עבודה זו נתמכה על ידי הקרן לפיתוח מדע וטכנולוגיה של פרויקט מפתח, מחלקת הבריאות של נאנג'ינג (No.YKK16098).

ניסויים בבעלי חיים אושרו על ידי הוועדה המוסדית לטיפול בבעלי חיים ושימוש (IACUC) של בית החולים מגדל התוף נאנג'ינג.
1. הכנת ריאגנטים ובעלי חיים
<ol><li>להמיס דוקסורוביצין הידרוכלוריד (פייזר, ארה"ב) במים מעוקרים. מערבולת כדי להשיג פתרון דוקס של 1 מ"ג / מ"ל ולשמור על 4 °C (70 °F).</li>
	<li>השתמש C57BL / 6 עכברים (8-10 שבועות; 25-30 גרם משקל). לצורך מחקר זה, עכברים נרכשו מהמרכז לחקר בעלי חיים מודל של אוניברסיטת נאנג'ינג ונשמרו בחדר החיות של בית החולים מגדל התוף נאנג'ינג.</li>
	<li>כלובי עכברים ללא פתוגן נשמרו תחת מחזור בהיר / כהה 12 שעות בטמפרטורה קבועה של 23 °C (70 °F). כל בעלי החיים ניזונים מדיאטת צ'או רגילה וקיבלו מזון ומים עד ליביטום.</li>
</ol>2. הקמת מודל בעלי חיים DCM
<ol><li>הפוך עכברים לקבוצה רגילה (n = 5) ולקבוצה דוקס (n = 5).</li>
	<li>לנהל את פתרון Dox תוך-פרטי באופן תוך-זמני במינון של 5 מ"ג/ק"ג באמצעות מזרק מעוקר 1 מ"ל 1x בשבוע עבור קבוצת Dox. לטפל בעכברים בקרה באותו אופן עם אותה כמות של תמיסת מלח.</li>
	<li>למדוד את משקל הגוף של שתי הקבוצות בשבוע. בהתאם, להתאים את מינון ההזרקה בהתבסס על משקל הגוף מדי שבוע במשך סך של 4 שבועות, עם מינון מצטבר של 20 מ"ג/ קילוגרם (איור 1). הערה: פרק זמן של 4 שבועות נבחר כי אקוקרדיוגרפיה ב 4 שבועות הראה הבדל משמעותי בתפקוד הלב בין שתי הקבוצות.</li>
</ol>3. בחינה אקוקרדיוגרפית
<ol><li>בסוף השבוע הרביעי, לערוך בדיקה אקוקרדיוגרפית של העכברים.</li>
	<li>להרידים את העכברים עם 2% איזופלוריין תוך גולגולתי. אם העכברים אינם מגיבים קמצוץ של העור עם פינצטה שיניים או מגרה את בהונות וזנבות, להמשיך עם הפרוטוקול.</li>
	<li>הנח את העכבר על פלטפורמה לטיפול בבעלי חיים בתנוחה העגמה. כדי לשמור על ההרדמה, לכסות את האף והפה של החיה עם חרוט האף ולספק 2% איזופלורן. הערה: עבור הרדמה IP, להזריק 4% הידרציה כלורל במינון של 0.2 מ"ל / 20 גרם.</li>
	<li>הסר פרוות חזה בזהירות עם מכונת גילוח חשמלית. להעריך את תפקוד הלב ב vivo באמצעות אקו-קרדיוגרפיה טרנסתורקטית.</li>
	<li>בצע את ההד-לב של LV הן בתצוגות ציר ארוך-צירי והן בתצוגות קצרות ציר בקצב פריימים של 233 הרץ. הממדים הסופיים-סיסטוליים והדיאסטוליים הסופיים הוגדרו כשלבים המתאימים לגל ה- ECG T ולגל ה- R, בהתאמה.</li>
	<li>במעקבי מצב M, מדוד את הקוטר הסיסטולי הממוצע של LV (LVIDd), קוטר קצה-דיאסטולי של LV (LVIDs), עובי מחיצה בין-חדרי (IVS) ועובי הקיר האחורי של LV (LVPW) מפעימות לב של 3-5. חשב גם את שבר הפליטה (EF) וקיצור השבר (FS) בהתבסס על אקו-קרדיוגרפיה.</li>
</ol>4. כתמים היסתולוגיים
<ol><li>לאחר הניתוח echocardiographic, להקריב את העכברים על ידי הזרקה תוך חדרית של 10% אשלגן כלורי.</li>
	<li>לטפס את הלב עם כ 30 מ"ל של תמיסת מלח לאחר הניתוח עד הכבד והריאה להיות חיוור.</li>
	<li>Excise את הלב ביסודיות לשטוף אותו בתמיסת חוצץ פוספט כדי להלל דם.</li>
	<li>תקן את הלב ב 4% פורמלין בטמפרטורת החדר במשך 24 שעות ולטפל ברקמה בקופסת פרפין, כך שעוות פרפין מתקררת ומתמצק.</li>
	<li>חותכים את הלבבות לפרוסות בעובי 5 מיקרומטר להכתמה פתולוגית.</li>
	<li>Dewax ו rehydrate חלקים המכילים שריר הפפילרי.<ol>
<li>מגלשות דגירה ב 55 °C (55 °F) במשך 30 דקות. לאחר מכן, דגירה קסילן 2x במשך 2 דקות כל אחד; 100% אתנול 2x במשך 2 דקות כל אחד; 95% אתנול 2x במשך 2 דקות כל אחד; 80% אתנול במשך 2 דקות; 75% אתנול במשך 2 דקות; ו-50% אתנול ל-2 דקות.</li>
	</ol>
</li>
	<li>כתם באמצעות המטוקסילין ואוסין (H&E) כמו גם הכתם של מאסון.</li>
</ol>

<ol>
	<li>Weintraub, R. G., Semsarian, C., MacDonald, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dilated+cardiomyopathy.">Dilated cardiomyopathy.</a> Lancet. 390 (10092), 400-414 (2017).</li><li>Ichihara, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+of+oxidative+stress+and+cardiac+dysfunction+by+bisoprolol+in+an+animal+model+of+dilated+cardiomyopathy.">Attenuation of oxidative stress and cardiac dysfunction by bisoprolol in an animal model of dilated cardiomyopathy.</a> Biochemical and Biophysical Research Communications. 350 (1), 105-113 (2006).</li><li>Fountoulakis, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+the+heart+mitochondrial+proteome+in+a+desmin+null+heart+failure+model.">Alterations in the heart mitochondrial proteome in a desmin null heart failure model.</a> Journal of Molecular and Cellular Cardiology. 38 (3), 461-474 (2005).</li><li>Fairweather, D., Rose, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coxsackievirus-induced+myocarditis+in+mice:+a+model+of+autoimmune+disease+for+studying+immunotoxicity.">Coxsackievirus-induced myocarditis in mice: a model of autoimmune disease for studying immunotoxicity.</a> Methods. 41 (1), 118-122 (2007).</li><li>Wang, Z. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+therapeutic+anti-CD4+monoclonal+antibody+inhibits+T+cell+receptor+signal+transduction+in+mouse+autoimmune+cardiomyopathy.">A therapeutic anti-CD4 monoclonal antibody inhibits T cell receptor signal transduction in mouse autoimmune cardiomyopathy.</a> Chinese Medical Journal. 120 (15), 1319-1325 (2007).</li><li>Riad, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor-4+deficiency+attenuates+doxorubicin-induced+cardiomyopathy+in+mice.">Toll-like receptor-4 deficiency attenuates doxorubicin-induced cardiomyopathy in mice.</a> European Journal of Heart Failure. 10 (3), 233-243 (2008).</li><li>Kankeu, C., Clarke, K., Passante, E., Huber, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin-induced+chronic+dilated+cardiomyopathy-the+apoptosis+hypothesis+revisited.">Doxorubicin-induced chronic dilated cardiomyopathy-the apoptosis hypothesis revisited.</a> Journal of Molecular Medicine. 95 (3), 239-248 (2017).</li><li>Zhao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-140-5p+aggravates+doxorubicin-induced+cardiotoxicity+by+promoting+myocardial+oxidative+stress+via+targeting+Nrf2+and+Sirt2.">MicroRNA-140-5p aggravates doxorubicin-induced cardiotoxicity by promoting myocardial oxidative stress via targeting Nrf2 and Sirt2.</a> Redox Biology. 15, 284-296 (2018).</li><li>O'Connell, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+and+long-term+models+of+doxorubicin-induced+cardiomyopathy+in+rats:+A+comparison+of+functional+and+histopathological+changes.">Short-term and long-term models of doxorubicin-induced cardiomyopathy in rats: A comparison of functional and histopathological changes.</a> Experimental and Toxicologic Pathology. 69 (4), 213-219 (2017).</li><li>Yuan, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTRP3+protected+against+doxorubicin-induced+cardiac+dysfunction,+inflammation+and+cell+death+via+activation+of+Sirt1.">CTRP3 protected against doxorubicin-induced cardiac dysfunction, inflammation and cell death via activation of Sirt1.</a> Journal of Molecular and Cellular Cardiology. 114, 38-47 (2018).</li><li>Sun, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TGF-beta+pathway+mediates+doxorubicin+effects+on+cardiac+endothelial+cells.">The TGF-beta pathway mediates doxorubicin effects on cardiac endothelial cells.</a> Journal of Molecular and Cellular Cardiology. 90, 129-138 (2016).</li><li>Minotti, G., Menna, P., Salvatorelli, E., Cairo, G., Gianni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthracyclines:+molecular+advances+and+pharmacologic+developments+in+antitumor+activity+and+cardiotoxicity.">Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity.</a> Pharmacological Reviews. 56 (2), 185-229 (2004).</li><li>Vejpongsa, P., Yeh, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+anthracycline-induced+cardiotoxicity:+challenges+and+opportunities.">Prevention of anthracycline-induced cardiotoxicity: challenges and opportunities.</a> Journal of The American College of Cardiology. 64 (9), 938-945 (2014).</li><li>Renu, K., V, G. A., P, B. T., Arunachalam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+of+doxorubicin-induced+cardiomyopathy+-+An+update.">Molecular mechanism of doxorubicin-induced cardiomyopathy - An update.</a> European Journal of Pharmacology. 818, 241-253 (2018).</li></ol>

לא הוכרזו ניגודי אינטרסים.

Dox היא תרופה אנטי-טמורית תקופתית לא-מדעית הנפוצה בפרקטיקה קלינית12. תופעת הלוואי העיקרית שלה היא cardiotoxicity, המאופיינת על ידי קרדיומיופתיה ואי ספיקת לב לאחר מכן13. המנגנון הבסיסי כולל נזק של peroxidation השומנים שריר הלב, עיכוב של רשתית סרקופלסמית שריר הלב Ca2 +- ATPase, והפ...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61158">A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo</a>. A figure was updated.
Figure 1 was updated from:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61158/61158fig1v2.jpg" /> 
Figure 1: Schematic diagram of a Dox-induced dilated cardiomyopathy. <a href="https://www.jove.com/files/ftp_upload/61158/61158fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61158/61158fig1v5.jpg" /> 
Figure 1: Schematic diagram of a Dox-induced dilated cardiomyopathy. <a href="https://www.jove.com/files/ftp_upload/61158/61158fig1v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61158">A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo</a>. A figure was updated.

Erratum: A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

אחד הגורמים הנפוצים ביותר לאי ספיקת לב, DCM מאופיין בהרחבת חדרית וירידה בתפקוד הלב והיא הסיבה הנפוצה ביותר להשתלת לב ברחבי העולם1. על מנת להמשיך לחקור את הפתוגנזה שלה ולמצוא טיפולים יעילים, גישה למודלים בעלי חיים בוגרים חשובה במיוחד. מטרת הניסויים המתוארים היא ליצור מודל עכבר יציב של DCM הדומה ל- DCM אנושי.
בשל הפתוגנזה המורכבת של DCM, ישנן שיטות רבות ושונות כדי להפוך מודלים בעלי חיים מתאימים. דגמי DCM ספונטניים2 יציבים יחסית, אך הם יקרים ולא זמינים בקלות. מודלים בעלי חיים מהונדסים גנטית3 אינם מבוססים היטב ודורשים שימוש ניסיוני יותר. מודלים בעלי חיים DCM המושרה על ידי זיהום ויראלי4 או פגמים אוטואימוניות5 קל להשיג, אבל הם לא מייצגים לחלוטין של DCM. מודלים הקשורים רעילות שריר הלב כוללים מודלים DCM הנגרמים מאלכוהול ומודלים בעלי חיים DCM המושרה Dox.
מודל קרדיומיופתיה המושרה דוקס מתקבל על ידי הזרקה תוך-איפריטונית של Dox6. המודל מנצל את תופעת הלוואי הכרונית החמורה ביותר של Dox: לאחר חשיפה של Dox, חולים לפתח סימפטומים DCM 12et מאוחר עם אחידות קלינית7. דוקס-המושרה סטרס חמצוני8 ונזק מיטוכונדריאלי9, אשר מובילים אפופטוזיס cardiomyocyte, הם סימפטומים בפתוגנזה של DCM. ישנם מודלים טיפול דוק אקוטי וכרוני: מינון גבוה יחיד של Dox (15 מ"ג/ק"ג) מעורר מודל לטווח קצר עבור קרדיומיופתיה10, בעוד זריקות דוקס במינון נמוך חוזרות ונשנות (שש שבועיות, 3 מ"ג/ קילוגרם) לגרום מודל לטווח ארוך עבור קרדיומיופתיה11. בהתבסס על המחקר שהוצג, עכברי סוג בר מוזרק תוך אלפיציונלי פעם בשבוע במשך חודש במינון של 5 מ"ג / קילוגרם תצוגה מורפולוגיה והיסתולוגיה של הלב עולה בקנה אחד עם המאפיינים של DCM עד סוף הטיפול, מתן דרך אידיאלית להקים מודל DCM.

<table>
	<tbody>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>servicebio</td>
			<td>CAS30525-89-4</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Model Animal Research Center of Nanjing University</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin hydrochloride</td>
			<td>Pfizer</td>
			<td>CAS25316-40-9</td>
			<td></td>
		</tr>
		<tr>
			<td>echocardiography</td>
			<td>Visualsonics</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin staining kit</td>
			<td>Solarbio</td>
			<td>G1120</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson staining kit</td>
			<td>Solarbio</td>
			<td>G1343</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffer solution</td>
			<td>Sigma</td>
			<td>P5368</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium chloride</td>
			<td>Sigma</td>
			<td>CAS7447-40-7</td>
			<td></td>
		</tr>
		<tr>
			<td>sterilized syringe</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
	</tbody>
</table>

a doxorubicin-induced murine model of dilated cardiomyopathy in vivo

קרדיומיופתיה מורחבת (DCM) מתייחסת לספקטרום של הפרעות שריר הלב הטרוגניות המאופיינת בהרחבת חדרי הלב וביצועי לב מדוכאים בהיעדר יתר לחץ דם, valvular, מולד, או איסכמי מחלות לב, אשר עשוי להיות קשור לזיהום, הפרעות אוטואימוניות או מטבוליות, או ירושה משפחתית. זה יכול להתקדם לתוך אי ספיקת לב עם פרוגנוזה גרועה. Doxorubicin (Dox) מועסק באופן נרחב כסם כימותרפי, אך השימוש בו מוגבל מכיוון שהוא גורם לשינויים דמויי DCM של שריר הלב. רעילות שריר הלב שלה מיוחסת עקה חמצונית, דלקת כרונית, אפופטוזיס cardiomyocyte. מודל של DCM ניצול אלה סימפטומים DCM הנגרמת Dox לא הוקם.

תפקוד הלב קרדיומיופתיה מתרחבת מאופיינת בהרחבת חדרים מתקדמת ותפקוד לקוי של התכווצות. איור 2 מציג תמונות אקו-קרדיוגרפיות מייצגות של שתי הקבוצות. עכברים שטופלו בדוקס הראו שבר פליטה מופחת משמעותית של החדר השמאלי וקיצור חלקי חדר שמאלי(איור 3A,B).</s...

Watch this Scientific Journal Video about A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo at JoVE.com

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

מתואר הוא פרוטוקול כדי להקים דוקסורוביצין המושרה קרדיומיופתיה המושרה (DCM) מודל בעכברים באמצעות הזרקה תוך-פטרייתונית לטווח ארוך של Doxorubicin.

דגם מורין המושרה על ידי דוקסורוביצין של קרדיומיופתיה מונפשת ב- Vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...

a-doxorubicin-induced-murine-model-of-dilated-cardiomyopathy-in-vivo

Research

JoVE Journal

Medicine

4.3K Views.  Clinical College of Nanjing Medical University. מתואר הוא פרוטוקול כדי להקים דוקסורוביצין המושרה קרדיומיופתיה המושרה (DCM) מודל בעכברים באמצעות הזרקה תוך-פטרייתונית לטווח ארוך של Doxorubicin.

Video: A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

מודל עכברי של ריפוי פצע

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

מודל Murine של תת עכבישי דימום

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

דגם Murine פרה של גרורות כבדית

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

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

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

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

חסך בתאי עכבר מן xenografts גידול האנושי משפר משמעותי ניתוח במורד זרם של תאי יעד

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

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

טכיקרדיה-Induced קרדיומיופתיה כמודל אי ספיקת לב כרונית, חזיר

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.

מודל דוקסורוביצין-induced קרדיומיופתיה בדג זברה למבוגרים

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

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.

Contractility איזומטרי מדידה של עורק מצע המעי העליון העכבר באמצעות חוט Myography

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

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

הערכת אנגיוגנזה תרפויטי במודל של מורטין של איסכמיה החוצה

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

In Vivo Mouse Model of Spinal Implant Infection

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The purpose of this method is to describe a novel mouse model of spinal implant infection (SII) that was created to provide an inexpensive, rapid, and accurate in vivo tool to test potential therapeutics and treatment strategies for spinal implant infections.
In this method, we present a model of posterior-approach spinal surgery in which a stainless-steel k-wire is transfixed into the L4 spinous process of 12-week old C57BL/6J wild-type mice and inoculated with 1 x 103 CFU of a bioluminescent strain of Staphylococcus aureus Xen36 bacteria. Mice are then longitudinally imaged for bioluminescence in vivo on post-operative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. Bioluminescence imaging (BLI) signals from a standardized field of view are quantified to measure in vivo bacterial burden.
To quantify bacteria adhering to implants and peri-implant tissue, mice are euthanized and the implant and surrounding soft tissue are harvested. Bacteria are detached from the implant by sonication, cultured overnight and then colony forming units (CFUs) are counted. The results acquired from this method include longitudinal bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux) and CFU counts following euthanasia.
While prior animal models of instrumented spine infection have involved invasive, ex vivo tissue analysis, the mouse model of SII presented in this paper leverages noninvasive, real time in vivo optical imaging of bioluminescent bacteria to replace static tissue study. Applications of the model are broad and may include utilizing alternative bioluminescent bacterial strains, incorporating other types of genetically engineered mice to contemporaneously study host immune response, and evaluating current or investigating new diagnostic and therapeutic modalities such as antibiotics or implant coatings.

The protocol describes a novel in vivo mouse model of spinal implant infection where a stainless-steel k-wire implant is infected with bioluminescent Staphylococcus aureus Xen36. Bacterial burden is monitored longitudinally with bioluminescent imaging and confirmed with colony forming unit counts after euthanasia.

במודל עכבר Vivo של זיהום שתל בעמוד השדרה

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

מורנה דיוק-גזור פרוסות הכבד כמודל Vivo Ex של ביולוגיה של הכבד

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...