وأيد هذا العمل من قبل ASSOCIAZIONE الإيطالية في لا يسر كل من Ricerca سول Cancro، الوزارة الإيطالية للجامعة والبحوث والمشاريع نسخ م وPROGETTI دي يسر كل من Ricerca دي أتينيو، منح N ° CPDA137871، FONDAZIONE Cariplo، منح N ° 2011-0485 إلى MDB، وFinanziamento جيوفاني Studiosi، جامعة بادوا، لغايا Codolo.

وقد تمت الموافقة على جميع إجراءات التعامل مع الحيوانات من قبل وزارة الصحة الإيطالية (DM 204/2011-B). 1. الحيوانات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تنمو C57BL6 / J الفئران الإناث إلى 8 أسابيع من العمر في أقفاص التهوية بشكل فردي مع مرشحات microisolation. ويفضل الإناث لأن التشكل التشريحي للجهاز-البولية التناسلية الخارجية لها يجعل قسطرة أسهل مما كانت عليه في الذكور: مذكرة. </li></ol> 2. خلية ثقافة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الحفاظ على الماوس الظهارة البولية خط خلية سرطان MB49 في RPMI 1640 متوسطة تستكمل مع 10٪ للحرارة المعطل مصل بقري جنيني و 50 ميكروغرام / مل الجنتاميسين على 37 درجة مئوية في ترطيب 5٪ CO 2. </li><li style=";text-align:right;direction:rtl"> زراعة خلايا MB49 في T-75 قوارير إلى 80٪ التقاء. يعرض للتريبسين و resuspend عند 0.5 × 10 5-0،5 × 10 6 خلايا في 150 ميكرولتر من برنامج تلفزيوني قبل الزرع في الفئران. </li></ol> 3. إينترفسكل الأورام زراعة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تخدير الفئران باستخدام مزيج من zoletil التخدير وxylor (33 ملغ / كغ و 20 ملغم / كغم، على التوالي)، حقن داخل الصفاق. </li><li style=";text-align:right;direction:rtl"> وضع الحيوانات في موقف ضعيف وإصلاح رجليه الخلفيتين الى منصة مع الاسكتلندي. الكفوف وإلى أن يفصل بما فيه الكفاية لجعل الصماخ الإحليل مرئية بشكل جيد ويمكن الوصول إليها بسهولة لإدخال القسطرة. </li><li style=";text-align:right;direction:rtl"> ملاحظة: بالنسبة للقسطرة، 24 G القسطرة الوريدية للأطفال هي مناسبة للفئران 8-10 أسابيع من العمر. اختيار القسطرة مهم للغاية من أجل تجنب تسرب السائل بين الجدار مجرى البول والقسطرة. يجب استخدام القسطرة تجويف أكبر للفئران أكبر أو كبار السن. </li><li style=";text-align:right;direction:rtl"> باستخدام كلاب صغيرة، قرصة حافة واحدة من الجزء الخارجي من مجرى البول وتطويعه بلطف شديد نحو &quot;نهاية الرأس&quot; من الفئران. هذا العمل يعرض الصماخ الإحليل. </li><li style=";text-align:right;direction:rtl"> إزالة الإبرة الداخلية للالقسطرة وتضاف هذه الأخيرة في مجرى البول في زاوية من 45 درجة مئوية، موجهة قليلانحو &quot;ذيل&quot; من الفئران. لا قوة مدخل القسطرة. في حالة المقاومة، مجرد تحريف القسطرة بلطف شديد، حتى أنه ينزلق داخل مجرى البول. عندما حول 0،5-0،8 سم من القسطرة في مجرى البول، ضع بعناية بالتوازي القسطرة إلى ذيل الفئران وأدخله تماما. </li><li style=";text-align:right;direction:rtl"> باستخدام حقنة 1 مل، الإبرة التي لابد من إدراجها في القسطرة، وغسل المثانة من البول المتبقي عن طريق حقن 200-300 ميكرولتر من برنامج تلفزيوني العقيمة ونضح كل السائل من المثانة. </li><li style=";text-align:right;direction:rtl"> ملاحظة: عندما حقن PBS، فإن المثانة تملأ وسوف تورم تكون واضحة بين رجليه الخلفيتين من الفئران. هذا يؤكد أن القسطرة هي الصحيحة. معظم القسطرة لديها حجم القتلى التي يجب أخذها في الاعتبار عند حساب حجم الحقن. </li><li style=";text-align:right;direction:rtl"> باستخدام معقمة 1 مل حقنة، وضخ 200 ميكرولتر من 0.1 N حمض الهيدروكلوريك. ملء المثانة من أجل حرق الجدار بأكمله. بعد 10 ثانية، وإزالة جميع الحامض وneutrali فورازي عن طريق حقن 200 ميكرولتر من 0.1 N هيدروكسيد الصوديوم مع محقنة معقمة. في حالة حقن أكبر عدد من الخلايا (0.5 × 10 6)، ولا يشترط الخطوة التحميض. </li><li style=";text-align:right;direction:rtl"> نضح كل السائل المتبقي وعلى الفور بمسح تجويف مع 300 ميكرولتر من برنامج تلفزيوني العقيمة. </li><li style=";text-align:right;direction:rtl"> إفراغ المثانة بواسطة الشفط، وحقن 150 ميكرولتر من PBS تحتوي على خلايا MB49 (المدى 0.5 × 10 5-0،5 × 10 6) في المثانة. ترك الحقنة تجميعها لالقسطرة لتجنب تسرب الخلايا. فمن الأفضل لتأمين حقنة عن طريق تحديد إلى لوحة مع الاسكتلندي. </li><li style=";text-align:right;direction:rtl"> بعد 1 ساعة، واستخراج بلطف القسطرة من مجرى البول، ووضع الفئران في قفص تحت مصباح وهاج حتى مستيقظا: هذا يحدث عادة بين 1 و 2 ساعة بعد نهاية العلاج. </li></ol> 4. التسليم إينترفسكل من HP-NAP <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ملاحظة: لا يقل عن 3 أيام بعد تقطير الخلية، فمن الممكن أن يبدأالعلاج. </li><li style=";text-align:right;direction:rtl"> يقثطر كما هو موضح في الخطوات 3،1-3،4. في هذه المرحلة، فإنه ليس من الضروري لحرق جدار المثانة مع حمض الهيدروكلوريك. </li><li style=";text-align:right;direction:rtl"> باستخدام حقنة 1 مل، وغسل المثانة من البول المتبقي عن طريق حقن 200-300 ميكرولتر من برنامج تلفزيوني العقيمة ونضح كل السائل من المثانة. </li><li style=";text-align:right;direction:rtl"> حقن 50 ميكروغرام من HP-NAP في 150 PBS ميكرولتر في المثانة. ترك الحقنة التي تعلق على القسطرة لتجنب تسرب من الحل البروتين. تأمين حقنة عن طريق تحديد إلى لوحة مع الاسكتلندي. </li><li style=";text-align:right;direction:rtl"> بعد 1 ساعة، واستخراج بلطف القسطرة من مجرى البول ووضع الفئران في قفص تحت مصباح وهاج، حتى يستيقظ. </li></ol> 5. الأورام إإكسبلنتس وتحليل النسيجي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في نهاية العلاج، والتضحية الحيوانات، ووضعها في مواقف مستلق وإصلاح رجليه الخلفيتين الى منصة مع الاسكتلندي. </li><li style=";text-align:right;direction:rtl"> باستخدام مشرط جراحي، وجعل شق عمودي عن 1.52 سم من خلال الجلد والبريتوني، بدأت للتو فوق الأعضاء التناسلية الخارجية إلى &quot;نهاية الرأس&quot; من الفئران. للحد من مخاطر الإضرار المثانة، إيلاء اهتمام خاص لعمق القطع. </li><li style=";text-align:right;direction:rtl"> سيتم وضع المثانة في يسار الوسط لقطع وسوف الورم تكون واضحة كما مظلمة كتلة الوردي / البنفسجي. على الختان لها، ضع المثانة بالكامل في قفص النسيجي وإصلاح في 10٪ من الفورمالين مخزنة قبل التضمين في البارافين. </li><li style=";text-align:right;direction:rtl"> لالنسيجية وتحليل النسيجية المناعي، وإعداد أقسام المسلسل من المثانة (46 سماكة ميكرون) باستخدام مشراح القياسية. </li><li style=";text-align:right;direction:rtl"> وصمة عار على المقاطع مع haematoxylin / يوزين والمضي قدما في حساب حجم الورم (D XD 2) وتحديد نسبة نخر في منطقة مستعرضة باستخدام بمساعدة الكمبيوتر محلل الصورة. </li><li style=";text-align:right;direction:rtl"> لسفينة العد، المضي قدما في طريق تلطيخ أقسام للعلامة البطانية CD31 / PECAM، وذلك باستخدام تقنية المناعي القياسية. مسح المقاطع لر طاقة منخفضة (4 ×) لتحديد منطقة أعلى كثافة الأوعية الدموية. داخل هذه المنطقة، عد microvessels الفردية في خمسة حقول عشوائية منفصلة في قوة عالية (20 ×). </li></ol>

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تعلن الكتاب أنه ليس لديهم المصالح المالية المتنافسة.

معظم السلف في علاج السرطان تتطلب اختبارات في النماذج الحيوانية قبل بدء التجارب السريرية. إمكانية دراسة بيولوجية الورم في الجسم الحي، والاستفادة من النماذج الحيوانية، يمثل أداة حاسمة للباحثين التحقيق التسبب بالسرطان، مما يتيح تقييم أساليب علاجية مختلفة. لا تز?...

سرطان المثانة هو واحد من أكثر أنواع السرطان شيوعا في الجهاز البولي التناسلي، مع ما يقرب من 75،000 حالة جديدة كل عام في الولايات المتحدة الأمريكية 1. تتطلب معدلات عالية من تكرار مدى الحياة المتابعة، مما يجعل سرطان المثانة واحدة من أغلى أنواع السرطان لعلاج. علاج الذهب القياسية لعالية الجودة NMIBC هو بتر عبر الإحليل، تليها العلاج المناعي إينترفسكل مع BCG. على الرغم من أن الآلية الدقيقة للنشاط المضادة للورم من BCG يبقى أن توضح تماما، ومن المتفق عليه أن تفعيل استجابة بوساطة الخلايا المناعية المخصب في T المساعد (ث) 1 والسامة للخلايا T (ح) 1 الخلايا هو أمر حاسم ل نجاح العلاج 2. على الرغم من أن BCG يبقى العلاج الأمثل لNMIBC، وهي نسبة عالية من المرضى لا يستجيبون للعلاج. علاوة على ذلك، فإنه يمكن أن يسبب عددا من الآثار الجانبية: حوالي 70٪ من الأورام تعامل تتكرر بعد مرور بعض الوقت والتقدم ~ 15٪ إلى النموذج العضلات الغازية للالمرض. الآثار الجانبية الأخرى المرتبطة العلاج BCG تشمل عسر البول، التهاب المثانة والتهاب الكلى 3-6. تطوير استراتيجيات علاجية جديدة يجب أن تأخذ في الاعتبار استخدام نماذج ما قبل السريرية، بعد الأولي في المختبر التقييم؛ هذا أهمية خاصة في الأورام التي يمكن أن تؤثر بشكل كبير تنميتها والاستجابة للعلاج المكروية. على مدى العقد الماضي، ونحن قد تناولت جوانب متعددة من النشاط تحوير المناعة من HP-NAP، وهو بروتين تنتجه بكتيريا هيليكوباكتر بيلوري، حدد في البداية بأنه قادر على تعزيز التصاق الخلايا البطانية النوى (PMNs) 7. هيكليا، HP-NAP ينتمي إلى البروتين لحماية الحمض النووي في ظل ظروف التجويع (DPS) الأسرة (8) ويتكون من 12 مفارز متطابقة مرتبة في قذيفة dodecameric. لقد أثبتنا أن HP-NAP هومستقبلات تول مثل (TLR) 2 ناهض، مع نشاط تحوير المناعة القوي التي تقف وراء تمايز اللمفاويات التائية نحو النمط الظاهري TH1، سواء في المختبر والمجراة 10/09. في فضيلة لهذا النشاط، HP-NAP غير قادرة على توجيه الاستجابة المناعية TH2 في الاستجابة أكثر فائدة TH1 في نموذج الفئران من الربو التحسسي 11. لتقييم القدرة المضادة للورم TH1 التي تعتمد من HP-NAP، اتخذنا الاستفادة من نموذج الفأر من سرطان المثانة وضعت قبل عدة سنوات من قبل أودونيل وزملاؤه (12) لتقييم أثر إدارة BCG. مع هذا البروتوكول، علينا أن نبرهن على HP-NAP لديها امكانات المضادة للورم قوية ضد سرطان المثانة وأن فعالية إدارة HP-NAP يوازي تراكم كبير، داخل الورم والغدد الإقليمي العقد، كل من Th1 و TC1 الخلايا الليمفاوية إنتاج الانترفيرون ( IFN) -γ 13 </سوب&gt;. وأظهرت أورام معزولة من الفئران التي عولجت HP-NAP-مزيد من نخر والأوعية الدموية أقل من نظيره غير المعالجة. يقدم هذا التقرير بروتوكول stepbystep، تفاصيل إعداد الحيوانات، قسطرة الإحليل، وحرق الكيميائية اللازمة لمرفق من الخلايا إلى الغشاء المخاطي المثانة، وحقن الخلايا السرطانية. نحن أيضا وصف الإدارة الموضعية من HP-NAP التي يمكن اعتبارها نموذجا أوليا من أي العوامل العلاجية المتقدمة لعلاج سرطان المثانة. الأدلة التي تم الحصول عليها عن طريق مقارنة أورام معزولة عن السيطرة وHP-المعالجة NAP الحيوانات ويؤكد ليس فقط حقيقة أن HP-NAP يمكن أن يكون مرشحا جيدا للعلاج مناعي سرطان المثانة، ولكن أيضا فعالية العامة للالتجريبية اقامة.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>C57BL/6J female mice</td>
			<td>Harlan Italy (Udine, Italy)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MB49 Cells</td>
			<td></td>
			<td></td>
			<td>Obtained from Prof. O&#39;Donnel, University of Iowa Carver College of Medicine, Iowa, USA</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Sigma-Aldrich (St. Louis, Missouri, USA)</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D1408</td>
			<td>10X, to be diluted in apyrogenic water</td>
		</tr>
		<tr>
			<td>Flask</td>
			<td>Becton Dickinson (Franklin Lakes, New Jersey, USA)</td>
			<td>353135</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1ml</td>
			<td>Becton Dickinson</td>
			<td>301358</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies (Waltham, Massachusetts, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Life Technologies</td>
			<td>15710-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Xilor</td>
			<td>Bio 98 s.r.l. (Milano, Italy)</td>
			<td></td>
			<td>2% Xylazin</td>
		</tr>
		<tr>
			<td>Zoletil</td>
			<td>Virbac (Carros, France)</td>
			<td>359713301992</td>
			<td>5% Zolazepam + 5% Tiletamine</td>
		</tr>
		<tr>
			<td>24G Catheter</td>
			<td>Terumo (Rome, Italy)</td>
			<td>SR+DM2419PX</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Carlo Erba Reagents (Milano, Italy)</td>
			<td>403871</td>
			<td>Liquid</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>JT Baker (Center Valley, Pennsylvania, USA)</td>
			<td>10095011</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Surgical Scalpel</td>
			<td>Albion Surgical Limited (Sheffield, England)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica Microsystem ( Wetzlar, Germany)</td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>VWR International (Radnor, Pennsylvania, USA)</td>
			<td>631-0108</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analyzer</td>
			<td>Zeiss (Jena, Germany)</td>
			<td>Cyres System</td>
			<td></td>
		</tr>
	</tbody>
</table>
Materials C57BL/6J female mice Harlan Italy (Udine, Italy) MB49 Cells Obtained from Prof. O&#39;Donnel, University of Iowa Carver College of Medicine, Iowa, USA RPMI Sigma-Aldrich (St. Louis, Missouri, USA) R8758 FBS Sigma-Aldrich F7524 PBS Sigma-Aldrich D1408 10X, to be diluted in apyrogenic water Flask Becton Dickinson (Franklin Lakes, New Jersey, USA) 353135 Syringe 1ml Becton Dickinson 301358 Trypsin Life Technologies (Waltham, Massachusetts, USA) Gentamycin Life Technologies 15710-049 Xilor Bio 98 s.r.l. (Milano, Italy) 2% Xylazin Zoletil Virbac (Carros, France) 359713301992 5% Zolazepam + 5% Tiletamine 24G Catheter Terumo (Rome, Italy) SR+DM2419PX HCl Carlo Erba Reagents (Milano, Italy) 403871 Liquid NaOH JT Baker (Center Valley, Pennsylvania, USA) 10095011 Powder Equipment Surgical Scalpel Albion Surgical Limited (Sheffield, England) Microtome Leica Microsystem ( Wetzlar, Germany) RM2235 Microscope Slides VWR International (Radnor, Pennsylvania, USA) 631-0108 Image Analyzer Zeiss (Jena, Germany) Cyres System

evaluation of the efficacy of the h. pylori protein hp-nap as a therapeutic tool for treatment of bladder cancer in an orthotopic murine model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

ويبين الشكل (1) وتقنية القسطرة. ومقسطر الماوس وتغرس مع 0.5 × 10 6 MB49 الخلايا. بدأ العلاج مع HP-NAP 3 أيام بعد حقن الخلايا السرطانية، لتمكينهم من تعلقها على جدار المثانة وتتكاثر. كل الحيوانات التي تنتمي إلى مجموعة مراقبة تطور الورم. البعض منهم قد يموتون قبل نهاي?...

Watch this Scientific Journal Video about Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model at JoVE.com

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

تقييم فعالية<em&gt; H. بيلوري</em&gt; البروتين HP-NAP كأداة علاجية لعلاج سرطان المثانة في مثلي فأري موديل

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

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Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

7.2K Views.  University of Padua. Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Video: Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

نموذج مثلي من سرطان المثانة الفئران

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

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 ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

المؤشرات الحيوية في نموذج حيواني لكشف الاقتران العصبية، الدموية، والسلوكية للPTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

ورم في المثانة Orthotopic النموذجي وتقييم المعالجة سارنا داخل المثانة

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

وسرطان المثانة نموذج مثلي للدراسات توصيل الجينات

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

على الفئران نموذج مثلي من سرطان البروستاتا البشرية الانبثاث

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

مؤسسة المناظير الفئران مثلي المثانة Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

excitotoxic المواد تحفيز الدماغ باعتبارها Microslices<em&gt; في المختبر</em&gt; نموذج من السكتة الدماغية

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

ما قبل السريرية مثلي فأري موديل سرطان البروستاتا البشرية

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

إشارات جيل التوافقي الثاني في أرنب الصلبة كأداة للتقييم للأنسجة العلاجي العابرة للربط (TXL) لقصر النظر

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...