המחברים מבקשים להודות לתכנית לביולוגית עכבר דייוויס UC לרבייה של העכברים. מחקר זה מומן על ידי מענק מהחברה Merck KGaA, דרמשטט, גרמניה.

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DPV, GTW, SMG, CJK, AMG, ומשרד GKH מצהירים שום אינטרסים מתחרים. MWD הוא החוקר הראשי של מענק שהתקבל מחברת Merck KGaA, וMW הנו עובד של חברת Merck KGaA.

האינדוקציה המוצלחת של קרצינומה של תאי מעבר בשלפוחית ​​השתן וקשקש פולשני בעכברי MUC1.Tg אדם מציעה מודל קליני לפיתוח חיסוני. מחקרי immunotherapeutic דורשים שימוש במודל ספונטני, ללא פגע חיסונית על מנת להעריך את התגובה הדלקתית להתקדמות גידול לאורך זמן, כמו גם את התגובה החיסונית ל?...

סרטן שלפוחית ​​השתן הוא הצורה הנפוצה ביותר של הסרטן הרביעית ושמיני גורם מוביל למוות מהסרטן בגברים אמריקאים. בארצות הברית, כ 72,500 מקרים חדשים ו -15,000 מקרי מוות מסרטן שלפוחית ​​השתן צפויים בקרב גברים ונשים גם יחד בשנת 2013 1. השכיחות של סרטן שלפוחית ​​השתן היא כשלוש פעמים כגבוהות אצל גברים בהשוואה לנשים. בארצות הברית, קרצינומה של תאי מעבר (TCC) הדין וחשבון על יותר מ -90% מהמקרים, בעוד קרצינומה של תאי קשקש (SCC) יש שכיחות של פחות מ -2% 2. שיעור ההישרדות היחסי ל -5 השנים הכולל לTCC פפילרי הוא 91.5% לעומת רק 30.9% עבור SCC 2. למרות TCCs papillary פולשנית מהווה כ 75% מהמקרים בזמן אבחנה, אפילו עם טיפול יותר מ -50% מהחולים חווים הישנות בתוך 5 שנים, עם עד 30% מהחולים הללו התקדמות למחלה פולשנית שריר 3,4 . משטרי טיפול אופייניים עבור inv הלא שרירהמחלה asive כוללת כריתה (טור) ואחריו כימותרפיה שלפוחית. בחולים עם טא בדרגה גבוהה או גידולי T1, TUR חוזר עשויה להתבצע לפני טיפול כימותרפי 3,4. עבור אותם חולים עם הישנויות Ta בדרגה נמוכה או בדרגה גבוהה Ta או נגעי T1, TUR ואחריו כימותרפיה adjuvant או טיפול חיסוני בצורה של Bacillus Calmette-גרן (BCG) עשוי לשמש 3,4. שלפוחית ​​BCG הוכח להיות עדיף על C mitomycin שלפוחית ​​ביחס לזמן עד להשנות 5. למחלה פולשנית שריר T2, כריתת שלפוחית ​​עם או בלי neoadjuvant כימותרפיה היא כמובן המומלץ של טיפול 3. בחולים עם SCC, כריתת שלפוחית ​​נראית כטיפול היעיל ביותר 6. בהתחשב בשיעורים הגבוהים מאוד של הישנות למרות הטיפולים הטובים ביותר הקיימים, יש בבירור צורך בטיפולים חדשים, יעילים יותר לסרטן שלפוחית ​​השתן. הרחבת immunotherapies החדש לbladdסרטן ER הוא גישה אפשרית אחת שעשוי לקיים הבטחה להארכת הישרדות ללא מחלה. מבחינה היסטורית, BCG כבר החיסוני היעילה רק לסרטן שלפוחית ​​השתן. מנגנון פעולה שלה הוא חשב לערב אינדוקציה ספציפי של תגובה חיסונית מסוג T-helper 1 (Th1) באמצעות הגדלת רמות של אינטרלויקין -2 (IL-2) ואינטרפרון גאמא (IFN-γ) 4. נייד, או Th1 חסינות, היא קריטי בטיפול חיסוני סרטן כלחות, או Th2, חסינות מעולם לא הוכח כיעיל נגד גידולים מוצקים, למעט של נוגדנים המכוונים נגד קולטני גורמי גדילה 7. בניסיון לשפר את היתרונות של טיפול יחיד BCG, אימונותרפיה שילוב 2B/BCG IFN-α הוערכה בניסוי שלב II בתרופה עם תוצאות חד משמעיות 8. גישה חלופית לטיפול חיסוני לסרטן שלפוחית ​​השתן עשויה להיות למקד אנטיגנים הקשורים גידולים (תעש), זיהוי שנעשה חיסוני סרטן ספציפיים יותר 7 </sעד&gt;. TAA אחד כזה הוא mucin 1 (MUC1), שהוא גליקופרוטאין פני תא ביטוי יתר בסרטן רבים תאי אפיתל, כגון שלפוחית ​​השתן, סרטן שד, סרטן ריאות, סרטן הלבלב ו9,10. הביטוי והשינוי של MUC1 גם שינו באופן משמעותי במהלך היווצרות הסרטן, underglycosylation כזה שחושף את הרצפים של חומצות אמינו אנטיגני המכונים מספר משתנה של חזרות טנדם (VNTR) על ליבת פפטיד. בעוד MUC1 הוא מולקולה עצמית, אזורי VNTR immunodominant אלה בדרך כלל לא נחשפו בשל glycosylation הנרחבת, וכך הם נתפסים על ידי המערכת החיסונית כ11,12 זר. ציטוטוקסיות לימפוציטים מסוג T (הרג) כי דווקא מכיר MUC1 epitopes היה מבודד מבלוטות הלימפה הניקוז-גידול של חולות סרטן שד 13, כמו גם את הדם ומח עצם של חולי מיאלומה 14,15, מה שהופך MUC1 יעד פוטנציאלי עבור תגובה חיסונית תאית. את VNTRs immunodominant של underglycosylateהטופס של ד MUC1 מוכר על ידי הרג, וכתוצאה מכך להרס של תאים סרטניים 16-19. תגובה חיסונית תאית ו / או הלחות יליד MUC1 הסרטנית, לעומת זאת, אינה חזקה מספיק כדי לחסל גידולים. כדי להגדיל את התגובה החיסונית החלשה שכבר קיימת לMUC1, יכולים להיות הציגו פפטידים סינטטיים immunodominant באמצעות חיסון ליצור תגובת CTL חזק מספיק כדי להיות של תועלת קלינית 18,20. חיסון liposomal MUC1 כבר הוכח להגדיל את ההישרדות בחולי סרטן ריאות 21,22, ליצור הרג מסוגל להרוג תאים סרטניים MUC1 חיוביים, ולייצר תגובת ציטוקינים Th1-מקוטבת 23,24. עם רמה גבוהה של MUC1 ביטוי 9,11,25, סרטן שלפוחית ​​השתן הוא מועמד הגיוני לבדיקת MUC1 מכוונת אימונותרפיה 26,27. יתר על כן, יש לו פוטנציאל MUC1 כגורם מנבא בשלפוחית ​​שתן הסרטן 28, MUC1 ביטוי בTCC קשור באופן משמעותי עם במה וכיתה, והגרורה TCCהוכח ממשיך להביע MUC1 29. על מנת להעריך את התועלת הפוטנציאלית של טיפול חיסוני MUC1 מכוון בסרטן שלפוחית ​​השתן, פיתחנו MUC1 חיסונית אנושי שלם (hMUC1) להביע מודל של סרטן שלפוחית ​​השתן מהונדס congenic (MUC1.Tg) עכבר על רקע C57BL / 6 30. MUC1 האנושי מתבטא כחלבון עצמי תחת השליטה של האמרגן שלה, וכתוצאה מדפוס ביטוי רקמה בקנה אחד עם זה שנצפה בבני האדם 30,31. העכברים היו מושרה עם הסרטן הידוע שלפוחית ​​N-בוטיל-N-(4-hydroxybutyl) ניטרוזמין (OH-BBN) 32, ולאחר מכן את הגידולים כתוצאה הוערכו על hMUC1 ביטוי וסוג ודרגת גידול. כדי להעריך את ההשפעה של החומר מהסרטן בTh1/Th2 רמות ציטוקינים במהלך התפתחות גידול, דגימות סרום שנאספו מעת לעת לצורך הניתוח זמנית. עכברים לאחר מכן טופלו בחיסון פפטיד MUC1 ממוקד, וציטוקינים בסרום ותגובות חיסוניות היו מסגרת הערכהטד על ידי immunoassay microbead fluorometric זמנית וELISPOT.

<table border="1">
	<tbody>
		<tr>
			<td>Reagent&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN)</td>
			<td>TCI America</td>
			<td>B0938</td>
			<td></td>
		</tr>
		<tr>
			<td>20 G Gavage Needles</td>
			<td>Popper &#38; Sons, Inc.</td>
			<td>7921</td>
			<td>Stainless steel</td>
		</tr>
		<tr>
			<td>Peptide Vaccine</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>investigational agent</td>
		</tr>
		<tr>
			<td>BD Microtainers</td>
			<td>BD</td>
			<td>365957</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Cassettes</td>
			<td>Simport</td>
			<td>M490-12</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>Fisher Scientific</td>
			<td>SF100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysis Buffer</td>
			<td>Pierce</td>
			<td>87787</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease &#38; Phosphatase inhibitor cocktail</td>
			<td>Thermo Scientific</td>
			<td>78444</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Cytokine 20plex Kit</td>
			<td>Invitrogen</td>
			<td>LMC006</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Microsphere Beads</td>
			<td>Luminex</td>
			<td>MC100xx-01</td>
			<td>xx is the bead region</td>
		</tr>
		<tr>
			<td>Anti-mouse TNF- Capture Antibody</td>
			<td>BD Pharmingen</td>
			<td>551225</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse TNF- Detection Antibody</td>
			<td>BD Pharmingen</td>
			<td>554415</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IFN- Capture Antibody</td>
			<td>Abcam</td>
			<td>ab10742</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IFN- Detection Antibody</td>
			<td>Abcam</td>
			<td>ab83136</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Sigma</td>
			<td>P3813-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay Buffer</td>
			<td>Millipore</td>
			<td>L-MAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokine Standard</td>
			<td>Millipore</td>
			<td>MXM8070</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-screen HTS 96well filter plates</td>
			<td>Millipore</td>
			<td>MSBVN1210</td>
			<td></td>
		</tr>
		<tr>
			<td>SA-PE</td>
			<td>Invitrogen</td>
			<td>SA10044</td>
			<td></td>
		</tr>
		<tr>
			<td>100 m Nylon Tissue Sieves</td>
			<td>BD</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Splenocyte Separation Media</td>
			<td>Lonza</td>
			<td>17-829E</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF- /IL-4 ELISpot plates</td>
			<td>R&#38;D Systems</td>
			<td>ELD5217</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-MUC1 monoclonal antibody</td>
			<td>Epitomics</td>
			<td>2900-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-actin monoclonal antibody</td>
			<td>Sigma</td>
			<td>A1978</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit HRP antibody</td>
			<td>Promega</td>
			<td>W401B</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse HRP antibody</td>
			<td>Santa Cruz Biotechnology, Inc.</td>
			<td>SC-2005</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>BioRad</td>
			<td>162-0174</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Protean TGX Precast Gels</td>
			<td>BioRad</td>
			<td>456-1083</td>
			<td></td>
		</tr>
		<tr>
			<td>Muse Count &#38; Viability Kit</td>
			<td>Millipore</td>
			<td>MCH100104</td>
			<td></td>
		</tr>
		<tr>
			<td>MUC1 Antibody</td>
			<td>BD Pharmingen</td>
			<td>550486</td>
			<td>IHC antibody</td>
		</tr>
		<tr>
			<td>Animal Research Peroxidase Kit</td>
			<td>Dako</td>
			<td>K3954</td>
			<td>IHC staining</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Equipment and Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore plate vaccum apparatus</td>
			<td>Millipore</td>
			<td>MSVMHTS00</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminex Lx200</td>
			<td>Millipore / Luminex</td>
			<td>40-013</td>
			<td>Manufactured by Luminex, distributed by Millipore</td>
		</tr>
		<tr>
			<td>Luminex Xponent Software</td>
			<td>Millipore / Luminex</td>
			<td>N/A</td>
			<td>Version 3.1; included with Luminex Lx200</td>
		</tr>
		<tr>
			<td>Milliple Analyst Software</td>
			<td>Milliplex / VigeneTech</td>
			<td>40-086</td>
			<td>Version 5.1</td>
		</tr>
		<tr>
			<td>Muse Cell Analyzer</td>
			<td>Millipore</td>
			<td>0500-3115</td>
			<td></td>
		</tr>
		<tr>
			<td>Muse Software</td>
			<td>Millipore</td>
			<td>N/A</td>
			<td>Version 1.1.0.0; included with Analyzer</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Unitron</td>
			<td>Z730</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism Software</td>
			<td>Graphpad Software Inc.</td>
			<td>N/A</td>
			<td>Version 5.1</td>
		</tr>
		<tr>
			<td>Mini Protean Tetra Cell Gel apparatus</td>
			<td>BioRad</td>
			<td>165-8001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans Blot SD Cell and PowerPac</td>
			<td>BioRad</td>
			<td>170-3849</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of invasive transitional cell bladder carcinoma in immune intact human muc1 transgenic mice:  a model for immunotherapy development

מודל קליני של סרטן שלפוחית ​​השתן פולשנית פותח בmucin האנושי 1 (MUC1) מהונדסים (MUC1.Tg) עכברים לצורך ההערכה חיסוני ו / או כימותרפיה רעילה לתאים. כדי לגרום לסרטן שלפוחית ​​השתן, עכברי C57BL / 6 (סוג MUC1.Tg ופראי) טופלו דרך הפה עם N-בוטיל-N-(4-hydroxybutyl) ניטרוזמין קרצינוגן (OH-BBN) על 3.0 מ&quot;ג / יום, 5 ימים / שבוע במשך 12 שבועות. כדי להעריך את ההשפעות של OH-BBN על פרופיל של ציטוקינים בסרום במהלך התפתחות גידול, כולה דם נאסף באמצעות מדמם submandibular לפני טיפול ואחת לארבעה שבועות. בנוסף, חיסון פפטיד MUC1 ממוקד ופלצבו ניתנו לקבוצות של עכברים לשבוע במשך שמונה שבועות. immunoanalyses microbead fluorometric זמנית של ציטוקינים בדם במהלך התפתחות וגידול החיסון הבא בוצעו. בסיום, אינטרפרון גאמא (IFN-γ) / אינטרלויקין 4 (IL-4) ניתוח ELISPOT לתגובת MUC1 ספציפי T-תא חיסון והערכות histopathological של סוג הגידולוכיתה בוצעה. התוצאות הראו כי: (1) את השכיחות של סרטן שלפוחית ​​השתן בעכברים מסוג שניהם MUC1.Tg ופראיים הייתה 67%, (2) קרצינומה של תאי מעבר (TCC) שפותחה בביחס של 2:1 בהשוואה לקרצינומה של תאי קשקש (SCC) (3) ציטוקינים דלקתיים מוגבר עם זמן במהלך התפתחות גידולים, וכן (4) מתן החיסון גורם לפפטיד פרופיל ציטוקינים סרום Th1-מקוטב ותגובת תא T MUC1 ספציפית. כל הגידולים בעכברי MUC1.Tg היו חיוביים לMUC1 ביטוי, ומחצית מכל הגידולים בעכברים מסוג MUC1.Tg ופראיים היו פולשני. לסיכום, שימוש בגישה צוות באמצעות התיאום של מאמציהם של רוקחים, אימונולוגים, פתולוגים, ביולוגים מולקולריים, פיתחנו מודל עכבר מהונדס חיסונית שלם של סרטן שלפוחית ​​השתן המבטא hMUC1.

ההערכה קלינית של השפעת immunotherapies רומן ושילובים בסרטן שלפוחית ​​השתן מחייבת הפיתוח של מודל חיה מתאים. במודל העכבר המהונדס שלנו, אינדוקציה עם החומר מהסרטן הכימי OH-BBN הביאה לשיעור גבוה של מקרי סרטן שלפוחית ​​השתן של TCC בעיקר עם כמה SCC, שהוא דומה לסרטן שלפוחית ​​השתן בבנ...

Watch this Scientific Journal Video about Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development at JoVE.com

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

N-בוטיל-N-(4-hydroxybutyl) ניטרוזמין-Induced מודל סרטן שלפוחית ​​השתן פותח בmucin האנושי 1 (MUC1) עכברים מהונדסים לצורך בדיקות MUC1 מכוון החיסוני. לאחר מתן חיסון פפטיד MUC1 ממוקד, תגובת הלימפוציטים T ציטוטוקסי לMUC1 אושרה על ידי רמות ציטוקינים בסרום ופעילות תאי T ספציפית מדידה.

אינדוקציה של קרצינומה של תאי מעבר שלפוחית ​​השתן פולשנית בMUC1 עכברים הטרנסגניים חיסוניים אנושיים שלמים: מודל לפיתוח חיסוני

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

induction-invasive-transitional-cell-bladder-carcinoma-immune-intact

Research

JoVE Journal

Medicine

21.1K Views.  University of California, Davis. N-בוטיל-N-(4-hydroxybutyl) ניטרוזמין-Induced מודל סרטן שלפוחית ​​השתן פותח בmucin האנושי 1 (MUC1) עכברים מהונדסים לצורך בדיקות MUC1 מכוון החיסוני. לאחר מתן חיסון פפטיד MUC1 ממוקד, תגובת הלימפוציטים T ציטוטוקסי לMUC1 אושרה על ידי רמות ציטוקינים בסרום ופעילות תאי T ספציפית מדידה.

Video: Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

הקמה התפשטות של גידולים רטינובלסטומה האדם עכברים החיסון לקויה

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

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.

Multi-פוטון הדמיה של הפלישה תאים סרטניים במודל עכבר orthotopic של קרצינומה של תאים קשקשיים אוראלי

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

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

פרוטוקול ליפרתרמיה הארוכה משך כל גוף בעכברים

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

השימוש בCystometry במכרסמים קטנים: חקר Chemosensation שלפוחית ​​השתן

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

דור של תת עורי וintrahepatic אדם xenografts קרצינומה hepatocellular בעכברים עם מערכת חיסון חלשים

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

תגובות שתן בשלפוחית ​​השתן ההתנפחות העוררת Visceromotor כמודל לשלפוחית ​​שתן כאב בעכברים

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

Axotomy פנים עצב של עכברים: דגם לחקר תגובת Motoneuron לפגיעה

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

אינדוקציה של טרשת עורקים מואצת בעכברים: מודל "פגיעת חוט"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

הטכניקה של פולשנית התרחבות אבי העורקים רוחבי בעכברים עבור אינדוקציה של גדילת

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

מודל פולשנית לניתוח שבר Endochondral ריפוי בעכברים בתנאים מתוקננים ביו-מכני

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

אינדוקציה של קרצינומה של תאי מעבר שלפוחית ​​השתן פולשנית בMUC1 עכברים הטרנסגניים חיסוניים אנושיים שלמים: מודל לפיתוח חיסוני

אינדוקציה של קרצינומה של תאי מעבר שלפוחית השתן פולשנית בMUC1 עכברים הטרנסגניים חיסוניים אנושיים שלמים: מודל לפיתוח חיסוני