Name: isolation of retinal arterioles for ex vivo cell physiology studies
Uploaded: 2018-07-14T15:00:00.000+00:00
Duration: 12 min 42 s
Description: Watch this Scientific Journal Video about Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies at JoVE.com

פיתוח מהפרוטוקולים כמתואר במאמר זה נתמך על ידי מענקים למימון חברות הטיולים הבאות: BBSRC (BB/I026359/1), הריב מטווח הראייה (1429 ו- 1822), לסוכרת נעורים (2-2003-525), טרסט (074648/Z/04), קרן לב הבריטי (PG/11/94/29169), מועדון הכדורגל מונפלייה R & D חטיבה (STL 4748 13), MRC (MC_PC_15026).

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ד ר ג'ואנה כור עכשיו עובד של מדטרוניק (טווין-ערים, מינסוטה, ארה ב), עבודתה הנוכחי אינו מתנגש עם זה המוצג כאן.

הפרוטוקולים שתוארו לעיל דורשים תרגול, אבל צריך להיות בר השגה בפתרון מינימלי. ביום ממוצע אנחנו להשיג רבים שמיש 6-8 של הבידוד ולהשיג ניסויים מוצלחים 3-4. אם יש בעיות, עם זאת, ישנם כמה צעדים שניתן לנקוט כדי לשפר את אחוזי ההצלחה. מדי פעם אנחנו גילינו, במיוחד בעת שימוש חולדות הצעיר (< 8 שבועות), כי התשואה של רבים יכול להיות נמוך. כדי לעקוף בעיה זו, ואנו ממליצים צריך שתוציאו את רקמת רשתית (10-30 s-500 x g) בין כל trituration שלבים שלבים 1.12-1.14. זה לעתים קרובות מסייע לשפר את התשואה של כלי אך יהיה גם להגדיל את כמות פסולת תא במסגרת ההכנה.
כאשר cannulating כלי ללימודי myography לחץ זה חשוב לבדוק רבים בקפידה עבור כל צד-סניפים ייתכן ביקע קרוב לאתר נגע הסתעפות. זה מייצג גורם נפוץ זליגת ואובדן הלחץ במהלך ניסויים. העניין המרכזי שעשויות להתרחש עם [Ca2 +]אני הפרוטוקולים היא הרמה של צבע טעינה. צבע עניים הטעינה מוביל יחס אות לרעש נמוך, בעת העמסת תוצאות שיבוש נורמלי Ca2 + הומאוסטזיס. כדי להפחית את הסבירות של אחד מנושאים אלה, ניתן להסיר aliquot קטן של homogenate ברשתית, ספינות בודדות לבדוק מעת לעת במהלך פרוטוקול טעינה. כאשר התחייבות תיקון-קלאמפ ניסויים, שיעור ההצלחה של היווצרות gigaseal תלויה מאוד איך טוב הנדן הבזליים עיכולו. כאשר בתחילה בדיקה פרוטוקול זה או באמצעות שימוש רב חדש של אנזימים יתכן צורך להתאים את הריכוז/המשך של אנזים עיכול. ניטור בקפידה על הפרדת השכבות אנדותל, שריר חלק יבטיח עיכול מספיקות כדי להסיר את הבסיס מספיק laminal כדי לקבל גישה. פיקוח קפדני זה נחוץ גם למנוע עיכול יתר, אשר יכולה להתבטא כמו הכלי מתחיל מתכווצים. אם הדבר יתרחש, תאי שריר חלק הם לעתים קרובות מדי שביר להקלטה מלחציים תיקון. בעת החלת אנזימים, חשוב לכלול DNAse אני כדי להבטיח הסרה של גדילי ה-DNA שחרור מכל התאים הפגועים בתהליך בידוד. שברי DNA הם קשיים ולגרום המלקחיים לדבוק arteriole במהלך השלבים האחרונים של תהליך הניקוי (שלב 4.4), לעיתים קרובות וכתוצאה מכך איבוד הספינה. ניקוי של כלי קשה מבחינה טכנית ומתבצעת בצורה הטובה ביותר בהגדלה גדולה (20 X) עם תנועות גורפת של מלקחיים סגור מקוטר המברשת הטוב ביותר האפשרי. בין הרייטינג, לנקות את המלקחיים עם המעבדה גליל.
כפי שמודגש קודם לכן, מניע המפתח מאחורי התפתחות הפרוטוקולים המפורטים בכתב יד זה היה כדי להבין טוב יותר למה זרימת הדם מופרת במהלך מחלות כלי הדם ברשתית. רוב העבודה שלנו עד כה התמקדה בעין סוכרתית המחלה28,33. רבים ניתן לבודד מפני באדמומיות של מכרסמים ניסיוניות של סוכרת בשיטות המתוארות בסעיף 1. כאשר ניסויים רבים ברשתית מבודד של בעלי חיים סוכרתיים, חשוב לנסות לשכפל מקרוב את התנאים hyperglycemic שחוו את כלי ויוו. מסיבה זו, שנגדל בדרך כלל רמות D-גלוקוז בידוד, פתרונות ניסיוני 25 מ מ. עיבוי של קרום המרתף כלי הדם היא תופעה מוכרת היטב בכלי ברשתית במהלך סוכרת34,35. בעת שימוש בבעלי חיים עם סוכרת ממושכת (> 1 חודשים משך המחלה), ריכוז האנזים מוגבר או עיכול פעמים נדרשים לעתים קרובות כדי להפעיל את היישום של תיקון-קלאמפ הקלטה שיטות.
הגבלה חשובה של שימוש שמחוץ מבודד רבים ברשתית ללמוד פיזיולוגיה של כלי הדם ברשתית, פתופיזיולוגיה אובדן neuropile ברשתית שמסביב. למרות הסרת של תאי גליה ו עצביים ברשתית מאפשר גישה קלה אל תאי השריר החלק בכלי הדם ברשתית ללימודי פיזיולוגיה של התא, התגובה של כלי מגשרים vasoactive ניתן לשנות באופן דרמטי היעדרות, נוכחות של הרשתית רקמות. הפעולות של אדנוזין טרי-פוספט (ATP), לדוגמה, לספק דוגמה טובה נקודה זו. ב מבודד עכברוש רבים ברשתית, תוספת של ATP גורמים חזקים כיווץ של כלי36, תוך נוכחות neuropile ללא פגע, כלי הדם מתרחבים37. לכן, בכל מקום אפשרי, אנחנו בדרך כלל מנסים לאמת את הממצאים העיקריים מן ההכנות שלנו arteriole מבודדים באמצעות שמחוץ ברשתית שלם-mounts ו ויוו מדידות של כלי השיט קוטר ודם זרם16,37 . ראוי לציין, שיטות חדשות שהתגלו לאחרונה לימוד רבים קטנים, נימים בתוך כל perfused הרשתיות חזירי שמחוץ38,39. הכנות צפויים לשפר במידה רבה את הבנתנו איך neuropile ברשתית מווסת את הטון arteriolar ו נימי ברשתית, כמה שינויים ברשתית haemodynamics לווסת את פעילות. עצבית ברשתית.
למרות ההליכים המתוארים במסמך זה מתמקדים על השימוש של רבים ברשתית עכברוש בודד עבור הבנה שריר חלק arteriolar תא פיזיולוגיה, אנו כעת מפתחים פרוטוקולים לאפשר גם המחקר של פונקצית תא אנדותל כלים אלה. עבודה ראשוני, אנחנו כבר מצליחים לשנות עיכול אנזימטי מקטעי רשתית arteriolar להניב צינורות תא אנדותל קיימא נתונות Ca2 + הדמיה ו- patchclamp הקלטה לימודי. תעלות רבים מבודד בשני קצותיו, הפעלת intraluminal משלוח תרופות, יכול בעתיד גם לאפשר אנדותל תלוית תגובות vasodilatory להיחקר בכלי אלה.

הבנת איך נשלטת זרימת הדם ברשתית היא מטרה חשובה, כיוון זרימת הדם נורמלי היה מעורב בפתוגנזה של מגוון רחב של סכנת הראייה מחלות רשתית1,2,3, 4. זרימת הדם ברשתית, המספקת את הנוירונים ברשתית הפנימי ותאי גלייה, יש הסדר עורק הקצה, עם כל הדם העורקים ברשתית, רבים עוברים דרך הנימים venules ברשתית, סוף סוף ורידים5. זרימת הדם ברשתית מוסדר על ידי הצליל של העורקים ברשתית, רבים, כמו גם את פעילות כויץ pericytes ממוקם על הקירות של נימי הדם ברשתית, venules נימי שלאחר6,7, 8. השליטה של הטון כלי הדם ברשתית היא מורכבת, הוא מווסת על ידי מגוון של תשומות במערכת הדם, רקמת רשתית שמסביב, כולל גזים בדם, במחזור מולקולות והורמונים, ושוחררה חומרים vasoactive מ ברשתית וסקולרית אנדותל, macroglia9,10,11. רבים ברשתית נמצאים הענפים עורקים קטנים של הרשתית, המורכבת של שכבה אחת של תאי השריר החלק בכלי הדם, הציפוי הפנימי של longitudinally לארגן תאי אנדותל12,13, 14. אלו כלי הטופס באתר הראשי של התנגדות כלי הדם בתוך מחזור הדם ברשתית, לכן יש תפקיד חשוב בשליטה מקומית של זרימת הדם ברשתית. רבים ברשתית לווסת זרימת נימי הדם ברשתית להרחבת או מתכווץ קוטר luminal שלהם, מתווכת על-ידי שינויים השריר החלק בכלי הדם contractility10,15,16. הבנת המנגנונים המולקולריים דרך הרשתית אשר רבים לווסת זלוף ברשתית ולכן דורש הכנות שבו תאי שריר חלק arteriolar יכולה להיות גישה ולמד בתנאים הכי קרוב פיזיולוגיים ככל האפשר.
Ex-vivo ההכנות של כלי הדם ברשתית מבודד מספקים גישה לתאי השריר החלק בכלי הדם, תוך שמירה על פונקציונליות, interconnectivity עם אנדותל הבסיסית שלהם. רוב המחקרים עד היום באמצעות ספינות בודדות התמקדו גדול שור או חזירי עורקי כלי (60-150 מיקרומטר). אלה יכול להיות מותקן זמינים מסחרית תיל או מערכות myograph לחץ כדי לאפשר חקירה תרופתי של השריר החלק בכלי הדם התא מנגנונים כויץ17,18. הכנות תרמו במידה רבה הידע שלנו על הפיזיולוגיה של כלי הדם ברשתית בתנאים רגילים. מחקרים מעטים השתמשו רבים ברשתית מבודד חיות מעבדה קטנה כמו שלהם קוטר קטן (~ 8-45 מיקרומטר) מונע את השימוש שלהם קונבנציונאלי מערכות myography19,20,21,22. יתרון חשוב, אולם, השימוש ספינות חיות מעבדה קטנה היא זמינותו רחב של מודלים מחלת ששונה גנטית, מהונדס, ברשתית. חיות מעבדה קטנה הם גם יותר בעניין ללימודי התערבות ' ויוו .
כאן נתאר פרוטוקולים פשוטה עבור בידוד, cannulating רבים ברשתית עכברוש לניסויים myography הלחץ. Ca2 + הדמיה electrophysiology פרוטוקולים באמצעות כלים אלה גם מפורטים. אלה יכולים לספק עוד תובנות ויסות contractility השריר החלק בכלי הדם ואת זרימת הדם ברשתית.

<table><tbody><tr><td>Beakers</td><td>Fisherbrand</td><td>15409083</td><td>Or any equilavent product</td></tr><tr><td>Curved Scissors</td><td>Fisher Scientific</td><td>50-109-3542</td><td>Or any equilavent product</td></tr><tr><td>Disposable plastic pipette/ transfer</td><td>Sarstedt</td><td>86.1174</td><td>6mL is best but other sizes are acceptable; remove tip to widen aperture</td></tr><tr><td>Dissecting microscope</td><td>Brunel Microscopes LTD</td><td></td><td>Or any equilavent product</td></tr><tr><td>Forceps</td><td>World Precision Instruments</td><td>Dumont #5 14095</td><td>Any equivalent fine forceps</td></tr><tr><td>Pasteur pipette</td><td>Fisherbrand</td><td>11546963</td><td>Fire polished to reduce friction but not enough to narrow the tip</td></tr><tr><td>Petri dish</td><td>Sigma-Aldrich</td><td>P7741</td><td>Or any equilavent 10cm product</td></tr><tr><td>Pipette teat</td><td>Fisherbrand</td><td>12426180</td><td>Or any equilavent product</td></tr><tr><td>Purified water supply</td><td>Merek</td><td>Milli-Q Integral Water Purification System</td><td>Or any equilavent system</td></tr><tr><td>Round bottomed test tube</td><td>Fisherbrand</td><td>14-958-10 B</td><td>5 mL; any equilavent product</td></tr><tr><td>Seratted forceps</td><td>Fisher Scientific</td><td>17-467-230</td><td>Or any equilavent product</td></tr><tr><td>Single edge blades</td><td>Agar Scientific</td><td>T585</td><td>Or any equilavent product</td></tr><tr><td>Sylgard</td><td>Dow Corning</td><td>184</td><td>Or any pliable surface&amp;nbsp;</td></tr><tr><td>Testtube rack</td><td>Fisherbrand Derlin</td><td>10257963</td><td>Or any equilavent product</td></tr><tr><td>Pressure myography</td><td></td><td></td><td></td></tr><tr><td>3-axis mechanical manipulator</td><td>Scientifica</td><td>LBM-7&amp;nbsp;</td><td>x2 (or any equilavent product)</td></tr><tr><td>3-way taps</td><td>Cole-Parmer</td><td>UY-30600-02</td><td>Or any equilavent product</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Analysis software&amp;nbsp;</td><td>Image J</td><td>https://downloads.imagej.net/fiji/</td><td>Free imaging software</td></tr><tr><td>Analysis plugin</td><td>Myotraker&amp;nbsp;</td><td>https://doi.org/10.1371/journal.pone.0091791.s002</td><td>Custom plugin for imageJ Freely available for download</td></tr><tr><td>Cannulation pipette glass</td><td>World Percision instruments</td><td>TW150F-4</td><td>Or any equilavent product</td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7010</td><td>Or any equilavent product</td></tr><tr><td>Digital Thermometer</td><td>RS</td><td>206-3722</td><td>Or any equilavent product</td></tr><tr><td>Fluo-4-AM</td><td>Thermo Fisher Scientific</td><td>F14201</td><td>Make 1 mM stock in DMSO</td></tr><tr><td>Forceps</td><td>World Precision Instruments</td><td>Dumont #7 14097</td><td>Any equivalent fine forceps</td></tr><tr><td>Fura-2-AM</td><td>Thermo Fisher Scientific</td><td>F1221</td><td>Make 1 mM stock in DMSO</td></tr><tr><td>Glass bottomed recording chamber</td><td>Warner Instruments</td><td>64-0759</td><td>Or any equilavent product</td></tr><tr><td>Helper pipette glass</td><td>World Percision instruments</td><td>IB150F-3</td><td>Or any equilavent product</td></tr><tr><td>In-line heater</td><td>Custom made</td><td></td><td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclypse TE 300</td><td>Or any equilavent product</td></tr><tr><td>Manometer</td><td>Riester</td><td>LF1459</td><td>Or any equilavent product</td></tr><tr><td>Microelectrode puller</td><td>Sutter instruments</td><td>P97</td><td>Or any equilavent product</td></tr><tr><td>Microforge +&amp;nbsp; Olympus CX 31 microscope</td><td>Glassworks</td><td>Fine Point F-550</td><td>Or any equilavent product</td></tr><tr><td>Micromanipulator</td><td>Sutter instruments</td><td>MP-285</td><td>Or any equilavent product</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</td><td>BD Plastipak</td><td>1mL</td><td>Syringe heated and pulled to internal diameter of pipette</td></tr><tr><td>Pipette holder</td><td>Molecular Devices</td><td>1-HL-U</td><td>Or any equilavent product</td></tr><tr><td>Suction pump</td><td>Interpet</td><td>AP1</td><td>Converted aeration pump by reversing bellows</td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>20mL Luer-lok</td><td>Or any equilavent product</td></tr><tr><td>Tungsten wire</td><td>Advent</td><td>W558818</td><td>75&amp;mu;m diameter</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>miscellaneous sizes</td><td>Or any equilavent product</td></tr><tr><td>USB camera</td><td>&amp;nbsp;Logitech&amp;nbsp;</td><td>HD Pro Webcam C920</td><td>Or any equilavent product</td></tr><tr><td>Video capture software</td><td>Hypercam</td><td>version 2.28.01</td><td>Or any equilavent product</td></tr><tr><td>Water bath</td><td>Grant</td><td>Sub Aqua 12 Plus</td><td>Or any equilavent product</td></tr><tr><td>x20 lens</td><td>Nikon</td><td>20x/0.40 WD 3.8, &amp;infin;/0.17</td><td>Or any equilavent product</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product</td></tr><tr><td>Y-connectors</td><td>World Percision Instruments</td><td>14012</td><td>Or any equilavent product</td></tr><tr><td>Patch clamping</td><td></td><td></td><td></td></tr><tr><td>3-way taps</td><td>Cole-Parmer</td><td>UY-30600-02</td><td>Or any equilavent product</td></tr><tr><td>3-axis mechanical manipulator</td><td>Scientifica</td><td>LBM-7&amp;nbsp;</td><td>Or any equilavent product</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A2411</td><td>3 mg disolved daily in 50 &amp;mu;L of DMSO (sonicate to dissolve)</td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Axopatch 200a</td><td>Or any equilavent product</td></tr><tr><td>Analogue digital converter</td><td>Axon</td><td>Digidata 1440A</td><td>Or any equilavent product</td></tr><tr><td>Collagenase Type 1A</td><td>Sigma-Aldrich</td><td>C9891&amp;nbsp;</td><td>0.1mg/mL in LCH</td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7010</td><td>Or any equilavent product</td></tr><tr><td>Digital Thermometer</td><td>RS</td><td>206-3722</td><td>Or any equilavent product</td></tr><tr><td>DNAse I</td><td>Millipore</td><td>260913</td><td>Working stock 1 MU mL-1 (10 MU diluted in 10 mL LCH)&amp;nbsp;</td></tr><tr><td>Faraday cage</td><td>Custom made</td><td></td><td>Any equilavent commerical product</td></tr><tr><td>Fine forceps</td><td>Dumont</td><td>No. 7</td><td>Any superfine forcep</td></tr><tr><td>Glass bottomed recording chamber</td><td>Warner Instruments</td><td>64-0759</td><td>Or any equilavent product</td></tr><tr><td>Headstage</td><td>Molecular Devices</td><td>CV203BU</td><td>Or any equilavent product</td></tr><tr><td>In-line heater</td><td>Custom made</td><td></td><td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclypse TE 300</td><td>Or any equilavent product</td></tr><tr><td>Microelectrode puller</td><td>Sutter instruments</td><td>P97</td><td>Or any equilavent product</td></tr><tr><td>Microforge +&amp;nbsp; Olympus CX 31 microscope</td><td>Glassworks</td><td>Fine Point F-550</td><td>Or any equilavent product</td></tr><tr><td>Micromanipulator</td><td>Sutter instruments</td><td>MP-285</td><td>Or any equilavent product</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Patching software</td><td>Molecular Devices</td><td>Pclamp v10.2</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</td><td>BD Plastipak</td><td>1mL</td><td>Syringe heated and pulled to internal diameter of pipette</td></tr><tr><td>Pipette holder</td><td>Molecular Devices</td><td>1-HL-U</td><td>Or any equilavent product</td></tr><tr><td>Protease Type XIV</td><td>Sigma-Aldrich</td><td>P5147&amp;nbsp;</td><td>0.01mg/mL in LCH</td></tr><tr><td>Single channel pipette glass</td><td>World Percision instruments</td><td>IB150F-3</td><td>Or any equilavent product</td></tr><tr><td>Suction pump</td><td>Interpet</td><td>AP1</td><td>Converted aeration pump by reversing bellows</td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>20mL Luer-lok</td><td>Or any equilavent product</td></tr><tr><td>Tungsten wire</td><td>Advent</td><td>W557418</td><td>50&amp;mu;m diameter</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>miscellaneous sizes</td><td>Any equilavent product to fit</td></tr><tr><td>USB camera</td><td>&amp;nbsp;Logitech&amp;nbsp;</td><td>HD Pro Webcam C920</td><td>Or any equilavent product</td></tr><tr><td>Water bath</td><td>Grant</td><td>Sub Aqua 12 Plus</td><td>Or any equilavent product</td></tr><tr><td>Whole cell pipette glass</td><td>Warner Instruments</td><td>GC150TF-7.5</td><td>Or any equilavent product</td></tr><tr><td>x20 lens</td><td>Nikon</td><td>20x/0.40 WD 3.8, &amp;infin;/0.17</td><td>Or any equilavent product</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product</td></tr><tr><td>x40 lens</td><td>Nikon</td><td>40x/0.55, WD 2.1, &amp;infin;/1.2</td><td>Or any equilavent product</td></tr><tr><td>Y-connectors</td><td>World Percision Instruments</td><td>14012</td><td>Or any equilavent product</td></tr><tr><td>Ca&lt;sup&gt;2+&lt;/sup&gt; imaging</td><td></td><td></td><td></td></tr><tr><td>3-way taps</td><td>Cole-Parmer</td><td>UY-30600-02</td><td>Or any equilavent product</td></tr><tr><td>3-axis mechanical manipulator</td><td>Scientifica</td><td>LBM-7&amp;nbsp;</td><td>Or any equilavent product</td></tr><tr><td>Acquisition software&amp;nbsp;</td><td>Cairn Research Ltd.</td><td>Acquisition Engine V1.1.5</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Analysis software for confocal imaging</td><td>Image J</td><td>https://downloads.imagej.net/fiji/</td><td>Free imaging software</td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7010</td><td>Or any equilavent product</td></tr><tr><td>Confocal microscope</td><td>Leica Geosystems</td><td>SP5&amp;nbsp;</td><td>Or any equilavent product; confocal</td></tr><tr><td>Digital Thermometer</td><td>RS</td><td>206-3722</td><td>Or any equilavent product</td></tr><tr><td>Fine forceps</td><td>Dumont</td><td>No. 7</td><td>Any superfine forcep</td></tr><tr><td>Glass bottomed recording chamber</td><td>Warner Instruments</td><td>64-0759</td><td>Or any equilavent product</td></tr><tr><td>In-line heater</td><td>Custom made</td><td></td><td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclipse TE2000</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Monochromator&amp;nbsp;</td><td>Cairn Research Ltd.</td><td>Optoscan</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</td><td>BD Plastipak</td><td>1mL</td><td>Syringe heated and pulled to internal diameter of pipette</td></tr><tr><td>Software for confocal microscope</td><td>Leica Geosystems</td><td>LAS-AF version 3.3.</td><td>Or any equilavent product; confocal</td></tr><tr><td>Suction pump</td><td>Interpet</td><td>AP1</td><td>Converted aeration pump by reversing bellows</td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>20mL Luer-lok</td><td>Or any equilavent product</td></tr><tr><td>Tungsten wire</td><td>Advent</td><td>W557418</td><td>50&amp;mu;m diameter</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>miscellaneous sizes</td><td>Any equilavent product to fit</td></tr><tr><td>Water bath</td><td>Grant</td><td>Sub Aqua 12 Plus</td><td>Or any equilavent product</td></tr><tr><td>x100 lens</td><td>Nikon</td><td>x100 N.A. 1.3 oil</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x20 lens</td><td>Nikon</td><td>20x/0.40 WD 3.8, &amp;infin;/0.17</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x20 lens</td><td>Leica Geosystems</td><td>HCX PL FLUOTAR, 20x/0.50, &amp;infin;/0.17/D</td><td>Or any equilavent product; confocal</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x63 lens</td><td>Leica Geosystems</td><td>HCX PL APO, 63x/1.40 - 0.60 OIL CS &amp;infin;/0.17/E</td><td>Or any equilavent product; confocal</td></tr><tr><td>Y-connectors</td><td>World Percision Instruments</td><td>14012</td><td>Or any equilavent product</td></tr><tr><td>&lt;strong&gt;Pipettes&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Specifications and settings for fabrication of pipettes for experimentation&lt;/strong&gt;</td></tr><tr><td>Helper pipette</td><td>World Percision instruments</td><td>IB150F-3</td><td>Inner diameter: 0.86 mm&lt;br/&gt; Ramp: 261&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 56&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 0.5-2 &amp;mu;m&lt;br/&gt; Polishing: yes&lt;br/&gt; Final Resistance: &gt;10 M&amp;Omega;</td></tr><tr><td>Cannulation pipette</td><td>World Percision instruments</td><td>TW150F-4</td><td>Inner diameter: 1.17 mm&lt;br/&gt; Ramp: 290&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 58&lt;br/&gt; Time: 150&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 3-10 &amp;mu;m&lt;br/&gt; Polishing: no&lt;br/&gt; Final Resistance: &amp;lt;1 M&amp;Omega;</td></tr><tr><td>On-cell patching</td><td>World Percision instruments</td><td>IB150F-3</td><td>Inner diameter: 0.86 mm&lt;br/&gt; Ramp: 261&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 56&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 0.5-2 &amp;mu;m&lt;br/&gt; Polishing: yes&lt;br/&gt; Final Resistance: &gt;5 M&amp;Omega;</td></tr><tr><td>Whole-cell patching</td><td>Warner Instruments</td><td>GC150TF-7.5</td><td>Inner diameter: 1.17 mm&lt;br/&gt; Ramp: 296&lt;br/&gt; Pressure: 200&lt;br/&gt; Heat: 287&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 50&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 2-3 &amp;mu;m&lt;br/&gt; Polishing: helpful but not necessary&lt;br/&gt; Final Resistance: 1-2 M&amp;Omega;</td></tr></tbody></table>

isolation of retinal arterioles for ex vivo cell physiology studies

הרשתית היא רקמה פעילה מאוד סמויה הדורשת הספקת דם ניכר. זרימת הדם ברשתית תומך הרשתית הפנימית, בעוד כלי חיבור לספק את photoreceptors. שינויים ברשתית זלוף לתרום רבות הפרעות מסכנות-הראייה, כולל רטינופתיה סוכרתית, גלאוקומה, סניף ברשתית הווריד occlusions. הבנת המנגנונים המולקולריים המעורבים בבקרה על זרימת הדם דרך הרשתית, איך אלה שונו במהלך מחלה עינית עלול להוביל הזיהוי של יעדים חדשים לטיפול של תנאים אלה. רבים ברשתית הן כלי ההתנגדות העיקרי של הרשתית, אפוא, להיות תפקיד מרכזי בוויסות ספיקת הדם ברשתית באמצעות שינויים בקוטר luminal. בשנים האחרונות פותחו שיטות בידוד רבים מן הרשתית חולדה אשר מתאימים למגוון רחב של יישומים כולל לימודי פיזיולוגיה של התא. ההכנה הזאת כבר החלה להניב תובנות חדשות כיצד זרימת הדם נשלטת ברשתית, אפשרה לנו לזהות חלק מהשינויים מפתח המתרחשים במהלך מחלה עינית. במאמר זה, אנו לתאר את שיטות בידודו של עכברוש רבים ברשתית, וכוללים פרוטוקולים לשימוש שלהם תיקון-קלאמפ אלקטרופיזיולוגיה, הדמיית סידן ולימודים לחץ myography. כלים אלה נתונות גם לשימוש ב- PCR-, מחקרים מערביים סופג אימונוהיסטוכימיה מבוססות.

Watch this Scientific Journal Video about Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies at JoVE.com

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

כתב יד זה מתאר פרוטוקול פשוטה עבור בידודו של רבים מן הרשתית עכברוש יכול לשמש אלקטרופיזיולוגיות, סידן הדמיה ואת הלחץ myography מחקרים.

בידוד של רבים ברשתית ללימודי פיזיולוגיה של התא Vivo לשעבר

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

isolation-of-retinal-arterioles-for-ex-vivo-cell-physiology-studies

Research

JoVE Journal

Biology

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

10.2K Views.  Queen's University of Belfast. כתב יד זה מתאר פרוטוקול פשוטה עבור בידודו של רבים מן הרשתית עכברוש יכול לשמש אלקטרופיזיולוגיות, סידן הדמיה ואת הלחץ myography מחקרים.

Video: Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

בידוד של תאים ראשיים Murine הרשתית גנגליון (RGCs) על ידי זרימת cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Bioengineering

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

בידוד תאי אנדותל רשתית מורין לריצוף הדור הבא

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

Developmental Biology

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

טיהור של כלי מוח עכבר

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Neuroscience

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

בידוד Cannulation של שיתוק Parenchymal arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

שלבי הכנה מדידה של תגובתיות ב העכבר רבים ברשתית Vivo לשעבר

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Medicine

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

טווח ארוך, טיפוח ללא סרום של אורגנוטיפי עכבר רשתית explants עם אפיתל פיגמנט רשתית שלם

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

כימות של פרמטרי כלי דם ברשתיות הר שלמות של עכברים עם רטינופתיות שאינן מתרבות ושגשוגות

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

השתלת הרשתית של הרשתית של עכבר בוגר כמודל ex vivo לחקר מחלות נוירו-וסקולריות ברשתית

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

הקפאה-מקטעי רשתית, שלם-טעינות, וההכנות להערכת מבודד היפוטוניק להמחשת Immunohistochemical של Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

בדיקה חדשנית להגנה על הרשתית עבור מחקר פתוגנזה של רטינופתיה סוכרתית

An Assay to Detect Protection of the Retinal Vasculature from Diabetes-Related Death in Mice

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves the loss of protection from diabetes-induced damage to the retina, while the second phase centers on the accumulation of this damage. Traditional assays primarily focus on evaluating capillary degeneration, which is indicative of the severity of damage, essentially addressing the second phase of DR. However, they only indirectly provide insights into whether the protective mechanisms of the retinal vasculature have been compromised. To address this limitation, a novel approach was developed to directly assess the retina's protective mechanisms - specifically, its resilience against diabetes-induced insults like oxidative stress and cytokines. This protection assay, although initially designed for diabetic retinopathy, holds the potential for broader applications in both physiological and pathological contexts. In summary, understanding the pathogenesis of diabetic retinopathy involves recognizing the dual phases of protection loss and damage accumulation, with this innovative protection assay offering a valuable tool for research and potentially extending to other medical conditions.

מחבר זרקור: הבנת עמידות כלי הרשתית והתקדמות המחלה

A protection assay was developed to monitor the retinal vasculature's resilience to death from diabetes/diabetic retinopathy-related insults such as oxidative stress and cytokines.

בדיקה לגילוי הגנה על כלי הדם ברשתית מפני מוות הקשור לסוכרת בעכברים

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves ...