";text-align:right;direction:rtl"> تحديد الجزء من الأمعاء إلى تقييم للنفاذية (الشكل 1) وقطع هذا القسم الحر عن بقية الأمعاء. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> من أجل الحفاظ على الاتساق بين الحيوانات، وقياس أجزاء من العفج والصائم نسبة إلى المعدة، وقياس أجزاء من القولون والدقاق نسبة إلى الأعور. </li><li style=";text-align:right;direction:rtl"> عند اختيار شرائح الأنسجة، لاحظ وجود الغشاء المخاطي المرتبطة الأنسجة اللمفاوية مثل بقع باير ل. ويمكن تحديد هذه كما إيماءة صغيرةules على الجانب المصلي من التجويف. </li><li style=";text-align:right;direction:rtl"> باستخدام حقنة 1 مل، تدفق بلطف محتويات اللمعية الجزء الأمعاء في طبق بتري مع برنامج تلفزيوني قبل ساخنة (37 درجة مئوية). قد يتم تجاهل هذه المحتويات البرازية أو تخزينها في -80 ° C لتحليل المستقبل كما تريد. </li></ol></li></ol> 3. إعداد المعوية الحويصلات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحضير حقنة 1 مل مع حجم 300 ميكرولتر من مركب اختبار أو الجزيء. لسلامة الغشاء المخاطي، 1 ملغ / مل حل من FITC-ديكستران M.Wt. 4400 يمكن استخدامها. تحقيقات تتراوح بين 4،400-70،000 دا في حجم يمكن أن تستخدم لزيادة حساسية. تناسب بشكل آمن صغيرة قسطرة الأوعية الدموية الحيوانية على الحقنة. </li><li style=";text-align:right;direction:rtl"> قياس 5 سم من افتتاح الجزء الأمعاء وربط القطاع مغلقة بإحكام مع خياطة حلقة في هذه المرحلة. وضع بلطف خياطة حلقة مرتبطة مسبقا حول فتحة الأمعاء وادخال القسطرة أضعفت. سحب الخناق مغلقة وذلك لتأمين جزء الأمعاءوالافراج عن حجم 300 ميكرولتر من الحقنة في الأمعاء، وضمان أن كل حل يتم حقن. </li><li style=";text-align:right;direction:rtl"> إزالة بلطف القسطرة في حين سحب في وقت واحد الخناق خياطة لتأمين إغلاق الكيس المعوي. قطع كيس معوي فضفاض من الأمعاء والمكان الى 50 مل أنبوب مخروطي الشكل مليئة 20 مل من المتوسط ​​الاوكسيجين، مسخن الى 37 ° C. </li></ol> 4. قياس النفاذية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مكان الأنابيب المخروطية التي تحتوي على الأكياس المعوية في حمام الماء الساخن لتعيين 37 ° C. في 0، 30، 60، 90، و 120 نقطة زمنية دقيقة، أخذ عينة 100 ميكرولتر من أنبوب مخروطي الشكل ونقل إلى 96 لوحة جيدا، لتحل محل وحدة التخزين مع 100 ميكرولتر من وسائل الإعلام الجديدة في كل حالة. </li><li style=";text-align:right;direction:rtl"> بعد أخذ العينة النهائية، وقطع الأكياس المفتوحة عند نقطة خياطة ونزولا على طول هذا الجزء، وتعريض سطح الغشاء المخاطي. </li><li style=";text-align:right;direction:rtl"> قياس طول وعرض كل جزء في الأمعاء. إذا دسالأحمر، والمفاجئة تجميد شرائح ومخزن في -80 ° C للبروتين أو تحليل الكيمياء الحيوية، أو بدلا من ذلك، مخزن في حل استقرار RNA لفحوصات الجزيئية. </li><li style=";text-align:right;direction:rtl"> بناء منحنى مستوى التخفيفات سجل لجزيئات ذات الكلمات الدلالية FITC النطاق 1-1 × 10 -6. </li><li style=";text-align:right;direction:rtl"> عينات قياس ومعايير لFITC على قارئ لوحة نيون، FITC الإثارة / الانبعاثات: 495 نانومتر / 519 نانومتر. </li></ol> 5. حساب نفاذية على ما يبدو، لكل المعوية كيس الفردية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحويل وحدات الوقت للثانية. </li><li style=";text-align:right;direction:rtl"> لكل نقطة زمنية، وحساب تركيز التراكمي، س س ر = (C ر * V ص) + (س ر المبلغ * V ق)، حيث: س ر = تركيز التراكمي في الوقت t C ر = تركيز في الوقت t V ص = حجم التداول في الجانب المتلقي س ر مبلغ = مجموع كل س ر السابق V ق =حجم عينات </li><li style=";text-align:right;direction:rtl"> مؤامرة س مقابل الوقت (T) وحساب المنحدر: δQ / δt </li><li style=";text-align:right;direction:rtl"> حساب نفاذية واضحة (التطبيق P) P التطبيق = (δQ / δt) / (A * التعاون)، حيث: A = مساحة من الأنسجة C 0 = تركيز الأولي </li></ol>

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الكتاب تعلن أنه ليس لديهم مصالح مالية المتنافسة.

هنا، نحن في تفصيل العزلة وإعداد الحويصلات في الأمعاء لتقييم وظيفة حاجز المخاطية خارج الحي. وقد استخدمت الاستعدادات كيس معوي في المقام الأول في مجال البحوث الدوائية، دراسة امتصاص الأدوية مرشح عبر الأمعاء. ومع ذلك، هذا الاختبار هو على قدم المساواة مناسبة تماما ل?...

حاجز الظهارية في الأمعاء الجهاز الهضمي هو منطقة سطح الغشاء المخاطي تقدر ب 400 م 2 في الإنسان البالغ. ونتيجة لذلك، يتعرض باستمرار للطعن من الميكروبات، والمخدرات بلعها، المواد الغذائية والسموم البكتيرية. المضيف يجب أن لا يميز فقط بين البكتيريا المتعايشة مقبولة ومسببات الأمراض المحتملة، ولكن يجب منع هذه الأنواع والجزيئات التي يفرزها من عبور حاجز الظهارية، بينما في نفس الوقت مما يتيح امتصاص المواد الغذائية. وهكذا، فإن دور ظهارة الأمعاء هو بمثابة حاجز انتقائي لمحتويات اللمعية 1. ويتحقق ذلك، في جزء منه، من قبل نظام الدفاع الظهارية الفطري في الغشاء المخاطي، الذي يعمل من خلال نظام بيولوجي استجابة تتألف من آليات التأسيسية ومحرض 2. فقدان وظيفة طلائي الحاجز هو علم الأمراض الذي هو سمة من عدد من أمراض الجهاز الهضمي. وفي الجسم الحييمكن تقدير فحص وظيفة حاجز الظهارية من خلال أنبوب تغذية عن طريق الفم من جزيء التتبع وتحليل مصل الدم لاحق 3. ومع ذلك، فإن هذا الأسلوب لا يقدم أي إشارة إلى موقع الخلل الحاجز. في المختبر، وبحكم فيفو تقييم المقاومة بطريق الظهارة باستخدام أنظمة Transwell 3 و غرفة أوسينج 4،5 على التوالي، يعملون عادة كعلامات بديلة من وظيفة حاجز الظهارية، ولكنها تفتقر إلى علم وظائف الأعضاء الأمراض المساهمة في النماذج الحيوانية 6. في هذا البروتوكول وصفنا نموذج إعداد الأنسجة خارج الحي الذي يسمح بتقييم مباشر ومترجم من سلامة الأمعاء والتي يمكن استخدامها لتقييم وظيفة حاجز المخاطية في عدد من المستويات. الأهم من ذلك، يمكن تطبيق هذه التقنية على نماذج حيوانية من المرض، أو قد يتم التلاعب بها دواء للسماح في عمق الاستجواب من ضعف الغشاء المخاطي الحاجز.

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			<td height="43" style="height:43px;width:227px;">Dekantel&#160; Non-absorbable Silk suture</td>
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			<td style="width:227px;">SUT-S 116</td>
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		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Media 199 (TC199)&#160;</td>
			<td style="width:227px;">Life Technologies</td>
			<td style="width:227px;">11043-023</td>
			<td style="width:227px;">No phenol red as this interferes with fluorescence</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:227px;">Life Technologies</td>
			<td style="width:227px;">21063-045</td>
			<td style="width:227px;">No phenol red as this interferes with fluorescence</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">N-acetylcysteine</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;"></td>
			<td style="width:227px;">Use at 10mM in media</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Small animal vascular cathether: Physiocath</td>
			<td style="width:227px;">Data Sciences International</td>
			<td style="width:227px;">277-1-002</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">FITC-Dextran 4400 MW</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;">FD-4</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">FITC-Dextran 20,000 MW</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;">FD-20</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">FITC-Dextran 70,000 MW</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;">FD-70</td>
			<td style="width:227px;"></td>
		</tr>
	</tbody>
</table>

ex vivo intestinal sacs to assess mucosal permeability in models of gastrointestinal disease

الحاجز الظهاري هو أول دفاع الفطرية في الجهاز الهضمي، وينظم بشكل انتقائي النقل من التجويف إلى المقصورات الأنسجة الكامنة، وتقييد نقل جزيئات أصغر عبر الظهارة وتقريبا تحظر تماما نقل الجزيئات الظهارية. يتم تحديد هذه الانتقائية من قبل طبقة جل المخاطية، مما يحد من نقل جزيئات محبة للدهون وكل من مستقبلات قمي والمجمعات البروتين صلي ضيقة من الظهارة. في المختبر نماذج ثقافة خلية من خلايا ظهارة هي مريحة، ولكن كنموذج، فإنها تفتقر إلى تعقيد التفاعلات بين الجراثيم، المخاطية هلام، ظهارة والجهاز المناعي. من ناحية أخرى، يمكن أداؤها في الجسم الحي تقييم امتصاص الأمعاء أو النفاذية، ولكن هذه المقايسات قياس امتصاص الجهاز الهضمي بشكل عام، مع عدم وجود دليل على خصوصية الموقع. فيفو السابقين فحوصات النفاذية باستخدام &quot;الحويصلات المعوية&quot;. هي طريقة أسرع وأكثر دقة لقياس إما سلامة المعوية العامة أو وسائل النقل المقارن من جزيء محدد، مع ميزة إضافية تتمثل في الأمعاء الموقع خصوصية. نحن هنا وصف إعداد الحويصلات المعوية للدراسات النفاذية وحساب نفاذية واضحة (P التطبيق) من جزيء عبر الحاجز المعوي. ويمكن استخدام هذه التقنية كوسيلة لتقييم امتصاص الدواء، أو لفحص الخلل حاجز الظهارية الإقليمي في النماذج الحيوانية من الأمراض المعوية.

ويمكن استخدام هذا البروتوكول لدراسة التغيرات الإقليمية في وظيفة حاجز المعوية في النماذج الحيوانية من الأمراض المعوية. عن طريق قياس تدفق تحقيق paracellular عبر سطح الغشاء المخاطي في مناطق مختلفة من الجهاز الهضمي 7، وسلامة منعطفات ضيقة الظهارية يمك?...

Watch this Scientific Journal Video about Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease at JoVE.com

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

فيفو السابقين المعوية الحويصلات لتقييم الأغشية المخاطية النفاذية في نماذج من الأمراض المعوية

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

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Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

20.9K Views.  University of Newcastle. This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

Video: Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

على<em&gt; في الجسم الحي</em&gt; الفحص لفحص الدم نفاذية سفينة

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

نماذج التفاعل بين الجينات والبيئة لكشف آليات الحساسية في مرض باركنسون

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

التكيف Videofluoroscopic طرق دراسة السنونو الإنسان لكشف وتوصيف عسر البلع في نماذج الفئران الأمراض

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

ل<em&gt; السابقين فيفو</em&gt; نموذج لدراسة هرمون العمل في الثدي البشري

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

في فيفو وفيفو السابقين المداخل لدراسة سرطان المبيض النقيلي الاستعمار من الهياكل التبانة بقعة في البريتوني الدهنية

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

استخدام فائقة MRI الميداني في القوارض الصغيرة نماذج من مرض الكلى المتعدد الكيسات ل<em&gt; في فيفو</em&gt; Phenotyping والدواء المراقبة

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Using an Ingestible Telemetric Temperature Pill to Assess Gastrointestinal Temperature During Exercise

Exercise results in an increase in core body temperature (Tc), which may reduce exercise performance and eventually can lead to the development of heat-related disorders. Therefore, accurate measurement of Tc during exercise is of great importance, especially in athletes who have to perform in challenging ambient conditions. In the current literature a number of methods have been described to measure the Tc (esophageal, external tympanic membrane, mouth or rectum). However, these methods are suboptimal to measure Tc during exercise since they are invasive, have a slow response or are influenced by environmental conditions. Studies described the use of an ingestible telemetric temperature pill as a reliable and valid method to assess gastrointestinal temperature (Tgi), which is a representative measurement of Tc. Therefore, the goal of this study was to provide a detailed description of the measurement of Tgi using an ingestible telemetric temperature pill. This study addresses important methodological factors that must be taken into account for an accurate measurement. It is recommended to read the instructions carefully in order to ensure that the ingestible telemetric temperature pill is a reliable method to assess Tgi at rest and during exercise.

This study describes an accurate, reliable and non-invasive technique to continuously measure gastrointestinal temperature during exercise. The ingestible telemetric temperature pill is suitable to measure gastrointestinal temperature in laboratory settings as well as in field based settings.

باستخدام درجة الحرارة حبوب منع الحمل هضم القياس عن بعد لتقييم درجة الحرارة الجهاز الهضمي أثناء التمرين

Exercise results in an increase in core body temperature (Tc), which may reduce exercise performance and eventually can lead to the development of ...

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 ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

تقييم لوظيفة الكلي في نماذج الماوس من المرض الكبيبي

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

في فيفو فوتولابيلينج الخلايا في القولون لتقييم إمكانية الهجرة من الخلايا المكونة للدم في الفئران حديثي الولادة

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

فيفو السابقين المعوية الحويصلات لتقييم الأغشية المخاطية النفاذية في نماذج من الأمراض المعوية

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