The authors would like to thank Drs. Stephen Wilson and Lisa Wilsbacher for helpful discussions. This work was supported in part by NIH HL054737.

הערה: נהלי שימוש בדג זברה אושרו על ידי הטיפול בבעלי חיים המוסדי של קליפורניה בסן פרנסיסקו וועדת שימוש. 1. הכנת כלים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנס סיכת פרטים קטנה לבעל סיכה ומהדק את הסיכה. </li><li style=";text-align:right;direction:rtl"> בעזרת מלקחיים טיפ נאה, לכופף בזהירות את קצה הפין כדי ליצור וו קל. </li><li style=";text-align:right;direction:rtl"> למניפולציה וייצוב של העובר במהלך פציעה, תכופף את הקצה של מד 28 ½ מחט אינץ &#39;רכובה על מזרק אינסולין באמצעות צבת מחט-האף. </li></ol> 2. הכנת עוברי דג הזברה לפגיעה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הגדרת זוגות רבייה דג הזברה ולאסוף ביצים במי ביצה (60ug / מלחי אקווריום מ&quot;ל) כפי שהוצג קודם 22. </li><li style=";text-align:right;direction:rtl"> להוסיף 0.003% N-Phenylthiourea (PTU) למים ביצה כאשר הם עובר כ -8 שעות לאחר הפריה (hpf) כדי למנוע melanization. </li><li style=";text-align:right;direction:rtl"> עובר dechorionate שתי פוסט יום הפריה (DPF) לפני הניסוי באמצעות מלקחיים טיפ נאה. <br/&gt; הערה: ניתן נפצע עוברים בכל עת לאחר שהמחזור מתחיל. הנתונים המוצגים כאן הוא עבור 2 DPF עוברים, אך הטכניקה יושמה בהצלחה בעוברים עד 5 DPF. </li><li style=";text-align:right;direction:rtl"> להרדים את העוברים עם 0.02% חומצה שנאגרו 3-aminobenzoic (Tricaine) כ 10 דקות לפני מניפולציות. </li></ol> 3. כלי מכניים פגיעה של עוברים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> העבר את העוברים בהרדמה לשקופית דיכאון בסטראו לנתח באמצעות pipet העברה. </li><li style=";text-align:right;direction:rtl"> בצד החלק הקצר של מחט המזרק כדי לתפעל את העובר עם היד הדומיננטית, למקם את העובר בצד שלה עם משטח הגחון מול מן המחט. </li><li style=";text-align:right;direction:rtl"> מקם את הפינים הפרטים הקטנים עם הקצה מחודד ישירות על משטח הגחון של הדגים האחוריים לפתיחה ואברי המין. מקם את הפינים פרטים הקטנים בזווית קלה, כך שהקצה המעוגל הוא מסוגל לחדור מבעד periderm ישירות לוריד הזנב (איור 1 &lt;/ Strong&gt;). </li><li style=";text-align:right;direction:rtl"> שימוש במחט המזרק כדי לתפעל את העובר, לנקב את וריד הזנב עם סיכת הפרטים הקטנה על ידי הקשה על העובר לסיכה להתחבר הסיכה לתוך הווריד מעט. </li><li style=";text-align:right;direction:rtl"> שימוש במחט המזרק, למשוך את העובר מפיני הפרטים הקטנים כדי ליצור קרע קטן בכלי. הערה: פגיעה מוצלחת תגרום לדימום מיידי מהווריד. </li></ol> 4. ניתוח של עצירת דימום <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בחר עוברים רק עם בעליל תאי דם להליך זה. </li><li style=";text-align:right;direction:rtl"> הכן להתחיל את הטיימר בהקדם סיכת הפרטים הקטן היא משכה מהספינה. </li><li style=";text-align:right;direction:rtl"> להפעיל את הטיימר בהקדם איבוד דם יכול להיות דמיין מהפצע. כאשר איבוד דם מהפצע מפסיק, לעצור את שעון העצר ולהקליט זמן כולל שזמן דימום. אם הקרישה היא עכבות, להקליט את הזמן כאשר יש כבר לא נראה תאי דם במחזור. </li></ol> 5. ניתוח של ריפוי פצעים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> העברת קופהבעלי חיים לא-פגיעה על מנות הדמיה זכוכית תחתונה למיקרוסקופיה. </li><li style=";text-align:right;direction:rtl"> הסר את רוב המים הביצה. </li><li style=";text-align:right;direction:rtl"> מכסה את העוברים ב.3-1.2% התכה נמוכה המומסים agarose במי ביצה, מחוממים לבין 42 ו -45 מעלות צלזיוס ותוספת של 0.02% Tricaine. </li><li style=";text-align:right;direction:rtl"> עוברי עמדה על צדם באמצעות מלקחיים. </li><li style=";text-align:right;direction:rtl"> לאחר agarose מתקרר, למלא את הצלחת עם Tricaine 0.02% במי ביצה. </li><li style=";text-align:right;direction:rtl"> לרכוש תמונות באמצעות brightfield, epifluorescence, או confocal. </li><li style=";text-align:right;direction:rtl"> הסר עובר מן agarose באמצעות מלקחיים ולהעביר חזרה למי ביצה. </li></ol>

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The authors declare that they have no competing financial interests.

דג הזברה שימשה בהצלחה כמודל לסוגים שונים של פצעים כולל פציעת לייזר 13-15, פקקת מושרה לייזר 16, ופצע 10 אפיתל. אנו מדווחים על שיטה של פציעה מכאנית שהיא פשוט לביצוע ומייצר פציעה מבוקרת במודל in vivo כי הוא נוח מאוד למיקרוסקופיה בזמן אמת. תוצאות פציעה בתגובה לעצרת דם מהירה וניתן למדידה ותכנית תיקון פצע שחזור שניתן לנטר באמצעות מיקרוסקופ וידאו וtimelapse. האנטומיה שלהם פשוטה והסטריאוטיפית של כלי דם, המאפשרת פגיעה לשחזור באתר מוגדר ומיקרוסקופי נגיש, ואת הנוכחות של רוב כלי דם וסוגי hematopoietic תא להפוך את עוברי דג הזברה שימושיים במיוחד ללימוד תגובות לפציעה. עם זאת, אין לי עוברי דג הזברה לימפוציטים פונקציונליים בשבועות הראשונים של התפתחות 5,6, מה שהופך את רוב ap המערכת זוpropriate ללימוד התרומה של חסינות מולדת בדלקת ותיקון. נכון לעכשיו, במגוון רחב של דג הזברה מהונדס קיים עם סמנים לתאים וחלבונים המשתתפים בהיווצרות קריש דם, קרישה, דלקת, ותיקון פצע, כולל קווים שתווית טרומבוציטים, פיברינוגן, אריתרוציטים, כדוריות דם לבנות והאנדותל של כלי דם 17,21,25- 31. כלים אלה ואחרים צריכים לעשות את זה אפשר לעקוב אחר התהליכים מעורבים בעצירת דימום ותיקון בפירוט משמעותי. פגיעה מכאנית משלימה פציעת לייזר לחקר עצירת הדימום בדג זברה. בעוד פגיעה הנגרם לייזר הייתה בשימוש כבר שנים כדי לעורר היווצרות קרישי דם בעוברי דג הזברה ומודלים של עכברים, מנגנונים שבאמצעותם פגיעת לייזר מפעילה קרישה והפעלת thrombocyte / טסיות דם אינן ידועות במלואם 16,32. פגיעה מכאנית מספקת שיטה רלוונטית מבחינה פיזיולוגית לגרימת קרישה על ידי הפרת כלי דם ו, ככל הנראה,רקמות גורם תלוי ייזום של מפל קרישה. הממצא שטיפול הירודין גדל באופן משמעותי פעמים דימום לאחר פציעה מצביע על כך שמודל זה הוא תרומבין תלוי. פגיעה מכאנית בנוסף משלימה פציעת לייזר על ידי מתן לתת הזדמנות למעקב תיקון כלי הפרעה מספקת של כלי דם. מחקרים קודמים השתמשו בהצלחה פגיעה מכאנית על ידי חתך אזמל ולנקב מחט להראות הבדלים בדימום פעמים בתנאים של מניפולציה תרופתית וגנטית 19,33. פציעת סיכת פרטים הקטנה ששמשה במודל הנוכחי עשויה להשלים מודלים פציעה אחרים על ידי מתן פגיעה לשחזור יותר בשל הגודל הקטן ומוגדר של הפצע שהיא מייצרת ועל ידי מתן הזדמנות ללמוד recanalization כלי ותיקון טוב יותר. אפיתל פצע בדג הזברה הוכיח להיות מודל רב עוצמה ללימוד דלקת ופצע תיקון 10. היכולתלהציג את פציעה של כלי דם מספקת הזדמנות להעריך תיקון של יותר פצעים מורכבים בהגדרות בי הפיברין מספק מטריקס, thrombi ופסולת זמניים נמחקים, וכלי להתחדש. כתהליכים אלה להשתתף בתיקון רקמות נורמלי ובדלקת אקוטית וכרונית ופתולוגיה של כלי הדם, בשיטה זו אמורה לעזור לדגמן היבטים של מחלה אנושית במערכת שבה ניתן לנטר התנהגויות סלולריות בזמן אמת במודל חיה שלם.

Zebrafish have been used extensively to study a variety of topics in vascular biology, including vascular development, angiogenesis, and hematopoietic development and pathology1-3. Embryos develop a functional circulation as well as leukocytes and other components of the innate immune system by 1 day post fertilization (dpf) 1,4,5. The conservation of the inflammatory and leukocyte response to injury has made the zebrafish embryo an informative model for such diverse inflammatory processes as tuberculous infection, enterocolitis, and tissue regeneration6-9. Zebrafish embryos have been used to study injury-related inflammation particularly in the context of epithelial wounding and the neutrophil response10,11. Injury to the embryo results in a highly conserved cellular response from cells at the injury site and the innate immune cells recruited to respond to the injury and regulate its resolution11,12. Other injury models have used focused laser pulses to spatially localize injury to specific cell types including neurons, muscle cells, and cardiomyocytes13-15. 
Zebrafish embryos have been used as a model to study hemostasis and thrombosis in conditions of pharmacological and genetic manipulation, using both mechanical and laser-induced thrombus formation16-19. Components of the coagulation cascade appear to be well-conserved and transgenics have allowed for detailed studies of thrombocyte and fibrin deposition at the site of coagulation17,20,21. The procedure presented in this paper complements these methods by providing a system for studying mechanical vessel injury resulting in vessel breach, thrombus formation and resolution, and vessel repair.

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mechanical vessel injury in zebrafish embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

פגיעת כלי מכאנית בוצעה על 2 DPF עוברים (איור 2 א - ג). תוצאות פציעה בתגובת קרישה מהירה ואמינה, כפי שנמדדו על ידי זמן הפסקת דימום (איור 2 ד). כדי לקבוע אם או לא הבדלים בתגובת הקרישה ניתן היו למדוד, הירודין נוגד הקרישה היה מנוהל לעוברים על ידי הזרקה לתוך Duct של קיביה מייד לפני פציעתם (5-10 NL של יחידה 1 לμl הירודין מומס במים) (ל הפגנה של זריקות לDuct של קיביה, ראה מאמר יופיטר קודם 23) 24. הממשל של הירודין לפני פציעה גרם לעלייה משמעותית בדימום פעמים לעומת שליטה ברכב (איור 2 ד). עדות לניזק ספינה וקרישה ניתן לראות מייד לאחר פציעה באמצעות קווים מהונדסים לאנדותל (kdrl: egfp) ותאי דם אדומים (gata1a: DsRed </em&gt;) מסומן 25,26. תמונות נרכשו ברצף כל 5 דקות לתקופה 12 שעות באמצעות epifluorescence. נציג עדיין תמונות מוצגות ברחבי שלבים שונים של תיקון פצע (איור 3). באמצעות שילוב של התערבות ההפרש לעומת זאת (DIC) ומיקרוסקופ פלואורסצנטי, ניתן למדוד פרמטרים שונים של תיקון פצע. כדי לקבוע אם או לא תיקון פצע אחרי דפוס לשחזור פני ניסויים, הזמן מחדש את זרימת דם נמדד ב -4 קבוצות של דגים. פגיעת כלי הביאה תגובה אמינה סטריאוטיפית של 253 ± 16 דקות להקמתה מחדש של זרימת דם בכלי הפצוע (n = 4-5 דגים בכל ניסוי, ממוצע ± SEM). <img alt="איור 1" src="/files/ftp_upload/52460/52460fig1highres.jpg" /> איור 1: תרשים של עובר 2 DPF מראה מיקום של פינים פרטים קטנים לPerf<html> orming פגיעה מכאנית של וריד הזנב (CV). תא דם מוצל באפור. <img alt="איור 2" src="/files/ftp_upload/52460/52460fig2highres.jpg" /> איור 2: פעמים דימום ניתן למדוד באופן חזותי לאחר פגיעה מכאנית סטילס מוידאו בזמן אמת של פגיעת כלי דג הזברה על 2 DPF עוברים.. תמונות מוצגות בעת פציעה (), במהלך איבוד דם מהפצע פעיל (B), ולאחר ההפסקה של איבוד דם (C). כל הזמנים שצוינו הם בשניות. עוברים מכוונים רוחבי עם קדמי במשטח עליון וגחון מול לשמאל. בר סולם 100 מיקרומטר. מנהל של הירודין נוגד הקרישה הוביל לעלייה משמעותית בזמני דימום לעומת שליטה ברכב (D) (p &lt;0.0001, מבחן t).pg &quot;target =&quot; _ blank &quot;&gt; לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו. <img alt="איור 3" src="/files/ftp_upload/52460/52460fig3highres.jpg" /> איור 3:. לדמיין פגיעה מכאנית ותיקון באמצעות סמנים מהונדסים סטילס מtimelapse DIC ומיקרוסקופ פלואורסצנטי לאחר פציעת הכלי באמצעות סמנים להאנדותל של כלי דם (kdrl: egfp) ותאי דם אדומים (gata1a: DsRed) ב 2 DPF עוברים. תמונות הראו פער בכלים והצטברות מקומית דם אדום תא (t = 25), תיקון חלקי עם זרימה הוקם מחדש דם (t = 210), ושחזור של מבנה נורמלי כלי (t = 615) מלא כנראה. זמן מצויינים בדקות. עוברים מכוונים רוחבי עם קדמי במשטח עליון וגחון מול לשמאל. * מציין את המיקום של אב העורקים הגב. הפציעה (ראש החץ) שיבש את הווריד וחלק ממקלעת הזנב הזנב. Scaleבר 25 מיקרומטר.

Watch this Scientific Journal Video about Mechanical Vessel Injury in Zebrafish Embryos at JoVE.com

Mechanical Vessel Injury in Zebrafish Embryos

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

פגיעה כלי מכאנית בעוברי דג הזברה

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

mechanical-vessel-injury-in-zebrafish-embryos

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.2K Views.  University of California. This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy. 

Video: Mechanical Vessel Injury in Zebrafish Embryos

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

התחדשות בכליות למבוגרי דג הזברה על ידי גנטמיצין מושרה פגיעה

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

בידוד ואפיון של תאים בודדים מעוברים דג הזברה

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

איתור תיוג זיקה של אנדוגני רצפטורים מעוברים דג הזברה

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

מניפולציה ploidy של עוברי דג הזברה עם חום 2 הלם טיפול

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

Biosensing מוטורית נוירון פוטנציאל ממברנה עוברי דג הזברה חיים

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

גישה אבולרית מדויק עבור דוגמנות פגיעה בכליות חריפה בפיתוח דג הזברה

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

ניתוח היסטולוגית של פגיעה חריפה בכבד אלכוהול דג הזברה

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

אינדוקציה של היפוקסיה צפרדע חיה דג הזברה עוברי

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

ויזואליזציה של הפעילות החשמלית הסלולר עוברי מוקדם דג זברה, גידולים

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

כל הר אימונוהיסטוכימיה בזיבפיש עוברים וזחלים

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...