Name: a doxorubicin-induced cardiomyopathy model in adult zebrafish
Uploaded: 2018-06-07T13:30:00
Description: Watch this Scientific Journal Video about A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish at JoVE.com

עבודה זו נתמכה בחלקו על ידי מענק פיתוח מדען של איגוד הלב האמריקני (14SDG18160021 כדי הסתייגות), R01 NIH ארה ב מעניקה HL 81753 HL 107304 ל XX ואת לקרן מאיו XX.

כל ההליכים המתוארים כאן בוצעו על פי המדריך על טיפוח ועל שימוש של חיות מעבדה (הלאומי באקדמיות העיתונות-2011), הן אושרו על ידי מאיו קליניק טיפול חיה מוסדיים ועל שימוש הוועדה.
1. הכנת דג זברה בגיר
<ol><li>להגדיר זוגות רבייה מספיק למעבר טנקים לרכוש לפחות כפול כמו הדגים הכולל לצורך הזר?...

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המודל של DIC מתקדמת, המינון של 20 מ"ג/ק"ג DOX נקבע ניסיוניים בתור המינון הגבוה גורם המוות דגים משמעותית במהלך 1 wpi אבל עדיין תוצאות במוות דגים וצמצום של תפקוד הלב לאחר wpi 4 (איור 3 ו איור 4C). מנה זו משולה אלה המשמשות לעתים קרובות במודלים DIC מכרסמים (15-25 מ"ג/ק"ג), המינון ה...

דוקסורוביצין (DOX), גם בשם Adriamycin, פותחה כמו תרופה אנטי-אמצעים מאז שנות ה-601,2. עכשיו זה עדיין בשימוש פעיל בשם כימותרפית חשוב עבור קשת רחבה של גידולים. עם זאת, יישום קליני של DOX היה הקשו על ידי שלה רעילות למינון, במיוחד cardiotoxicity מאופיין על ידי הסימפטומים משתנים החל שינויים electrocardiographic ללא תסמינים דלקת כרום הלב ולא מפוצה קרדיומיופתיה 1 , 2. עד כה, הועלו השערות הגדולות לפחות שלושה להסביר DIC, כולל חמצן תגובתי מופעל מינים (ROS)1,3,4,5, עיכוב של טופואיזומראז II-β ( 6,TOP2β)7, ו אפנון של סידן תאיים שחרור1,8,9. לצבירת ראיות גם מרמזת על נטייה גנטית מהווה גורם סיכון מרכזי DIC10,11,12,13. זהויות גנים הקשורים נטיות אלה DIC, לעומת זאת, נותרים אינו מודע לקיומם. Dexrazoxane הוא היחיד אדג'וונט סוכן שאושרו על ידי מינהל המזון והתרופות האמריקני (FDA) לטיפול DIC, אבל עם יישום מוגבל14,15,16, דבר המלמד את הצורך לזהות נוספים אסטרטגיות טיפוליות. מודלים חייתיים של DIC נידונות ולכן למטרות אלו. בשל הנגישות שלהם ואת הפשטות, מחקרים מכניסטית בדגמים DIC יכול באופן פוטנציאלי יש רחבה יותר השפעות על סוגים אחרים של cardiomyopathies: פתוגנזה נפוצה עשוי להיות משותף בין cardiomyopathies של חבלות מסיבות שונות, במיוחד בזמן מאוחר יותר שלבים פתולוגיים17,18,19,20.
בנוסף מודלים מכרסם של DIC, פותחו מודלים DIC דג זברה עם תפוקה גבוהה יותר כדי להקל על הגילוי של גורמים גנטיים חדשים והן הרפוי. מודל DIC עובריים הוקם העוברים דג זברה שקוף לסינון תרכובות טיפולית21. בהתחשב בכך cardiomyopathies הן מחלות תחילת למבוגרים עם הפתוגנזה פרוגרסיב, קרדיומיופתיה דג זברה בוגרת מודלים כבר פיתח22,23,24,25,26. אנחנו שנוצר המודל רכשה הראשון הנובע אנמיה כרונית24, ואחריו DIC כמודל קרדיומיופתיה רכשה השני דג זברה בוגרת23של שריר הלב. מצאנו כי הזרקה של סיילין יחיד DOX לתוך דג זברה בוגרת המניע cardiotoxicity המורכב שלב אקוטי בערך בתוך 1 בשבוע שלאחר ההזרקה (wpi), בעקבות שלב כרונית של שריר הלב עד 6 חודשים לאחר ההזרקה. בעוד haploinsufficiency של מטרת rapamycin מכניסטית(mtor) המשפרת את שריר הלב בשלב כרוני, היא מגזימה תמותת דגים בשלב אקוטי, דבר המלמד את הערך של המודל DIC למבוגרים להבחין תלויי-שלב מנגנונים23. אנחנו עוד יותר הוכיח כי המודל DIC למבוגרים יכול לשמש כדי להדגיש אוסף של דג זברה insertional לב (ZIC) המוטנטים שנוצרו באמצעות גישה המבוסס על transposon מוטגנזה מכוונת insertional27. מסך פיילוט זיהה 3 גנים ידועים של שריר הלב, כמו גם DnaJ (Hsp40) homolog, תת משפחה B וחבר 6b (dnajb6b) כמו חדש DIC הרגישות גנים28. לכן, הדור של המודל DIC למבוגרים בדג זברה הוביל מתודולוגיה חדשה המאפשרת זיהוי ממגבילי גנטיים עבור DIC, אשר משלים את הגנום כולו האגודה קיים מחקר (GWAS) ואת לוקוס תכונה כמותית (QTL באופן שיטתי ) ניתוח.
בתקופת הדור, יישומו של המודל DIC דג זברה למבוגרים, הבחנו וריאציות משמעותית בקרב חוקרים שונים ו/או אפילו בין זריקות שונות שביצע החוקר אותו. הטבע האורך של המודל מטיל אתגרים רישום תוצאות של חוקרים שונים, את תהליך פתרון הבעיות רציפים. כדי להקל על השימוש פשוט בתדר קרדיומיופתיה מתח שיטה זו על ידי קהילת המחקר, אנו מתארים את הפרוטוקול שלנו בפירוט, נוכח שני סוגים של הזרקת ה-IP, ולדון שיקולים להפחית וריאציות בקרב חוקרים שונים.

<table>
	<tbody>
		<tr>
			<td>Crossing tank</td>
			<td>Aquaneering</td>
			<td>ZHCT100</td>
			<td>Fish breeding</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>ThermoFisher</td>
			<td></td>
			<td>Maintaining embryo</td>
		</tr>
		<tr>
			<td>3 L medium tank</td>
			<td>Aquaneering</td>
			<td>ZT280</td>
			<td>Maintaining fish</td>
		</tr>
		<tr>
			<td>Paramecia</td>
			<td>Carolina</td>
			<td>131560</td>
			<td>Food for juvenile fish</td>
		</tr>
		<tr>
			<td>Live hatched brine shrimp</td>
			<td>in house</td>
			<td></td>
			<td>Food for adult fish</td>
		</tr>
		<tr>
			<td>Doxorubicin hydrochloride</td>
			<td>Sigma</td>
			<td>D1515-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml safe-lock tube</td>
			<td>Eppendorf</td>
			<td>No. 022363204</td>
			<td>For drug storage</td>
		</tr>
		<tr>
			<td>Aluminum foil paper</td>
			<td>Fisher</td>
			<td>1213104</td>
			<td>For preventing light exposure</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Roche</td>
			<td>No. 03115887001</td>
			<td>For dechorionating embryo</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (HBBS)</td>
			<td>ThermoFisher</td>
			<td>14025076</td>
			<td>Vehicle for DOX</td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>Falcon</td>
			<td>431741</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;L NanoFil micro-syringe</td>
			<td>WPI</td>
			<td>NANOFIL</td>
			<td>For injection</td>
		</tr>
		<tr>
			<td>34 gauge needle&#160;</td>
			<td>WPI</td>
			<td>NF34BV-2</td>
			<td>For injection</td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Argent</td>
			<td>MS-222</td>
			<td>Anesthetizing fish</td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Costar</td>
			<td>3539</td>
			<td>For embryo drug treatment</td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Bel-art product</td>
			<td>F37898</td>
			<td>For transfering embryo</td>
		</tr>
	</tbody>
</table>

a doxorubicin-induced cardiomyopathy model in adult zebrafish

דג זברה בוגרת גנטית נגיש (רזבורה rerio) יותר ויותר שימש כמודל חוליות להבנת מחלות אנושיות כגון שריר הלב. בשל הנוחות amenability כדי מניפולציות גנטיות תפוקה גבוהה, הדור של קרדיומיופתיה רכשה מודלים, כגון המודל דוקסורוביצין-induced קרדיומיופתיה (DIC) בדג זברה למבוגרים, פותח את דלתות השדרות מחקר חדש, לרבות גילוי סימני פיסוק שריר הלב באמצעות בדיקה גנטית קדימה. שונה מהמודל DIC דג זברה עובריים, חריפה הראשונית והן לאחר מכן כרונית השלבים של שריר הלב יכול להיקבע במודל DIC דג זברה למבוגרים, המאפשרת לימוד מנגנוני איתות תלויי-הבמה אסטרטגיות טיפוליות. עם זאת, ניתן להשיג תוצאות משתנה עם המודל הנוכחי, אפילו בידי חוקרים מנוסים. כדי להקל על יישום העתידי של המודל DIC, אנו מציגים פרוטוקול מפורט כיצד ליצור מודל זה DIC בדג זברה בוגרת. ומתארות שתי דרכים חלופיות בקרום הבטן הזרקה (IP). בהמשך נדון אפשרויות כיצד להפחית וריאציות כדי להשיג תוצאות אמינות ואת לספק הצעות כיצד לפרש את התוצאות בהתאם.

כאן, שתי שיטות לביצוע הזרקה IP למודל DIC בדג זברה בוגרת מוצגים. תוך שימוש קלאסי, מבוססת IP הזרקת שיטת29, צוין כי הפתרון DOX מוזרק (צבע אדום) יכול לפעמים מתריסה החוצה מהמיקום שבו חדרו המחט. הזריקה IP חלופית משתמש במיקום שונה לתקוע מחט זה 3-4 מ מ בלבד הצפק איפה DOX הוא שוחרר ...

Watch this Scientific Journal Video about A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish at JoVE.com

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

מודל דוקסורוביצין-induced קרדיומיופתיה בדג זברה למבוגרים

שיטה לייצר מודל קרדיומיופתיה דוקסורוביצין-induced למבוגרים דג זברה (רזבורה rerio) מתואר כאן. שתי דרכים חלופיות של הזרקת בקרום הבטן מוצגים ואת התנאים כדי להפחית וריאציות בין קבוצות שונות ניסיוני הנדונים.

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

This method can help determine key factors affecting viral agents during the development of an adaptive model for ensocycline induced catatoxicity. The main advantage of this technique is that it is a simple ensocycline induced catatoxicity model which can be used for high-throughput genetic and drug-based screenings. When making DOX stock solution, thoroughly dissolve the DOX powder in deionized water until absolutely no clumps are visible.For quality control testing, collect a wild-type Zebra fish embryos from at least two pair of fish in two separate collections. Manually dechorionate the embryos 24 hours post-fertilization using fine needles. After the dechorionation, refresh the embryo water and remove any dead embryos.Each collection of embryos must have at least 36 embryos for a quality control test. Now, dilute the stock DOX solution in fresh embryo water to a final concentration of 100 micromolar. Prepare at least 100 microliters per three embryos.Mix the dilution using a vortex. The final dilution should have a light red color. Next, load the wells of a 96 well plate with 100 microliters of diluted DOX solution.Prepare one well per three embryos. Then, transfer the embryos to the wells using a plastic transfer pipette. While transferring embryos, keep the embryos close to the end of the pipette tip.Then, put the pipette tip into solution and allow the embryos to swim naturally into the well. Do not eject the embryos with solution as this will alter the DOX concentration. 48 hours post-fertilization, refresh the DOX solution.At this time, look under 10X magnification to identify dead embryos, which have no heartbeat and identify embryos with edema. Be quick to count and remove any dead embryos, otherwise the remaining embryos in the well will also die. Repeat the dead count at 72 hours post-fertilization.If there is at least 25%death then the DOX solution has a good drug efficacy and can be used for the experiment. Prior to injecting the fish with DOX, fast the fish for 24 hours. Next, group the fish.While anesthetized, use a clean filter paper to dry each fish and measure its body weight. Make groups of fish that are all within 10%of the same body weight, so each group can be injected with the same dose of DOX. Next, calculate the working concentration of DOX for each group so that each group is injected with five microliters of solution and receives the same dose of DOX by body weight.Then, dilute the stock of DOX in 1X HBSS. Mix the dilution with a vortex and then pulse spin the dilution to keep it cooled together in the tube. For the injection, prepare an injecting platform.In a clean, 100 milliliter Petri dish, place a sponge. With the aid of a dissection microscope, cut a cavity into the sponge to hold one fish. Four centimeters usually works.Next, unto a 34-gage needle, attach a 10 microliter microsyringe. Then, rinse the needle with one 1X HBSS to remove any bubbles or blocks from the syringe and tubing. Now, briefly anesthetize the adult fish with tricaine.Then, soak the sponge in the embryo water with tricaine and transfer a fish onto the sponge with its abdomen up for the injection. Now, intraperitoneally inject DOX solution by quickly inserting the needle at a 45 degree angle into the midline between the pelvic fins about one to two millimeters deep. Then, slowly release all of the DOX solution.Before retracting the needle, wait five seconds. Alternatively, the fish can be positioned laterally, with the anterior to the right. Then, inject at the lateral line above the pelvic fin, with the bevel up pointing towards seven o'clock at a 45 degree angle and three to four millimeters deep.If either method for injection was successful, there will be a red tint in the belly. After the injection, quickly transfer the fish to a clean crossing tank filled with fresh system water where it can recover. Then, rinse the needle with HBSS and proceed to the inject the next fish.Later, return the injected fish to a system with running circulation, but separate from the main system to avoid cross-contamination. For the next 24 hours, continue to fast the fish. Over the first week post-injection, be certain to check the fish daily and remove any dead fish.When using the classic belly-up IP injection method, it was noted that the injected DOX solution could sometimes ooze out from the location where the needle penetrated. The alternative lateral IP injection effectively prevented the leakage. Injection of DOX dosed at 50 milligrams per kilogram led to severe toxicity where the majority of fish died within one week.However, at a lower dose of 20 milligrams per kilogram, the injected Zebra fish could be maintained for chronic observation. With the alternative injection, there was no fish death during the first two weeks, and only about 10%death at four weeks. The classic method for injection at the same dose led to about 30%dead by four weeks.Next, transgenic fish with a casper background expressing DsRed and cardiomyocytes were used to assess the progression of cardiac dysfunction in the DIC model. Their red heart could be viewed at both the systolic and diastolic stages under a fluorescent microscope. After injection of 20 milligrams per kilogram of DOX using the alternative lateral injection method, ventricular function decline was detected beginning after four weeks.Once mastered, delivery of Doxorubicin with either technique can be performed on about 30 to 50 fish an hour. Following this procedure, other methods like echocardiography, electrocardiography or ex vivo heart function essay can be performed in order to profile heart function of this DIC model at different stages. While attempting this procedure, it is important to remember to use freshly prepared Doxorubicin and to test its drug efficacy after long-term storage, before the injection.Also, always handle Doxorubicin taking the appropriate safety precautions.

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Research

JoVE Journal

Medicine

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

אינדוקציה של אוטם שריר הלב ב דג הזברה למבוגרים באמצעות Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

הערכת שינויים teratogenic במודל דג הזברה של חשיפה אלכוהול עוברית

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

הקרנה למכפילים מלנומה תוך שימוש במודל גידול כבני מקום דג זברה

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

דגם דג זברה של סוכרת וזכרון מטבולית

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

מודל אפשרי דג הזברה של מחלת כליות פוליציסטיות: מציאה של<em&gt; Wnt5a</em&gt; גורם ציסטות בכליות דג הזברה

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

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

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

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

טכיקרדיה-Induced קרדיומיופתיה כמודל אי ספיקת לב כרונית, חזיר

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

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

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

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

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

מודל אוטם שריר הלב באמצעות הארכה קבועה של עורק שמאלי קדמי כלילי יורד

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac performance in the absence of hypertension, valvular, congenital, or ischemic heart diseases, and which may be related to infection, autoimmune or metabolic abnormalities, or family inheritance. It can progress into congestive heart failure with a poor prognosis. Doxorubicin (Dox) is widely employed as a chemotherapeutic drug, but its use is limited because it causes DCM-like changes of the myocardium. Its myocardial toxicity is attributed to oxidative stress, chronic inflammation, and cardiomyocyte apoptosis. A model of DCM exploiting these Dox-induced DCM symptoms has not been established.

Described is a protocol to establish a Doxorubicin-induced dilated cardiomyopathy (DCM) model in mice via long-term intraperitoneal injection of Doxorubicin.

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

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

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

מודל פגיעה בכליות חריפה המושרה על ידי ציספלטין בדג זברה למבוגרים

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...