This work was supported by the KU Leuven PF &#8216;IMIR&#8217;, the FWO Research community on biology and ecology of bacterial and fungal biofilms (FWO: WO.026.11N) and by the FWO project G.0804.11. SK gratefully acknowledges KU Leuven for the PDMK 11/089 fellowship and FWO for the postdoctoral fellowship. We are grateful to Nico Vangoethem for his assistance with preparation of the figures. We would like to acknowledge Celia Lobo Romero for technical assistance during in vivo experimental procedures.

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تعلن المؤلفين أنه ليس لديهم المصالح المالية المتنافسة.

استخدام النماذج الحيوانية، ونماذج القوارض وخاصة، للدراسات مخصصة لالأغشية الحيوية الميكروبية مهم جدا لجهاز المناعة المضيف هو عامل أساسي في تشكيل بيوفيلم أن النماذج في المختبر لا يمكن حساب ل. في هذه الدراسة، نحن تصف تحت الجلد C. واضحة نسبيا البيض نموذج ?...

المبيضات البيض هو كائن المتعايشة، التي يمكن العثور عليها في مواقع مختلفة من الأفراد الأصحاء، على سبيل المثال على الجلد أو جزء من الجهاز الهضمي والنباتات المهبلية. ومع ذلك، في المستشفى، وخصوصا مرضى نقص المناعة، فإنه قد يسبب مجموعة واسعة من الالتهابات 1. في هؤلاء الأفراد، وضعف الجهاز المناعي يسمح خلايا المبيضات لنشر إلى مجرى الدم وتغزو الأنسجة العميقة يسبب العدوى المهددة للحياة. وبالإضافة إلى ذلك، فإن وجود ركائز غير الحيوية مثل القسطرة الوريدية المركزية والمسالك البولية، وصمامات القلب الاصطناعية والمفاصل قد توفر مكانا لالمبيضات مرفق 2. الانضمام إلى مثل هذه ركائز هو شرط أساسي لمزيد من التطوير بيوفيلم، وهو ما يمثل طبقة من الخميرة وخوطي خلايا جزءا لا يتجزأ من المواد البوليمرية خارج الخلية، التي تتألف أساسا من السكريات 2. C. البيض قسطرة -associated العدوىوترتبط مع ارتفاع معدل الوفيات. والسمة العامة للالأغشية الحيوية هي قابلية انخفضت لمضادات الفطريات المعروفة، مثل الأزولات 3،4. أثبتت أحدث فصول فقط من الأدوية المضادة للفطريات، مثل echinocandins وصياغة liposomal من الأمفوتريسين B لتكون فعالة ضد العدوى المرتبطة القسطرة 5-7. بسبب بيوفيلم القدرة على مقاومة مضادات الفطريات، والنهج العلاجية محدودة جدا، وغالبا ما يؤدي إلى إزالة القسطرة واستبدال لاحقا كحل وحيد. أكثر من الفهم الحالي للC. لدينا تنبع تطوير بيوفيلم البيض من الدراسات في المختبر على ركائز غير الحيوية مثل البوليسترين، أو المواد البلاستيكية المستخدمة لتصنيع الأجهزة المذكورة أعلاه، أي سيليكون، البولي يوريثين 2. هذه النماذج هي متقدمة جدا ومحاولة لتقليد الوضع في الجسم الحي بأكبر قدر ممكن. ومع ذلك، فإن هذه الأنظمة لا تنطوي على تدفق الدم المستمر وركان الجهاز المناعي للمضيف. وأدى ذلك إلى تطوير نظم نموذج في الجسم الحي، مثل القسطرة الوريدية المركزية (سي) نموذج 8-10، ونموذج التهاب الفم أسنان من داء المبيضات الفموي 11 ونموذج الفئران لالمصاحب قسطرة بيلة المبيضات 12. بالإضافة إلى ذلك، C. تمت دراسة تطوير بيوفيلم البيض في الجسم الحي على الأسطح المخاطية، مثل تلك التي من المهبل عن طريق الفم تجويف 13 و 14. ساهم مختبرنا مع إنشاء C. تحت الجلد البيض نموذج بيوفيلم، والتي تقوم على زرع من القطع القسطرة المصابة على الجزء الخلفي من الفئران سبراغ داولي 15. وقد استخدم هذا النموذج بنجاح في المختبر لدينا لاختبار قابلية بيوفيلم لفلوكونازول وechinocandin المخدرات 5،16، لدراسة تأثير العلاج اندماجي من ديكلوفيناك وcaspofungin 17. وفي الآونة الأخيرة، فإننا تكييفها هذا النظام لاستخدامها في BALB / ج الفئران 18،19. فيمقارنة مع غيرها في نماذج الجسم الحي، والميزة الرئيسية لهذا النموذج تحت الجلد هو إمكانية لدراسة الأغشية الحيوية متعددة لكل حيوان وضعت داخل التجويف من القطع قسطرة مزروع. للحد من عدد الحيوانات المختبرية، وتكيفنا هذا النموذج لدراسة تطوير C. البيض الأغشية الحيوية غير جراحية باستخدام التصوير تلألؤ بيولوجي (BLI) 18،19. أثبتت هذه الطريقة أن تكون تقنية قوية، والتي يمكن استخدامها لقياس الأغشية الحيوية عن طريق قياس إشارة محددة BLI في المنطقة ذات الاهتمام (في حالتنا مجال القسطرة زرعها)، وتجنب التضحية الحيوانية. وبالمقارنة مع البكتيريا، والتي يمكن التعبير عن كل من الجينات والركيزة اللازمة للتفاعل تلألؤ بيولوجي نظرا لإدخال لوكس الاوبرون محدد 20، ومعظم الكائنات الحية حقيقية النواة، بما في ذلك C. البيض، تعتمد على التعبير مغاير من الجين luciferase المراسل إلى جانبالإدارة الخارجية للركيزة محددة، مثل D-وسيفيرين أو coelenterazine 21. وربما يرجع ذلك إلى وجود جدار الخلية الفطرية وC. كان البيض التشكل، والتسليم داخل الخلايا الركيزة للانزيم luciferase المراسل تحديا الرئيسي 21. من أجل حل هذه المشكلة، Enjalbert وآخرون 22 هندسيا سلالة حيث C. الاصطناعية وقد تنصهر البيض الإصدار الأمثل كودون من الجينات ليفرز بشكل طبيعي Gaussia princeps luciferase المراسل (gLuc) للC. البيض PGA59 الجينات، ترتكز على GPI- بروتين جدار الخلية. بسبب وجود luciferase المراسل في جدار الخلية، ويمكن تجنب المشاكل المتعلقة توافر داخل الخلايا الركيزة. وقد استخدم هذا النظام بشكل خاص لدراسة التهابات سطحية الناجمة عن C. البيض 22. قريب جدا، كان يستخدم BLI أيضا لمتابعة تطور داء المبيضات الفموي البلعومي وامكن لهاالعلاج ه 23. هذه النتائج تدعم استخدام BLI باعتبارها تقنية واعدة لدراسة الالتهابات التي تسببها الخلايا التي تعيش بحرية ولكن أيضا العدوى المرتبطة الجهاز. في هذه الدراسة، فإننا وصف C. تطوير بيوفيلم البيض على قطع القسطرة البولي يوريثان في الفئران BALB / ج وتقدير لها باستخدام BLI. ونحن نقدم بروتوكول مفصلة في المختبر استعمار القسطرة البولي يوريثين خلال الفترة من التصاق تليها زرع في الفئران والتنمية بيوفيلم اللاحقة في الحيوانات الحية. وبصرف النظر عن قياس إشارة BLI المنبعثة من C. خلايا البيض، ونحن أيضا تحديد مستعمرة للمقارنة مع تقنية قياسية لبيوفيلم الفطرية الحمل الكمي.

<table border="0" cellpadding="0" cellspacing="0" width="877">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Name of Reagent</td>
			<td style="width:199px;">Company</td>
			<td style="width:113px;">Catalog Number</td>
			<td style="width:236px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Yeast extract granulated</td>
			<td style="width:199px;">Merck</td>
			<td style="width:113px;">MERC1.03753.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Bacteriological peptone</td>
			<td style="width:199px;">Oxoid</td>
			<td style="width:113px;">LP037B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Agar granulated</td>
			<td style="width:199px;">Difco</td>
			<td style="width:113px;">214530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">D-(+)-glucose</td>
			<td style="width:199px;">Fluka</td>
			<td style="width:113px;">49159-5KG</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Phosphate buffered saline&#160;</td>
			<td style="width:199px;">Prepared in the laboratory</td>
			<td style="width:113px;"></td>
			<td>for 1L of 10x PBS: 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">RPMI1640 with L-glutamine and without sodium carbonate&#160;</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:113px;">R6504-1L</td>
			<td>Prepare according the protocol for Candida albicans drug susceptibility testing&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">3-(N-Morpholino)propanesulfonic acid (MOPS)</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:113px;">M1254</td>
			<td>MOPS is used to adjust the pH of RPMI medium (pH 7.0)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">fetal bovine serum (FBS)</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:113px;">F7524</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Polyurethane tripe-lumen intravenous catheter piece (2.4 mm diameter, Certofix Trio S730)&#160;</td>
			<td style="width:199px;">BBraun</td>
			<td style="width:113px;">CV-15703</td>
			<td>Polyurethane part cut into 1 cm pieces</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Dexamethasone</td>
			<td style="width:199px;">Fagron SAS, France</td>
			<td style="width:113px;">611139</td>
			<td>Immunosuppressant (stock solution 10 mg/ml)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Ampicillin&#160;</td>
			<td style="width:199px;">Duchefa Biochemie, The Netherlands</td>
			<td style="width:113px;">A0104</td>
			<td>Antibacterial prophylaxis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Ketamine 1000</td>
			<td>Pfizer</td>
			<td style="width:113px;">804 119</td>
			<td>Anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Domitor</td>
			<td>Pfizer</td>
			<td style="width:113px;">134737-1</td>
			<td>Anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Antisedan</td>
			<td>Pfizer</td>
			<td style="width:113px;">134783-2</td>
			<td>Reversal of anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Xylocaine gel (2%) - this is Linisol</td>
			<td style="width:199px;">AstraZeneca</td>
			<td style="width:113px;">352 1206</td>
			<td>Local anesthetic for the skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Terramycin/ polymyxin-b ophthalmic ointment</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td>To prevent drying and infection of eyes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Coelenterazine&#160;</td>
			<td style="width:199px;">Prolume (Nanolight)</td>
			<td style="width:113px;">NF-CTZ-FB</td>
			<td>Light sensitive agent (must be kept in the dark)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iodine isopropanol (1%)</td>
			<td style="width:199px;">3M&#8482; DuraPrep&#8482;&#160;</td>
			<td style="width:113px;"></td>
			<td>Disinfectant for the skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 % chlorhexidine in 70 % alcohol.&#160;&#160;</td>
			<td style="width:199px;">Cedium</td>
			<td style="width:113px;"></td>
			<td>Disinfectant for the skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Equipment</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Cell counting chamber</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Insulin syringes (0.3 ml)</td>
			<td style="width:199px;">Terumo Myjector 29G</td>
			<td style="width:113px;">324826</td>
			<td>For injection of coelenterazine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Electric razor</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td>For small animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Sterile surgical tools</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td>Scissors, 2 pairs of tweezers, scalpel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Heating pad</td>
			<td style="width:199px;">Leica</td>
			<td style="width:113px;">14042321474</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Skin suture</td>
			<td style="width:199px;">Johnson&#38;Johnson</td>
			<td style="width:113px;">K890H</td>
			<td>Surgical thread, needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath sonicator</td>
			<td>Branson 2210</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">BLI camera (IVIS Spectrum)&#160;</td>
			<td style="width:199px;">Perkin Elmer, Alameda</td>
			<td style="width:113px;">IVISSPE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Living Image software&#160;</td>
			<td style="width:199px;">Perkin Elmer, Alameda</td>
			<td style="width:113px;"></td>
			<td>(version 4.2)&#160;</td>
		</tr>
	</tbody>
</table>

candida albicans biofilm development on medically-relevant foreign bodies in a mouse subcutaneous model followed by bioluminescence imaging

المبيضات البيض تطوير بيوفيلم على الأسطح الحيوية و / أو غير الحيوية تمثل تهديدا محددا لمرضى المستشفى. وحتى الآن، C. وقد تم دراسة الأغشية الحيوية البيض في الغالب في المختبر ولكن هناك حاجة ماسة لفهم أفضل لهذه العملية الديناميكية في ظل ظروف في الجسم الحي. قمنا بتطوير نموذج الفئران تحت الجلد في الجسم الحي لدراسة C. تشكيل بيوفيلم البيض. في نموذجنا، متعددة (ما يصل إلى 9) وزرع أجهزة -infected المبيضات إلى الجزء الخلفي من الحيوان. هذا يعطينا ميزة كبيرة على نظام نموذجي القسطرة الوريدية المركزية لأنها تسمح لنا لدراسة عدة الأغشية الحيوية المستقلة في حيوان واحد. مؤخرا، ونحن تكييفها هذا النموذج لدراسة C. تطوير بيوفيلم البيض في BALB / ج الفئران. في هذا النموذج، تنضج C. الأغشية الحيوية البيض تتطور خلال 48 ساعة وتظهر العمارة بيوفيلم ثلاثية الأبعاد النموذجية. القياس الكمي لبيوفيلم الفطريةويتم تحليل تقليديا بعد الوفاة ويتطلب التضحية المضيف. لأن هذا يتطلب استخدام العديد من الحيوانات لإجراء دراسات الحركية، طبقنا غير الغازية تلألؤ بيولوجي التصوير (BLI) لمتابعة طولي يصل في الجسم الحي تنضج C. البيض الأغشية الحيوية النامية في نموذج تحت الجلد لدينا. C. تم هندستها خلايا البيض للتعبير عن Gaussia princeps luciferase المراسل الجين (gLuc) تعلق على جدار الخلية. ويتم إنتاج إشارة تلألؤ بيولوجي من قبل luciferase المراسل أن يحول coelenterazine الركيزة أضاف في الضوء الذي يمكن قياسها. إشارة BLI تشبه عدد الخلايا التي تم الحصول عليها من القسطرة explanted. التصوير غير الغازية لقياس في تشكيل الجسم الحي بيوفيلم يوفر تطبيقات فورية لفحص والتحقق من الأدوية المضادة للفطريات في ظل ظروف في الجسم الحي، فضلا عن الدراسات التي تعتمد على التفاعلات المضيف الممرض، تساهم بموجبه إلى فهم أفضل لجي من التسبب في العدوى المرتبطة القسطرة.

في هذه الدراسة، وتبين لنا الإجراء الجراحي لزرع قسطرة ويزدرع خلال المجراة C. تنمية البيض بيوفيلم في الماوس. وعلاوة على ذلك، فإننا عرض الكمي من الأغشية الحيوية الناضجة ليس فقط من قبل CFUs الكلاسيكية تعداد ولكن أيضا من قبل BLI. ...

Watch this Scientific Journal Video about Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging at JoVE.com

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

المبيضات البيض بيوفيلم التنمية على الهيئات الأجنبية طبيا ذات الصلة في الماوس تحت الجلد نموذج تلاه ضوء بارد التصوير

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

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Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

12.9K Views.  Institute of Botany and Microbiology, VIB, KU Leuven. We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Video: Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

رصد الانبثاث ورم والآفات الحالة للعظم مع ضوء بارد ، والتصوير المقطعي مايكرو

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

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 ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

التصوير بالموجات فوق الصوتية الموجهة داخل القلب حقن لتطوير نموذج الفأر من الثدي الانبثاث سرطان الدماغ تلاه الطولية التصوير بالرنين المغناطيسي

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

الصدمة الحادة الدماغ في الفئران تليها الطولية التصوير ثنائي الفوتون

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

تقييم-التسلل ورم خلايا الدم البيضاء مجموعات فرعية في ورم تحت الجلد نموذج

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

التصوير تلألؤ بيولوجي لنموذج مناعيا الحيوان ورم أرومي دبقي

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

وحديثي الولادة ماوس الحبل الشوكي ضغط إصابة نموذج

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

التصور الكمي من التسلل الكريات البيض في نموذج الفئران من التهاب عضلة القلب مداهم من قبل ورقة ضوء المجهري

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

متساوي القياس كونتراكتيليتي القياس الشريان المساريقي العلوي الماوس باستخدام أسلاك ميوجرافي

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...