עבודה זו מומנה דרך קורק בדרום מרפאת קופת ויקטוריה אוניברסיטת השד בבית החולים, האגודה למלחמה בסרטן אירי (CRI07TAN) לבין סרטן קורק מרכז המחקר. תא לואיס ריאות קו ביציבות להביע בלוציפראז הייתה מתנה סוג של ד&quot;ר קארין יוס, Cell Genesys, Inc, סן פרנסיסקו, ארה&quot;ב.

<html> הקדמה הכבד הוא לעתים קרובות את האתר הראשון של מחלה גרורתית וניתן האתר היחיד של התפשטות% כמספר 30 של 40 חולים עם מחלה מתקדמת. עם זאת אפשרויות לטיפול בגידולים בכבד גרורתי מעטים עם מיעוט מהחולים מתאים כריתה ורק 23% המגיבים העיקריים chemotherapeutics. מחקר קליני בתחום זה הוגבל על ידי בהעדר העכבר מודל מתאים ללימוד מחלת כבד מבודד. אנו מתארים גישה מפוצל הטחול כי ניתן להשתמש באופן אמין להקים אחיד, גרורות בכבד מפוזר. כריתה של מחצית זו של הטחול ימנע את ההשלכות של הגידול הטחול ואת החצי השני של הטחול הוא חזר עם מחזור פורטל שלם כדי לשמור על הטחול מאפייני הגנה חיסונית. המודל שאנו מתארים הוא די פשוט לביצוע, ניתן לעשות זאת במהירות דרך חתך אחד בלבד. המאפיינים bioluminescent של קו תאים סרטניים מאפשר כימות מדויק של התגובה לטיפול מבלי להקריב בעלי חיים. פרוטוקול I. הכנה Cell <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לואיס לתאי ריאה קרצינומה גדלים שונה של Dulbecco בינוני חיוניים בתוספת סרום 10% עגל העובר על 37 מעלות צלזיוס, 5% CO 2 ו שנקטפו על ידי trypsinisation כאשר תת ומחוברות. </li><li style=";text-align:right;direction:rtl"> תאים נספרו באמצעות Nucleocounter (חמו-metec, דנמרק) ואת השעיית תא בודד של 1 X 10 6 תאים μl 200 הוכן תקשורת חופשית בסרום. </li></ol> השנייה. עכבר הכנה הערה: כל הנהלים אושרו על ידי ועדת האתיקה של מכללת אוניברסיטת קורק. במעבדה שלנו אנו משתמשים בזריקות הרדמה כדי להרדים את העכבר, לחילופין, ניתן להשתמש הרדמה נשאפים כדי להשיג את אותו אפקט. המתח עכבר השתמשנו הוא שיער MF1 נו / נו המתח המתקבל הרלן מעבדות (אוקספורדשייר, אנגליה). נקבה MF1 נו / נו עכברים במשקל 16-22 גרם של 6-8 שבועות של גיל נכללו בניסוי זה. זנים אחרים כגון C57 ו Balb / C יכול לשמש גם עם שורות תאים מתאים סרטניים. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אלחוש נמסר על ידי הזרקת intraperitoneal של קטמין 100ul של 1.5 מ&quot;ג ו 300 xylazine UG. </li><li style=";text-align:right;direction:rtl"> עומק ההרדמה היא הוערכה באמצעות צביטה הבוהן. לא צריך להיות שום נסיגה רפלקס עם קמצוץ הבוהן. </li><li style=";text-align:right;direction:rtl"> עכבר הרדים ממוקם כראוי מודבק על כרית חימום. </li><li style=";text-align:right;direction:rtl"> הבטן היא prepped עם פתרון בבטאדין 10%. </li><li style=";text-align:right;direction:rtl"> הבטן אתר כירורגי הם עטופים בצורה סטרילית. </li></ol> ג. בטן החתך <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חתך subcostal קטן עשוי בעור. </li><li style=";text-align:right;direction:rtl"> החתך מורחב 2-3 ס&quot;מ. </li><li style=";text-align:right;direction:rtl"> בטן קיר השרירים חולקו ואת חלל הצפק נכנס. </li></ol> IV. חשיפה אגף הטחול <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הטחול הוא לגייס exteriorized בעדינות על גזה מראש לחתוך סטרילית. </li><li style=";text-align:right;direction:rtl"> שני חיבורי אותיות vicryl 4-0 קשורות סביב הגוף באמצע של הטחול. </li><li style=";text-align:right;direction:rtl"> הטחול מחולק בין שני חיבורי אותיות יצירת שני חמי spleens. </li></ol> V. הזרקת תאים לתוך הטחול, חמי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> G 27 משמש להזריק נפח 100 μL של תאים לתוך אחד חמי הטחול. </li><li style=";text-align:right;direction:rtl"> תאים Tumour מותר לנקז אל הכבד במשך עשר דקות מתן זמן מספיק עבור רוב התאים מוזרקים להגיע אל הכבד. </li></ol> VI. כריתה של הטחול, חמי, סגר שחזור בטן <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מוזרק חמי הטחול הוא נכרת. </li><li style=";text-align:right;direction:rtl"> השני חמי הטחול הוא חזר חלל הבטן. </li><li style=";text-align:right;direction:rtl"> שכבת השריר נסגר עם תפר PDS 4-0 בעזרת תפר פועל פשוט. </li><li style=";text-align:right;direction:rtl"> עור הבטן נסגר עם Prolene 4-0 באמצעות תפרים קטע. </li><li style=";text-align:right;direction:rtl"> בולוס נוזלים מנוהל ואת שלאחר הניתוח כאבים, Carpofen (5 מ&quot;ג / ק&quot;ג) מנוהל על ידי הזרקת IP לניתוח שלאחר 24 שעות. </li><li style=";text-align:right;direction:rtl"> העכבר הוא מותר להתאושש מן ההרדמה </li></ol> VII. IVIS כל הגוף Bioluminescent הדמיה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עכבר הוא מורדם באמצעות הרדמה בזריקות. </li><li style=";text-align:right;direction:rtl"> המצע Luciferin ניתנת על ידי התוך צפקי עשר דקות לפני הזריקה הדמיה </li><li style=";text-align:right;direction:rtl"> עכברים מוצבים על הבמה צילמו דקות 3 IVIS באמצעות המערכת כולה הדמיה הגוף </li><li style=";text-align:right;direction:rtl"> הארה מהכבד ניתן למדוד באמצעות אזור של כלי הריבית </li></ol> VIII. חשיפה של הכבד העכבר <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עכבר הוא מורדם באמצעות הרדמה בזריקות. </li><li style=";text-align:right;direction:rtl"> חתך גג בטן מאפשר הדמיה של גרורות בכבד מפוזר in vivo. </li><li style=";text-align:right;direction:rtl"> העכבר הוא מורדמים על ידי נקע בצוואר הרחם והכבד ניתן נכרת לניתוח נוסף. </li></ol>_upload/1977/1977fig1.jpg &quot;alt =&quot; איור 1 &quot;/&gt; באיור 1. מטופלים Murine נכרת טרי ואת הגידול הסתננו הכבדים. <img src="/files/ftp_upload/1977/1977fig2.jpg" alt="איור 2" /> איור 2. קינטיקה של גידול בכבד הגידול כפי שמעידים הדמיה IVIS הגוף כולו.

<ol>
	<li>Kasuya, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+subcutaneous+spleen+reservoir+for+multiple+portal+venous+injections+to+treat+liver+malignancies.">Mouse models of subcutaneous spleen reservoir for multiple portal venous injections to treat liver malignancies.</a> Cancer Res. 65, 3823-3827 (2005).</li><li>Liu, Q. Z., Tuo, C. W., Wang, B., Wu, B. Q., Zhang, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+metastasis+models+of+human+colorectal+carcinoma+established+in+nude+mice+by+orthotopic+transplantation+and+their+biologic+characteristic.">Liver metastasis models of human colorectal carcinoma established in nude mice by orthotopic transplantation and their biologic characteristic.</a> World J Gastroenterol. 4, 409-411 (1998).</li><li>Li, Z. Y., Ni, S., Yang, X., Kiviat, N., Lieber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenograft+models+for+liver+metastasis:+Relationship+between+tumor+morphology+and+adenovirus+vector+transduction.">Xenograft models for liver metastasis: Relationship between tumor morphology and adenovirus vector transduction.</a> Mol Ther. 9, 650-657 (2004).</li><li>Cashman, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+gene+therapy+as+a+neoadjuvant+to+surgical+excision+to+control+metastatic+cancers.">Immune gene therapy as a neoadjuvant to surgical excision to control metastatic cancers.</a> Cancer Lett. , (2008).</li></ol>

אין ניגודי אינטרסים הכריז.

התפתחות אמינה במודלים vivo לחקר גרורות בכבד הופך הגדלת חשוב עם כניסתו של אסטרטגיות טיפוליות חדשות. לצורך המחקר מדויק של אסטרטגיות חדשות, במודלים של בעלי חיים צריכים להיות קל יחסית לביצוע, טופס מספר משמעותי של הגרורות, לערב את כל השלבים של מפל גרורתי יש מערכת כתב המאפשר איתור הספירה של מספר מצומצם של תאים גרורתי. הגישה מפוצל טחול כמתואר כרוכה בפעולה אחת היא טכניקה קלה יחסית ללמוד ליצור גרורות בכבד אחיד מפוזר עם סיכון נמוך מאוד של carcinomatosis ו פקקת וריד הפורטל 1. חלוקת הטחול לשתי חמי spleens לאפשר הזרקת תאים סרטניים לתוך אחד חמי הטחול. תאים Tumour להזין את זרימת הפורטל ולנסוע אל הכבד, מחקה את הצעד הראשון של מפל גרורתי. כריתה של הטחול, חמי מוזרק ימנע את צמיחת הגידול הטחול ואת חזרתם של הטחול, חמי אחרים מבטיחה את השמירה על הנכסים החיסונית הטחול איזו חיה כבר לפני הליך .. מודל זה עשוי להיות מנוצל עבור מגוון רחב של סוגי גידולים. בלוציפראז ריאות לואיס קו התא מאפשרת כימות bioluminescent של מחלת כבד לאורך זמן. רגישות מוגברת של מערכת פליטת אור מאפשר איתור וכימות של מספר קטן של תאים גרורתי. זה מאפשר תזמון מדויק לייזום של פרוטוקולי הטיפול ומאפשר כימות של מחלת כבד ללא הקרבה של החיה. פשטות, עמידות reproducibility דגם זה מתאים ללימוד אסטרטגיות טיפול שונות, כולל רומן chemotherapeutics 2, ריפוי גנטי 3 ו 1,4 חיסוני.

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		<th>Material Name</th>
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<td>DMEM</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D6429</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D8537</td>
<td>&nbsp;</td>
<tr>
<td>Ketamine</td>
<td>&nbsp;</td>
<td>Vetoquinol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Xylazine</td>
<td>&nbsp;</td>
<td>Vetoquinol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Carprofen</td>
<td>&nbsp;</td>
<td>Vetoquinol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Videne</td>
<td>&nbsp;</td>
<td>Ecolab, Leeds, UK</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Vicryl 4-0 suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Ethicon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Prolene 4-0 suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Firefly Luciferin</td>
<td>&nbsp;</td>
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murine bioluminescent hepatic tumour model

וידאו זה מתאר את הקמתו של גרורות בכבד במודל של עכברים, כי ניתן לנתח לאחר מכן על ידי הדמיה bioluminescent. תאים Tumour מנוהלים במיוחד כדי לכבד לגרום גידול בכבד מקומי, באמצעות גיוס של הטחול ואת פיצול לשני, והשאיר על כנו את כלי הדם pedicle עבור כל מחצית של הטחול. לואיס לתאי ריאה קרצינומה כי constitutively לבטא את הגן בלוציפראז גחלילית (luc1) הם מחוסן אל הטחול, חמי אחד שהוא resected אז 10 דקות מאוחר יותר. השני חמי הטחול נשאר ללא פגע וחזר הבטן. הגידול בצמיחה הכבד יכול להיות פיקוח על ידי פליטת אור הדמיה באמצעות דימות כולו IVIS מערכת הגוף. הדמיה כמותי של הגידול באמצעות IVIS מספק quantitation המדויק של תאים סרטניים קיימא. תא Tumour מוות נמק עקב טיפול תרופתי מצוין מוקדם על ידי הפחתה של האות bioluminescent. זה מודל העכבר מאפשר לחקור את המנגנונים הישרדות גרורתי גידול תאים וגדילה והוא יכול לשמש לצורך הערכה של הרפוי של גרורות בכבד.

Watch this Scientific Journal Video about Murine Bioluminescent Hepatic Tumour Model at JoVE.com

Murine Bioluminescent Hepatic Tumour Model

מאמר זה מתאר את הליך הגיוס של orthotopic גידולים בכבד bioluminescent בעכברים, ניתוח בדיעבד של הגידול מוגבל בכבד באמצעות הארה חיים שלמים הדמיה הגוף.

Tumour Murine Bioluminescent כבדית דגם

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

murine-bioluminescent-hepatic-tumour-model

Nanyang Technological University

Research

JoVE Journal

Medicine

17.1K Views.  University College Cork. מאמר זה מתאר את הליך הגיוס של orthotopic גידולים בכבד bioluminescent בעכברים, ניתוח בדיעבד של הגידול מוגבל בכבד באמצעות הארה חיים שלמים הדמיה הגוף.

Video: Murine Bioluminescent Hepatic Tumour Model

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

הדור ניסויית של קרצינומה-Associated Fibroblasts (CAFs) מ Fibroblasts החלב אנוש

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

במצע כפולה vivo bioluminescent ההדמיה

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

השרשה תוך גולגולתי עם 3D לאחר In vivo Bioluminescent הדמיה של gliomas Murine

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

ניתוחים למודל חולדה של השתלה כבדה orthotopic חלקית עם שחזור עורקית כבדיה

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

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. ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

דגם orthotopic bioluminescent של התקדמות סרטן הלבלב

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

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, ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

דגם Murine של פגיעת המעיים איסכמיה reperfusion

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

שיטה של ​​המגזרים הישירים Intra-כבדית משלוח באמצעות מודל קלאמפ עכברוש הכבד hilar

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...