המחברים מודים ג&#39;יידן סמית ללאור וההוכחה עורכת את כתב היד. עבודה זו קיבלה תמיכה מהאגודה הלאומית הטרשת הנפוצה (NMSS, מענקים חלקיים RG-4001-A1), הטרשת הנפוצה האגודה האיטלקית (AISM, להעניק 2010/R/31), המשרד האיטלקי לבריאות (GR08-7), כנפיים לחיים, Banca Agricola Popolare di Ragusa (BAPR), המועצה האירופית למחקר (ERC) על פי הסכם ERC-2010-STG גרנט לא 260,511-SEM_SEM ותכנית מסגרת ה -7 של הקהילה האירופית (EC) (FP7/2007-2013) תחת גרנט הסכם n * מעלות; 280,772 - Ione.

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יש המחברים אין לחשוף.

טיפולים בתאי גזע סומטיים מבוססים הם מתעוררים כאחד מהאסטרטגיות המבטיחות ביותר לטיפול בהפרעות במערכת העצבים המרכזית דלקתיות כרוניות כגון 11 MS2. בעוד שמנגנוני שמירה על ההשפעות הטיפוליות שלהם עדיין צריכה להיות הובהר לגמרי, ההשפעה המשמעותית של השתלת NPC במודלי ניסו?...

ראיות חזקות שקמה במחקרי vivo המעידים על היעילות הטיפולית של השתלת תאים סומטיים גזע עצביים / מבשר (NPCs) במודלים של בעלי חיים של הפרעות במערכת העצבים המרכזית 1-8. עם זאת, מספר הסוגיות הנוגעות לאספקה ​​של תאי גזע למארח דורש שיקול דעת לפני שניתן לתרגם את תוצאות ניסויים אלו ליישומים קליניים. המשוכה במיוחד משמעותית לקראת הפיתוח של טיפולים (nonhematopoietic) משקמים בתאי גזע לטיפול במחלות multifocal, דלקתיות כרוניות במוח היא זיהוי של המסלול האידיאלי של הזרקת NPC. הבנה מוצקה של הפתופיזיולוגיה של המחלה ממוקדת (מוקד או multifocal; ניווניות דלקתיות או עיקריות ראשוניות), וניתוח זהיר של נושאי היתכנות וסיכונים הקשורים בטכניקות המסירה הן בזיהוי הפרוטוקול האופטימלי למסירה בתאי גזע. בעוד המוקד ( <em&gt; למשל. לparenchyma מערכת העצבים) השתלת תאי גזע היא גישה הגיונית לטיפול במחלות של מערכת העצבים המרכזית מאופיינות באזורים מוגבלים מרחבית של נזק (למשל פרקינסון ומחלת הנטינגטון, פגיעות טראומטיות במוח ובחוט השדרה, ושבץ), אותו הגישה עשויה להוכיח להיות כמעט לא ריאלי במצבים כגון טרשת נפוצה, שבו נזק במערכת העצבים המרכזית multifocal, כרוני, והופץ במרחב מצטבר לאורך זמן. במקרה אחרון זה, מיקוד זריקות תא מוקד לנגעים בודדים גם הפריע הקיבולת המוגבלת של NPCs המושתל להגר למרחקים ארוכים בתוך parenchyma מערכת העצבים המרכזית, ובכך גרם לזיהוי של שיטות חלופיות, מתאימות יותר של מערכת העצבים המרכזית מיקוד עם השתלות NPC פחות פולשני . הבטחה גדולה יצאה מהתצפיות שNPCs למקד את הגידול תוך גולגולתי (למשל. גליומה) בעכברים כאשר הוא מוזרק intravascularly מחוץ CNS9. בעקבות זאת זרעבראיות vivo של pathotrophism תאי גזע 10, נתונים נרחבים שנצברו בנוגע להיתכנות והיעילות טיפולית של ההשתלה המערכתית של NPCs בחיות מעבדה עם Encephalomyelitis הניסיוני אוטואימוניות (EAE), כמודל לניזק במערכת העצבים המרכזית דלקתי, באמצעות או דרך הווריד (Iv) או intracerebroventricular הזרקת NPC 1,2,5,6,8 (ICV.) .. הראנו ראשון שזה תלוי ביכולת של NPCs המושתל למקד ולהזין את מערכת העצבים המרכזית המודלקות, ולעסוק בין תאית מרובה לאחר מכן תוכניות תקשורת בתוך microenvironments ספציפי in vivo 11. על מנת למקד במיוחד במערכת העצבים המרכזית, NPCs מועברים ישירות לתוך נוזל השדרה מחזור (CSF) על ידי הזרקת ICV, או למחזור הדם באמצעות הזרקת iv. ברגע שנכנס גם למחזור הדם או CSF, NPCs מושתל באופן פעיל באינטראקציה עםדם המוח (BBB) ​​או נוזל השדרתי דם מחסומים (BCSFB) והזן את parenchyma מערכת העצבים המרכזית. אינטראקציה זו בין שתל NPC וBBB (או BCSFB) מוסדרת על ידי קבוצה מסוימת של מולקולות פני השטח NPC הידבקות תא (מצלמות) והנחייתם של הביטוי של רמות גבוהות של Counter-ligands CAM על תאי אנדותל / מופעלים ependymal 12-14. דוגמאות למצלמות אלה כוללות את הקולטן לhyaluronate, CD44, ומולקולת ההידבקות בין תאית (ICAM) -1 אנטיגן יגנד מאוחר מאוד (VLA) -4 5,15,16 (כי, בלויקוציטים, אחראים של האינטראקציה עם ependymal הופעל ותאי האנדותל), ולאנטיגן הרבה יותר נמוך במידת ימפוציטים הקשורים פונקציה (LFA) -1 ויגנד P-גליקופרוטאין selectin (PSGL) -1. NPCs גם להביע מגוון רחב של קולטני chemokine, כולל CCR1, CCR2, CCR5, CXCR3, וCXCR4 (אבל לא מבטא CCR3 וCCR7), שהם בחינה תפקודית פעילים, הן במבחנה in vivo 5,16. לפיכך, systemicaNPCs הזריק lly להשתמש מצלמות אלה, יחד עם קולטן מצמידים G-חלבון (GPCRs), לצבור ברמה של מערכת העצבים המרכזית המודלקות. לעומת זאת, NPCs הזריק מערכתי לעכברים בריאים לא נכנסים למערכת העצבים המרכזית דרך כלי דם או מסלולי שטח נוזל השדרתי 2. דלקת במערכת העצבים המרכזית, או אנדותל / הפעלת תא ependymal הבאה ציטוקינים מערכתיים או lypopolisaccharide הזרקה (LPS) כמודל של דלקת קרום המוח כימי שגרם, הוא הכרחית ולכן להצטברות של NPCs הזריק מערכתית למוח וחוט השדרה 2. לכן, מיקוד מוצלח של מערכת העצבים המרכזית עם טיפולי NPC מערכתיים תלוי בזיהוי של חלון ספציפי מחלה של הזדמנות (WOO) שבה המוח וסביבת חוט השדרה הם תורמים להצטברות וההגירה transendothelial של NPCs. בתנאים כאלה, בדרך כלל מתעוררים בהקשר של דלקת חריפה וsubacute 17. ברגע שיש נכנס למערכת העצבים המרכזית, מושתל NPCs מובחןהוכחו לשפר את תכונות clinico-פתולוגית של עכברים, כמו גם פרימאטים גדולים יותר, לא אנושיים עם EAE. זו תוארה להיות תלויה מהחלפה המינימלית תא 2 והפרשה יוצאת דופן של גורמי חיסון רגולציה והגנה עצביים אוטוקריני בתוך מערכת העצבים המרכזית perivascular 2,5,6,18 לעומת אזורים שאינה במערכת העצבים המרכזית מודלקים 19,20 (למשל בלוטות לימפה) בתגובה ל איתות תא דלקתית שהושרו על ידי חדירת תאי מערכת החיסון 5. במסמך זה אנו מתארים את ההיבטים המתודולוגיים העיקריים של ההזרקה המערכתית של NPCs הגופני למודל של עכברים של EAE הכרוני. באופן ספציפי יותר, אנו מגדירים את הפרוטוקולים שהקמנו (i) נובע, להרחיב ולהתכונן לNPCs סומטיים השתלה מאזור subventricular (SVZ) של מבוגרים עכברי C57BL / 6; (Ii) לגרום EAE הכרוני בעכברים כאלה וכן (iii) לבצע טיפולי יעיל מערכתי (iv או ICV) השתלת NPC iעכברי n כדי EAE.

<table border="0" cellpadding="0" cellspacing="0" width="1474">
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			<td height="21" style="height:21px;width:753px;">Cell culture</td>
			<td style="width:300px;"></td>
			<td style="width:149px;"></td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EBSS</td>
			<td>Sigma</td>
			<td>E2888</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Cystein</td>
			<td>SIGMA-ALDRICH CO LTD</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papain</td>
			<td>WORTHINGTON</td>
			<td>30H11965</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Fisher scientific</td>
			<td>D/0700/50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse NeuroCult basal medium</td>
			<td>Stem Cell technologies</td>
			<td>05700</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NeuroCult proliferation supplements</td>
			<td>Stem Cell technologies</td>
			<td>05701</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Basic fibroblast growth factor</td>
			<td>Peprotech</td>
			<td>100-18B-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epidermal growth factor</td>
			<td>Peprotech</td>
			<td>AF-100-15-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td>1514012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel (coating solution)</td>
			<td>BD biosciences</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NeuroCult&#174; Differentiation Kit (Mouse)</td>
			<td>Stem cell technologies</td>
			<td>05704</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accumax</td>
			<td>eBioscience</td>
			<td>00-4666-56</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s PBS (DPBS) (10x) without Ca&#38; Mg</td>
			<td>PAA LABORATORIES LTD</td>
			<td>H15-011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Myco trace</td>
			<td>PAA LABORATORIES LTD</td>
			<td>Q052-020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide (DMSO)</td>
			<td>SIGMA</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">immunofluorescence</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal goat serum</td>
			<td>PAA LABORATORIES LTD</td>
			<td>B11-035</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether</td>
			<td>SIGMA-ALDRICH CO LTD</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti Nestin</td>
			<td>Abcam</td>
			<td>ab11306</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti GFAP</td>
			<td>DAKO</td>
			<td>203344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti Histone H3 (phospho S10)&#160;</td>
			<td>Abcam</td>
			<td>ab14955</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti MAP-2</td>
			<td>Abcam</td>
			<td>ab32454</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat anti MBP</td>
			<td>AbD SEROTEC</td>
			<td>MCA409S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-O4 Antibody, clone 81 | MAB345</td>
			<td>Millipore</td>
			<td>MAB345</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mounting solution</td>
			<td>DAKO</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EAE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Freund&#39;s Adjuvant Incomplete</td>
			<td>SIGMA-ALDRICH CO LTD</td>
			<td>F5506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mycobacterium tuberculosis&#160;</td>
			<td>DIFCO</td>
			<td>H37Ra</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MOG(35&#8211;55)&#160;</td>
			<td>Espikem</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pertussis toxin</td>
			<td>List Biological Laboratories</td>
			<td>181</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue processing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iris scissor straight</td>
			<td>Fine Sciences Tolls</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Blunt/bended forceps</td>
			<td>Fine Sciences Tolls</td>
			<td>11080-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Brain slicer</td>
			<td>Zivic instruments</td>
			<td>BSMAS005-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical blades</td>
			<td>Swann-Morton</td>
			<td>324</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P200, P1000 pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamine (Vetalar)</td>
			<td style="width:300px;">Boehringer Ingelheim</td>
			<td>01LC0030&#160;&#160;</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylazine (Rompun)</td>
			<td>Bayer</td>
			<td>32371</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereotaxic frame</td>
			<td>KOPF</td>
			<td>Model 900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hamilton syringe</td>
			<td>Hamilton</td>
			<td>7762-04</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde (PFA)</td>
			<td>SIGMA</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VECTASTAIN Elite ABC Kit</td>
			<td>vector laboratories</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
	</tbody>
</table>

systemic injection of neural stem/progenitor cells in mice with chronic eae

תאי גזע עצביים / מבשר (NPCs) הם מקור תאי גזע מבטיח לגישות השתלה המכוונות לתיקון מוח או שיקום בנוירולוגיה רגנרטיבית. הוראה זו עלתה מחומר הראיות רבות שתיקון המוח מושגת לאחר השתלת NPC מוקד או מערכתית בכמה מודלים פרה של מחלות נוירולוגיות. נתוני ניסוי אלה זיהו את מסלול משלוח תא כאחד המכשולים העיקריים של טיפולים בתאי גזע לטיפול במחלות המוח משקמים שדורשת הערכה דחופה. השתלת תאי גזע intraparenchymal מייצגת גישה הגיונית לאלו פתולוגיות מאופיינות בנגעים במוח מבודדים ונגישים, כגון פגיעות בעמוד השדרה ומחלת פרקינסון. למרבה הצער, עיקרון זה הוא גרוע החלים על תנאים מאופיינים בטבע multifocal, דלקתי והפיץ (שניהם בזמן ובמרחב), כוללים טרשת נפוצה (MS). ככזה, מוח מיקוד על ידי systמשלוח NPC Emic הפך פרוטוקול פולשנית וטיפולי יעיל נמוך כדי לספק לתאי המוח וחוט השדרה של מכרסמים וקופים לא אנושיים מושפעים מניזק דלקתי כרוני ניסיוני של מערכת העצבים המרכזית (CNS). זו שיטה החלופית של משלוח התא מסתמכת על pathotropism NPC, במיוחד היכולת המולדת שלהם (i) לחוש את הסביבה באמצעות מולקולות הידבקות תא פונקציונליות וקולטניים ציטוקינים וchemokine דלקתית; (Ii) לחצות את המחסומים דולפים אנטומיים לאחר תוך ורידי (iv). או intracerebroventricular הזרקה (ICV); (Iii) מצטבר ברמה של אתר מרובה perivascular (ים) של נזק מוח וחוט השדרה דלקתי; וכן (iv) להפעיל תזונתי רקמה מדהימה ואפקטי רגולציה חיסוניים על תאי יעד מארח שונים in vivo. כאן אנו מתארים את השיטות שפיתחנו לiv. ו&lt;em&gt; משלוח ICV של NPCs syngeneic בעכברים עם Encephalomyelitis הניסיוני אוטואימוניות (EAE), כמודל של פגיעה במיאלין הדלקתית של מערכת העצבים המרכזית הכרונית, ולדמיין את אספקת תאי גזע המערכתית כטכניקה חשובה למיקוד סלקטיבי של המוח המודלק בנוירולוגיה רגנרטיבית.

גזירת NPC ואפיון ניתוחי SVZ מתבצעים בברכות (n = עכברים / בריכה 5-7) של C57Bl / 6 עכברים 6-8 בשבוע שישנים בדרך של ניתוק המכני והאנזימטית (איור 1 א). אחרי כמה ימים של culturing בCGM, ריחוף ללא neurospheres להתחיל להרכיב (איורים 1 א ו 1 ב &#39;). התחומים עיקריים נאספים ומכ...

Watch this Scientific Journal Video about Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE at JoVE.com

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

ההשתלה של תאי גזע עצביים / אב (NPCs) מחזיקה בהבטחות גדולות בתחום הנוירולוגיה רגנרטיבית. המשלוח המערכתי של NPCs הפך לפרוטוקול יעיל, נמוך פולשנית וטיפולית יעיל מאוד כדי לספק תאי גזע במוח ובחוט השדרה של מכרסמים וקופים לא אנושיים מושפעים מניזק דלקתי כרוני ניסיוני של מערכת העצבים המרכזית.

הזרקה מערכתית של תאי גזע / אב עצביים בעכברים עם EAE כרוני

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

systemic-injection-neural-stemprogenitor-cells-mice-with-chronic

Research

JoVE Journal

Immunology and Infection

17.5K Views.  University of Cambridge, UK.  ההשתלה של תאי גזע עצביים / אב (NPCs) מחזיקה בהבטחות גדולות בתחום הנוירולוגיה רגנרטיבית. המשלוח המערכתי של NPCs הפך לפרוטוקול יעיל, נמוך פולשנית וטיפולית יעיל מאוד כדי לספק תאי גזע במוח ובחוט השדרה של מכרסמים וקופים לא אנושיים מושפעים מניזק דלקת...

Video: Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

זיהוי מוטציות DNA בתאי גזע / אב המטוהר Hematopoietic

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

כרוני לטווח ארוך<em&gt; Pseudomonas aeruginosa</em&gt; זיהום דרכי הנשימה בעכברים

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

המיאלין oligodendrocyte גליקופרוטאין (MOG<sub&gt; 35-55</sub&gt;) מושרה ניסויי אוטואימוניות Encephalomyelitis (EAE) בעכברים C57BL / 6

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

מודל של עכברים של דלקת כרונית הנגרם על ידי פתוגן באתרים מקומיים ומערכתיים

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

פנוטיפי אנליזה פונקציונלית של תאים תקינה T הופעל מבודדת מן עכברים הנגועים בנגיף לימפוציטית כרונית Choriomeningitis

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

ניתוח של לימפוציטים extravasation שימוש<em&gt; במבחנה</em&gt; דגם של מחסום אנושי דם-מוח

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable dry eye animal model to test novel therapeutics to treat autoimmune dry eye conditions. This protocol describes a chronic autoimmune dry eye rat model. Lewis rats were immunized with an emulsion containing lacrimal gland extract, ovalbumin, and complete Freund&#39;s adjuvant. A second immunization with the same antigens in incomplete Freund&#39;s adjuvant was administered two weeks later. These immunizations were administered subcutaneously at the base of the tail. To boost the immune response at the ocular surface and lacrimal glands, lacrimal gland extract and ovalbumin were injected into the forniceal subconjunctiva and lacrimal glands 6 weeks after the first immunization. The rats developed dry eye features, including reduced tear production, decreased tear stability, and increased corneal damage. Immune profiling by flow cytometry showed a preponderance of CD3+ effector memory T cells in the eyeball.

This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later.

כרוני אוטואימונית יבש עין עכברוש דגם עם הגדלת זיכרון תאי זיכרון T ב Eyeball רקמות

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

חיסוני המאמצת של תאים iNKT ב גלוקוז-6-פוספט איזומראז (G6PI)-הנגרמת עכברים RA

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to draining lymph nodes (LNs). Interruption of lymph flow is an important method when studying the function of lymphatic vessels. The afferent lymphatic vessels from the murine footpad to the popliteal lymph nodes (pLNs) are well-defined as the only routes for lymph drainage into the pLNs. Suturing these afferent lymphatic vessels can selectively prevent lymph flow to the pLNs. This method allows for interference in lymph flow with minimal damage to the lymphatic endothelial cells in the draining pLN, the afferent lymphatic vessels, as well as other lymphatic vessels around the area. This method has been used to study how lymph impacts high endothelial venules (HEV) and chemokine expression in the LN, and how lymph flows through the adipose tissue surrounding the LN in the absence of functional lymphatic vessels. With the growing recognition of the importance of lymphatic function, this method will have broader applications to further unravel the function of lymphatic vessels in regulating the LN microenvironment and immune responses.

A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented.

חסימת זרימת הלימפה על ידי תפירת כלי לימפה אפילנט בעכברים

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to ...

A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice

Intrathymic injection in mouse models is an important technique for studying thymic and immune function, including genetic and acquired T cell disorders. This requires methods for the direct deposition of reagents and/or cells into the thymus of living mice. Traditional methods of intrathymic injection include thoracic surgery or minimally invasive percutaneous blind injections, both of which have significant limitations. Ultra-high frequency ultrasound imaging devices have made image-guided percutaneous injections possible in mice, greatly improving the injection accuracy of the percutaneous injection approach and enabling the injection of smaller targets. However, image-guided injections rely on the utilization of an integrated rail system, making this a rigid and time-consuming procedure. A unique, safe, and efficient method for percutaneous intrathymic injections in mice is presented here, eliminating reliance on the rail system for injections. The technique relies on using a high-resolution micro-ultrasound unit to image the mouse thymus noninvasively. Using a free-hand technique, a radiologist can place a needle tip directly into the mouse thymus under sonographic guidance. Mice are cleaned and anesthetized before imaging. For an experienced radiologist adept at ultrasound-guided procedures, the learning period for the stated technique is quite short, typically within one session. The method has a low morbidity and mortality rate for the mice and is much faster than current mechanically assisted techniques for percutaneous injection. It allows the investigator to efficiently perform precise and reliable percutaneous injections of thymuses of any size (including very small organs such as the thymus of aged or immunodeficient mice) with minimal stress on the animal. This method enables the injection of individual lobes if desired and facilitates large-scale experiments due to the time-saving nature of the procedure.

The present protocol describes an interventional radiology procedure established for intrathymic injection in mice to avoid the risk of open surgery and improve the accuracy of blind percutaneous injections.

טכניקה זעיר פולשנית, מדויקת ויעילה להזרקה תוך-תימית בעכברים

Intrathymic injection in mouse models is an important technique for studying thymic and immune function, including genetic and acquired T cell disorders. ...