We thank Monika Kilchenmann, Federica Bisignani and Agathe Duda for their excellent technical assistance.

כל הניסויים דבקו בהצהרה לשימוש בבעלי חיים ברפואת עיניים וחזון מחקר של האגודה לחקר חזון אופתלמולוגיה (ארוו). .1 בעלי חיים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשמור על דג הזברה wild-type (Danio rerio) של זן AB (אורגון) בגילים שבין 6-12 חודשים בתנאים סטנדרטיים במים עם טמפרטורה של 26.5 מעלות צלזיוס ו14/10 שעה מחזור אור / 15 כהה. </li><li style=";text-align:right;direction:rtl"> פעל בהתאם להנחיות הטיפול בבעלי החיים של המוסדות המעורבים לניסויים בבעלי החיים לאחר שאושר על ידי לשכת הווטרינרית המחוזית. </li></ol> .2 MNU טיפול <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן מים המכילים חומר יבש 150 מ&quot;ג / L של -nitrosourea -methyl- N N (MNU). זהירות! MNU הוא רעיל; עלול לגרום לסרטן, נזק גנטי תורשתי או לפגיעה בעובר. </li><li style=";text-align:right;direction:rtl"> דגירה דג הזברה במים המכילים MNU ל60 דקות בטמפרטורת חדר. </li><li style=";text-align:right;direction:rtl"> יש לשטוף את דג הזברה במהירות עםמים וטריים לשים את הדגים לאקווריום חדש בלי MNU. לשמור על דגים בתנאים סטנדרטיים כל עוד רצוי לניסויים. </li></ol> .3 מדידת חדות ראייה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להפעיל את מערכת optomotor, לבחור &#39;בדיקה&#39; מהתפריט ולהגדיר את האפשרויות ל&quot; מדרגות פשוטות &quot;,&quot; חדות (תדר) &quot;ו16&quot; אקראיים / נפרדים &quot;. </li><li style=";text-align:right;direction:rtl"> התקן בקבוק עירוי מלא ב500 מ&quot;ל מים על 1 מ &#39;מעל מערכת optomotor. </li><li style=";text-align:right;direction:rtl"> הנח דג הזברה אחד בחדר הבדיקה ולחבר אותו לבקבוק העירוי. מניחים את חדר הבדיקה במערכת optomotor. </li><li style=";text-align:right;direction:rtl"> התחל את המדידה ולבחון את תנועות עיניים בזמן אמת על מסך המחשב. וכך, תגובה חיובית (&quot;כן&quot;) מוגדרת כקפיצות האלה ברציפות בכיוון הנכון, ואילו תשובה שלילית (&quot;לא&quot;) מייצג תנועות עיניים אקראיות דומות לאלה שנצפו עם תחנהשבכות האר&quot;י. אם העיניים של תערוכת דג הזברה שלוש או יותר תגובות optokinetic שלאחר מכן (OKR), לחץ על &#39;כן&#39;; אם לא, לחץ על &#39;לא&#39;. </li><li style=";text-align:right;direction:rtl"> חלץ את חדות הראייה, שחושב על ידי התוכנה על ידי קביעת הסף של התדר המרחבי של גירוי optokinetic, תחת &quot;תוצאות&quot; בתפריט. </li></ol> .4 היסטולוגיה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים את דג הזברה על ידי מנת יתר של אתיל methanesulfonate 3-aminobenzoate (200-300 מ&quot;ג / ליטר) וenucleate העיניים. </li><li style=";text-align:right;direction:rtl"> לתקן את כל העיניים בparaformaldehyde 4% (PFA) שבנאגר מלוח פוספט (PBS) בשעה 4 מעלות צלזיוס במשך שעה 12 ולאחר מכן מייבשים את הדגימות בסדרת אלכוהול מדורגת. </li><li style=";text-align:right;direction:rtl"> להטביע את הדגימות בפרפין, לחתוך 5 מיקרומטר סעיפים דרך ראש עצב הראייה (ONH) ולעגן אותן בשקופיות זכוכית. </li><li style=";text-align:right;direction:rtl"> כתם חלקי פרפין deparaffized עם hemalum למשך 4 דקות וטובלים את השקופיות פעמיים במים מזוקקים ואחריו 0.2% ACI הידרוכלוריתאמוניה ד ו0.8%. כתם החלקים עם eosin במשך 3 דקות לאחר הפיתוח של הכתמת hemalum במים ברז לדקות לפחות 10. (H &amp; E). </li><li style=";text-align:right;direction:rtl"> להטביע את השקופיות מיובשים בהרכבה בינונית ולבחון את השקופיות מתחת למיקרוסקופ האור. </li></ol> .5 אימונוהיסטוכימיה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> transferase deoxynucleotidyl מסוף תיוג סוף dUTP ניק (TUNEL) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דגירה סעיפי deparaffinized עם 20 מיקרוגרם / מ&quot;ל ​​proteinase K בטמפרטורת חדר במשך 15 דקות. שטוף את החלקים שלוש פעמים עם טריס שנאגרו מלוח (TBS) במשך 5 דקות כל אחד. </li><li style=";text-align:right;direction:rtl"> דגירה הסעיפים בתערובת 50 μl תגובת TUNEL (10% פתרון תיוג ו90% פתרון אנזים) בתא humidified על 37 מעלות צלזיוס למשך 60 דקות. לשטוף שלוש פעמים עם כפות במשך 5 דקות כל אחד. </li><li style=";text-align:right;direction:rtl"> הר השקופיות עם ההרכבה בינונית המכיל DAPI ולבחון את השקופיות מתחת למיקרוסקופ פלואורסצנטי. </li></ol></li><li style=";text-align:right;direction:rtl"> הגרעיני Antigen מכתים תא מתרבים (PCNA) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מרתיחים את חלקי deparaffinized במאגר אחזור אנטיגן (טריס-EDTA 0.05 + Tween% 20, pH 9.0) במשך 3 דקות ולשטוף שלוש פעמים עם כפות במשך 5 דקות כל אחד. לחסום עם 100 μl פתרון החסימה (TBS + 10% + 1% אלבומין בסרום שור העז הנורמלי בסרום, pH7.6) בטמפרטורת חדר למשך שעה 1. </li><li style=";text-align:right;direction:rtl"> כתם עם נוגדנים ראשוניים אנטי PCNA בדילול 1: 200 בתא humidified על 4 מעלות צלזיוס למשך הלילה. לשטוף שלוש פעמים עם כפות + Tween 20 ל5 דקות כל אחד. </li><li style=";text-align:right;direction:rtl"> לסיים את זיהוי עם הנוגדנים משני המתאימים בדילול 1: 500 בטמפרטורת חדר למשך שעה 1. </li><li style=";text-align:right;direction:rtl"> הר השקופיות עם ההרכבה בינונית המכיל DAPI ולבחון את השקופיות מתחת למיקרוסקופ פלואורסצנטי. </li></ol></li></ol>

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The authors have nothing to disclose.

בעבר, הקבוצה שלנו העבירה את מודל MNU של ניוון קולטי אור ממכרסמים למערכת דג הזברה 9. האירועים שלאחר מכן תוארו ואחריו לתקופה של עד 30 ימים. בפרק זמן זה ניוון רשתית מלא מורפולוגיים והתחדשות התרחשו לאחר הטיפול הראשוני. ראשית, היסטולוגיה מגלה תא מוט מופחת לספור מיום 3 בע?...

חזון הוא התחושה חיונית ביותר לאדם ויש לו ירידת הערך שלה השפעה חברתית וכלכלית גבוהה. בעולם המפותח, מחלות ניווניות ברשתית הן הסיבה המובילה לאיבוד ראייה ועיוורון בקרב אנשים מבוגרים 1. הסיבה למרבית המחלות רשתית ניווניות מובנת רק בחלקו ופתרונות טיפוליים כדי להחזיר את החזון אבוד הם מאוד מוגבלים. רטיניטיס פיגמנטוזה הוא דוגמא אופיינית למחלה ניוונית של רשתית עם אובדן קולטי אור העיקרי 2-3. N -methyl- -nitrosourea N (MNU) גורם לניוון רשתית ולכן בשימוש נרחב במכרסמים מודל מחלות עם מוות של תאי קולטי אור עיקריים 4. זה סוכן אלקילציה ומוביל לגידולים שפירים וממאירים, אשר מופיעים בדרך כלל מספר חודשים לאחר החשיפה 5-7. בנוסף, הוא גורם למוות של תאי קולטי אור ספציפיים בתוך תקופת תצפית לטווח קצר. וכך, אובדן structu שכבת הרשתיתמחדש ודילול רשתית משמעותי נצפה באופן תלוי ריכוז. תאי גליה רשתית הופעלו, אך לא חלה שינויים באפיתל הפיגמנט ברשתית (RPE) נמצאו. אפופטוזיס הקשורות במתח reticulum (ER) endoplasmic נראה המסלול העיקרי של פעולת MNU ברשתית 8. יש לנו לאחרונה הצגתי MNU כמודל כימי כדי לגרום לניוון קולטי אור בדג הזברה 9. , בין שאר, דג הזברה (Danio rerio) הפכה להיות חשובה במחקר חזותי בגלל הדמיון של מערכת הראייה שלה לזה של בעלי חוליות אחרות 10. הרשתית החיצונית מכילה קולטני האור, שניתן לקבץ לארבעה סוגים שונים קונוס עם רגישויות שיא באולטרה הסגול, קצר, בינוני, ואורך גל של הספקטרום הנראה לעין ארוכה וסוג קולטי אור מוט אחד. בשכבת הגרעין הפנימית (INL), גופי התא של הפרעה דו קוטבית, וinterneurons אופקיים, amacrine נמצאים, כמו well כסומה התא של תאי גליה מולר. בשכבת plexiform החיצוני (OPL) הקשרים סינפטיים בין קולטני אור והרשתית הפנימית נוצרים, ואילו שכבת התא הקרובה ביותר לעדשה היא שכבת תא גנגליון (GC), שרכיבים יוצר אקסונים ארוכים המרכיבים את עצב הראייה ומערכת הראייה . קשרים סינפטיים בין תאי הגנגליון והתאים בשכבת הגרעין הפנימי נוצרים בשכבה הפנימית plexiform (IPL) 11. הרשתית נמצאת מחוץ לרשתית נוירו ומקיפה מגזרים חיצוניים קולטי האור עם microvilli הפסגה ארוך 12. יתר על כן, דג הזברה הוא משובי ומסוגלים להצמיח מחדש מוח פצוע, רשתית, חוט השדרה, לב ורקמות אחרות 13 מאוד. כאשר נזק לרשתית מתרחשת, תאים בINL, אשר נחשבו לתאי מולר, מופעלים ויש להם הפוטנציאל להתמיין לסוגי תאים ברשתית שונים. בנוסף, הם גם ליצור אבות מוט, אשר ממוקמים בONL. כך אחרurce שמספק את הרשתית של דג הזברה מבוגרת עם תאים חדשים הוא האזור השולי הריסים. יש צורך במקור זה כדי להשיג צפיפות קבועה של קולטני אור מוט בעין דג הזברה גדלה ברציפות 14. מודל MNU יכול לשמש כגישת ניוון / התחדשות פשוטה לשחזור לרקמת רשתית. בשל דמיון מסוים של תהליכים ביולוגיים בדג זברה ובבני אדם זה יכול לפתוח את הדלתות כדי לזהות מסלולים מעורבים מוות של תאים ולהקרין תרופות נוירו פוטנציאליות. בהתבסס על מחקר קודם מהקבוצה שלנו, אנחנו עכשיו להמחיש את השיטות של מודל דג הזברה-Induced MNU זו של דה ברשתית והתחדשות כתוצאה מכך לרבות שינויים פונקציונליים עם פי מעבדה קטעי וידאו 9.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Acetic acid</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr >
			<td >Ammonia</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>294993</td>
			<td>0.80%</td>
		</tr>
		<tr >
			<td >Bovine serum albumine (BSA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>05470</td>
			<td></td>
		</tr>
		<tr >
			<td >Dako Pen</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr >
			<td >DAPI mounting medium</td>
			<td>Vector Labs, Burlingame, CA, USA</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr >
			<td >Eosin</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>45260</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethanol</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>2860</td>
			<td>100%, 96%, 70%</td>
		</tr>
		<tr >
			<td >Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>ED</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethyl 3-aminobenzoate methanesulfonate salt</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>E10521</td>
			<td>Tricaine</td>
		</tr>
		<tr >
			<td >Eukitt</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>3989</td>
			<td></td>
		</tr>
		<tr >
			<td >Goat anti-rabbit Alexa 594</td>
			<td>Life Technologies, Zug, Switzerland</td>
			<td>A11012</td>
			<td></td>
		</tr>
		<tr >
			<td >Goat normal serum</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>X0907</td>
			<td></td>
		</tr>
		<tr >
			<td >Hydrochloric acid</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>320331</td>
			<td>0.20%</td>
		</tr>
		<tr >
			<td >In situ Cell Death Detection Kit</td>
			<td>Roche Applied Sciences, Rotkreuz, Switzerland</td>
			<td>11684795910</td>
			<td>TUNEL Kit</td>
		</tr>
		<tr >
			<td >Mayer&#39;s hemalum solution</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>109249</td>
			<td></td>
		</tr>
		<tr >
			<td >Methylnitrosourea (MNU)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>N4766</td>
			<td>Toxic</td>
		</tr>
		<tr >
			<td >OptoMotry</td>
			<td>CerebralMechanics, Lethbridge, AB, Canada</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr >
			<td >Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5368</td>
			<td></td>
		</tr>
		<tr >
			<td >Proteinase K</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>S3004</td>
			<td></td>
		</tr>
		<tr >
			<td >Rabbit anti-PCNA</td>
			<td>Santa Cruz Biotechnology, Santa Cruz, USA</td>
			<td>sc-33756</td>
			<td></td>
		</tr>
		<tr >
			<td >Superfrost Plus glass slides</td>
			<td>Gehard Menzel GmbH, Braunschweig, Germany</td>
			<td>10149870</td>
			<td></td>
		</tr>
		<tr >
			<td >Tris buffered saline (TBS)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5912</td>
			<td></td>
		</tr>
		<tr >
			<td >Trizma&#210; base</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr >
			<td >Tween 20</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr >
			<td >Xylene</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>534056</td>
			<td></td>
		</tr>
		<tr >
			<td >Zebrafish (Danio rerio) AB (Oregon) strain</td>
			<td>University of Fribourg, Dept. of Biology</td>
			<td>n.a.</td>
			<td>Own fish facility</td>
		</tr>
	</tbody>
</table>

methylnitrosourea (mnu)-induced retinal degeneration and regeneration in the zebrafish: histological and functional characteristics

מחלות רשתית ניווניות, רטיניטיס פיגמנטוזה למשל, עם וכתוצאה מכך חשבון נזק קולטי אור עבור רוב אובדן הראייה בעולם התעשייתי. מודלים של בעלי החיים הם בעלי חשיבות מכרעת ללימודי מחלות כגון. בהקשר זה רעלן קולטי האור ספציפי -nitrosourea N -methyl- N (MNU) כבר בשימוש נרחב במכרסמים כדי לגרום פרמקולוגית ניוון רשתית. בעבר, הקמנו מודל מושרה MNU רשתית ניוון בדג הזברה, עוד מערכת מודל פופולרית במחקר חזותי. הבדל מרתק ליונקים הוא נוירוגנזה המתמשכת ברשתית דג הזברה מבוגרת והתחדשותו לאחר פגיעה. כדי לכמת תצפית זו יש לנו עובדי מדידות חדות ראייה בדג הזברה המבוגר. וכך, רפלקס optokinetic שימש למעקב שינויים תפקודיים בדגים שאינם מורדמים. זה היה בתוספת היסטולוגיה, כמו גם staini immunohistochemicalng לאפופטוזיס (TUNEL) וההתפשטות (PCNA) כדי לתאם שינויים מורפולוגיים פיתוח. לסיכום, אפופטוזיס של קולטני אור מתרחש שלושה ימים לאחר טיפול MNU, אשר מלווה ירידה ניכרת של תאים בשכבה החיצונית הגרעינית (ONL). לאחר מכן, התפשטות של תאים בשכבת הגרעין הפנימי (INL) וONL הוא ציין. בזאת, אנו מגלים שלא רק היסטולוגית אך מלאים גם התחדשות תפקודית מתרחשת על פני מסלול של 30 ימים זמן. עכשיו אנחנו ממחישים את השיטות לכמת ומעקב דה דג הזברה רשתית והתחדשות באמצעות MNU בוידאו בפורמט.

חדות ראייה: ניסיוני ההגדרה [תדר המרחבי: 0.042 חוגים / תואר (ג / ד); ניגודיות: 100%; מהירות סחיפה: 20 מעלות / שני (ד / src); בחזרה בהיקות אור: 152 cd / m 2] של מחקר זה אפשרה הערכת OKR של דג הזברה מבוגרת. משך הממוצע של מדידת VA היה על 5 - 10 דקות לכל דג הזב...

Watch this Scientific Journal Video about Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics at JoVE.com

Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

במסמך זה אנו מדגימים כימות של דה ברשתית והתחדשות והשפעתה על תפקוד ראייה באמצעות -nitrosourea N -methyl- N בדג הזברה המבוגר. מספרי קולטי אור אובדן של חדות ראייה והירידה היו במעקב על ידי התפשטות בשכבת הגרעין הפנימית. התחדשות מורפולוגית ופונקציונלית מלאה התרחשה 30 ימים לאחר הטיפול הראשוני.

Methylnitrosourea (MNU) -induced רשתית ניוון והתחדשות בדג הזברה: היסטולוגית ומאפיינים פונקציונליים

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial ...

methylnitrosourea-mnu-induced-retinal-degeneration-regeneration

Research

JoVE Journal

Biology

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

11.4K Views.  University of Bern.  במסמך זה אנו מדגימים כימות של דה ברשתית והתחדשות והשפעתה על תפקוד ראייה באמצעות -nitrosourea N -methyl- N בדג הזברה המבוגר. מספרי קולטי אור אובדן של חדות ראייה והירידה היו במעקב על ידי התפשטות בשכבת הגרעין הפנימית. התחדשות מורפולוגית ופונקצ...

Video: Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

אבלציה בלייזר של דג הזברה כלית ראשית לחקר התחדשות אפיתל הכליה

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

מבחני גנטית תרופתי ו פונקציונלית לתפעל התחדשות של Planarian Japonica Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

ב electroporation vivo של Morpholinos לתוך הרשתית דג הזברה למבוגרים

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

ב electroporation vivo של Morpholinos לתוך למבוגרים התחדשות זנב סנפיר דג הזברה

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging דג הזברה למבוגר

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Assay עבור התחדשות קו לרוחב בדג הזברה למבוגר

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

גאפ junctional אינטר תקשורת: ביומרקר פונקציונלית להערכת תופעות לוואי של toxicants ורעלים, ואת היתרונות הבריאותיים של מוצרים טבעיים

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.

אימונוולבלינג מיוכי ניוון שרירים ביופסיות

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

ניטרורדוקטאז/אבלציה בתיווך מטרונידזול ופלטפורמת MATLAB (RpEGEN) לחקר התחדשות של אפיתל פיגמנט הרשתית של דג הזברה

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

ייצור יעיל ועקבי של אפיתל פיגמנט רשתית/כורואיד שטוח מעיני אדם לניתוח היסטולוגי

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...