המחברים מבקשים להודות לדמיאן Gaillard לחיסונים ומעקב של ניסויים בבעלי חיים. עבודה זו נתמכה על ידי ANSES (הסוכנות הצרפתית למזון, סביבה ובריאות תעסוקתית ובטיחות) ועל ידי מענק מהקרן צרפת פרקינסון.

כל הנהלים והפרוטוקולים מעורבים בעלי חיים היו בהתאם לEC Directive 86/609 / EEC ואושרר על ידי יבוא, הוועדה הלאומית הצרפתית לשיקול אתי בניסויים בבעלי חיים (פרוטוקול 11-0043). בעלי החיים שוכנו וטיפלו בANSES של אושר מתקנים ניסיוניים בליון (אישור B 69,387 0801). 1. הכנת עכברים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים עכברים על ידי הזרקת intraperitoneal של מנה הקטלנית של pentobarbital נתרן. </li><li style=";text-align:right;direction:rtl"> אחזר את המוח כולו מגולגולת העכבר ולמקם אותו בצלחת פטרי פלסטיק 35 מ&quot;מ על קרח עד מיצוי. </li><li style=";text-align:right;direction:rtl"> לחלץ את חוט השדרה הצווארי. הערה: αS או מאחד חצאים המוח לאחר חתך sagittal או ממוח עכבר גזור, זמין לאחר הניסויים מפורטים בטבלה 1 תמצית. </li></ol><table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td> Experimenלא </td><td> עכברים הבידוד (שווה ערך מוח) </td><td> תקופת הישרדות (Dpi) </td><td> חציון / הישרדות מקסימלי (ימים) </td><td> αS זיהוי ד ידי ELISA / WB / IHC </td></tr><tr><td> 1 </td><td> עכברי Uninoculated </td><td> 441 ± 166 </td><td> 419/736 </td><td> 8/8 </td></tr><tr><td> 2 </td><td> עכברים מחוסן (0.2 מ&quot;ג) </td><td> 150 ± 52 </td><td> 140/241 </td><td> 9/9 </td></tr></tbody></table> רשימת טבלת 1. של ניסויים שבוצעו על עכברי M83. חיסונים בוצעו לאחר 6 שבועות לניסוי 2 באזור striato-קליפת המוח עם 20 μl של homogenate מוח של עכבר חולה (כרך / WT 1% ברמת סוכר 5%), לאחר הרדמה של עכברים הומוזיגוטים בן 6 שבועות M83 ב -3% שאיפת isoflurane. dpi: ימי הודעה inoculני. 2. αS הפקה ממוח חצאים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Sagittally לחתוך את המוח כדי להשיג שני חצאים. לשקול כל מחצית בצינור ribolysis מכיל כדורי טחינה. </li><li style=";text-align:right;direction:rtl"> הכן חיץ מלח גבוה (HS) המכיל 50 מ&quot;מ טריס-HCl, pH 7.5, 750 מ&quot;מ NaCl, 5 מ&quot;מ EDTA, 1 מ&quot;מ DTT, קוקטיילים phosphatase ומעכבי פרוטאז 1%. הוסף חוצץ מלח הגבוה לחצאים המוח כדי להשיג 20% homogenates (משקל / נפח). </li><li style=";text-align:right;direction:rtl"> הכן דגימות מהחצאים המוח באמצעות homogenizer מכאני על 6.0 מ &#39;/ s במשך 23 שניות פעמיים. לאחר הומוגניזציה 23 שניות הראשונות, למקם את הצינורות המכילים את homogenates על קרח למשך 2 דקות לפני מחזור 23-שניות השניה. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה דגימות XG ב 1000 במשך 5 דקות כדי לחסל ברי מוח unground. לשחזר את supernatants, מתחלק לaliquots 200 μl ולשמור אותם ב -80 ° C לניתוח ELISA שלאחר מכן. </li></ol> 3. αS הפקה מאזורי המוח גזור <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לנתחהמוח כולו בצלחת פטרי פלסטיק 35 מ&quot;מ על קרח עם זכוכית מגדלת חשמל נמוכה (הגדלה 8X) באמצעות שתי forcipes שקצוות נשמרים יחד למעט כאשר לנתח את ההיפוקמפוס. לא יעלה על 10 דקות כדי לשמור על שלמות מוח. מניחים את המוח בצד ימין למעלה ולאחזר את האזורים במוח הבאים לפי סדר הבא: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> נפרד אחד משתי נורות חוש הריח באמצעות מלקחיים להציב מאחורי הנורה. לנתק אותו מהמוח על ידי תנועה כלפי מטה. חזור על פעולה זו עבור הנורה השנייה. </li><li style=";text-align:right;direction:rtl"> בעדינות טריז המלקחיים בין שני cortexes ולהעביר אותה קדימה כדי להקל על ניתוק של שני cortexes. שמירה על המוח במקום אחד עם מלקחיים, להשתמש עוד להפריד את הקליפה מן ההיפוקמפוס. </li><li style=";text-align:right;direction:rtl"> מקם את המלקחיים 2 מ&quot;מ מתחת לקליפת המוח. לשמור על לחץ עדין על המלקחיים עד החלק העליון של ההיפוקמפוס הוא גלוי. לקלף את החלק הראשון של קליפת המוח, ולחזור עם החלק השני. השתמש במלקחיים כדי להפריד בין שני cortexesהחל משעת ההיפוקמפוס ונע לכיוון החלק הקדמי של המוח. </li><li style=";text-align:right;direction:rtl"> מקם את המלקחיים הפתוחים סביב אחד hippocampi. סגור את המלקחיים בתחתית של ההיפוקמפוס אז בעדינות להסיר אותו, מתאושש ככל האפשר. חזור על התהליך עבור ההיפוקמפוס השני. </li><li style=";text-align:right;direction:rtl"> מקם את המלקחיים הפתוחים מתחת לאחת מstriata ועדינות להפריד אותו מהמוח. השתמש במלקחיים כדי להסיר כל קליפה שנותרה מסטריאטום. חזור על תהליך זה עבור סטריאטום השני. </li><li style=";text-align:right;direction:rtl"> השתמש במלקחיים כדי לדכא בעדינות על ידי 2 מ&quot;מ קווי המתאר של המוח הקטן כדי להקל על הפרדה של המוח הקטן מהמוח. מניחים את המלקחיים ממש מאחורי המוח הקטן ולהסיר אותו על ידי העברת המלקחיים קדימה. </li><li style=";text-align:right;direction:rtl"> השתמש בחלק הרחב של המלקחיים כדי להעלות את mesencephalon כדי לראות בבירור שבו מצטרף לגזע המוח. לעשות חתך אנכי בצומת לאחר מכן להסיר את גזע המוח. </li><li style=";text-align:right;direction:rtl"> מקם את המלקחיים מאחורי mesencephalon, שבו אנישעות מורכבות מארבעה מבנים מעוגלים. לחתוך בצורה אנכית עד mesencephalon הופרד לחלוטין מהמוח שנותר. </li></ol></li><li style=";text-align:right;direction:rtl"> הכן homogenates של% משתנים (משקל / נפח) במאגר HS, בהתאם לכמות של רקמות זמינות, כלומר, homogenate 5% למשקל בין 10 ו -30 מ&quot;ג, 10% למשקל בין 30 ל 80 מ&quot;ג, ו -20% למשקל מעל 80 מ&quot;ג. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להוסיף נפח מתאים של חיץ HS לאזורים במוח גזור להשגת% הצפויים של homogenate. </li><li style=";text-align:right;direction:rtl"> וורטקס ולבדוק שהרקמות שקועים לחלוטין במאגר HS. </li><li style=";text-align:right;direction:rtl"> Homogenize דגימות שהוכנו מאזורי המוח גזור או חוט השדרה הצווארי עם מטחנת רקמה המורכבת משפופרת זכוכית בורוסיליקט ושני עליים, A ו- B. </li><li style=";text-align:right;direction:rtl"> יוצקים כל אזור במוח שנמחץ ישירות לתוך הצינור. הכנס העלי לתוך הצינור ולחזור בו. חזור על תנועה זו כעשר פעמים ללנתק את הרקמה. לאחר מכן להשתמש העלי &#39;לגontinue טחינת הרקמה עם 20 תנועות נוספות. העבר את homogenates לתוך צינור 1.5 מיליליטר עם טפטפת העברה 1 מיליליטר. </li></ol></li><li style=";text-align:right;direction:rtl"> צנטריפוגה דגימות XG ב 1000 במשך 5 דקות על 4 מעלות צלזיוס לחסל את כל שברי מוח unground. אחזר את supernatants, לחלק אותם לaliquots 200 μl ולשמור אותם ב -80 ° C לניתוח ELISA שלאחר מכן. </li></ol> 4. איתור של αS ידי ELISA <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לדלל את נוגדני ציפוי ל0.01 ng / ml. השתמש באחת אנטי-αS ארנב polyclonal או נוגדן חד שבטי בשיבוט 42 50 2 CO 3 / NaHCO חיץ מ&quot;מ Na 3 (pH 9.6). </li><li style=";text-align:right;direction:rtl"> מעיל עם כל טוב של פתרון ציפוי זה 100 μl, ולהשאיר 96 גם microplates על 4 מעלות CO / N. השתמש בנוגדן polyclonal הארנב נגד αS בפתרון הציפוי לELISAs באמצעות נוגדני זיהוי syn514, לשכפל 42, LB509, AS11, 4D6 או 8A5. השתמש בשיבוט הנוגדן חד שבטי נגד αS 42 כפתרון ציפויבשילוב עם נוגדני זיהוי αS אנטי-pSer129. הערה: אם יש צורך, הצלחות עשויות להיות כל הזמן על 4 מעלות צלזיוס במשך שבוע אחד לפני ELISA מתבצע. </li><li style=";text-align:right;direction:rtl"> השתמש מכונת כביסה צלחת לשטוף את הצלחות חמש פעמים עם 300 μl של פוספט שנאגרו מלוח עם 0.05% Tween 20 (PBST) בכל טוב. מהצעד הזה והלאה, דגירה היא על RT. </li><li style=";text-align:right;direction:rtl"> הוסף 200 μl של T20 PBS חיץ חסימה בכל טוב. Shake עבור שעה 1 ב 150 סל&quot;ד. לשטוף את הצלחות חמש פעמים עם PBST. </li><li style=";text-align:right;direction:rtl"> לדלל את homogenates המוח (דילול 1: 100 של homogenates 20%, 01:50 של homogenates% 10 ו01:25 של homogenates 5% בPBST BSA 1%), ולהוסיף 100 μl היטב כל אחד. אז דגירה של 2 שעות בזמן שלוחץ על 150 סל&quot;ד. לשטוף את הצלחות חמש פעמים עם PBST. </li><li style=";text-align:right;direction:rtl"> הוסף את נוגדני זיהוי αS השונים בPBST עם BSA 1% בדילולים מוזכרים ברשימת חומרים. דגירה עבור שעה 1 ב 150 סל&quot;ד. לשטוף את הצלחות חמש פעמים עם PBST. </li><li style=";text-align:right;direction:rtl"> להוסיף או אנטי-mouse או conjugates HRP IgG נגד ארנב בדילול 1: 8,000 בPBST בתוספת 1% BSA עבור שעה 1 בעוד הרועד בסל&quot;ד 150. לשטוף את הצלחות חמש פעמים עם PBST. </li><li style=";text-align:right;direction:rtl"> הוסף ל3,3 &quot;, פתרון 5,5&#39;-tetramethylbenzidine (TMB) 100 μl לכל היטב דגירה במשך 15 דקות בחושך בזמן רועד בסל&quot;ד 150. </li><li style=";text-align:right;direction:rtl"> לעצור את התגובה על ידי הוספה של 1 N HCl 100 μl לכל גם אז למדוד את הספיגה ב 450 ננומטר עם קורא microplate. </li><li style=";text-align:right;direction:rtl"> לניתוח נתונים, לחסר ערך OD שהתקבל בהיטב עם כל חומרים כימיים, למעט כל דגימות מוח עכבר (גם ריק) מערכי OD נמדדו עבור כל אחת מהדגימות נותחו. </li></ol> 5. epitope מיפוי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לבצע מיפוי epitope לפי השיטה שתוארה על ידי אוסמן 16. בקצרה, פפטידים מקום של רצף α-סינוקלאין האנושי המכיל 12 חומצות אמינו על nitrocellulose עם 10 חומצות אמינו חופפות. </li><li style=";text-align:right;direction:rtl"> לחסום עם 50 מ&quot;מ טריס / pH חיץ 150 מ&quot;מ NaCl 10 המכיל 0.05% Tween 20 ו 5% אבקת חלב. דגירה הנוגדן בחסימת פתרון בריכוז של 2 נוגדן מיקרוגרם למ&quot;ל ב2-10 ° CO / N. </li><li style=";text-align:right;direction:rtl"> לשטוף את הממברנה שלוש פעמים באמצעות 50 מ&quot;מ טריס / 150 pH חיץ מ&quot;מ NaCl 10 המכיל 0.05% 20. דגירה Tween עם המצומד אנטי עכבר IgG HRP העז. שטוף את הקרום חמש פעמים נוספות תוך שימוש באותה החיץ אז כתם באמצעות ערכת צביעת הכתם TMB מערבית. </li></ol> 6. ניתוח סטטיסטי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש בתוכנת R וnlme החבילה לבצע רגרסיות מעורבת לוואי מודל OD. עבור כל השוואה, לבצע מודל רגרסיה השפעה מעורב. השתמש השפעה קבועה להבחין סימפטומטי מקבוצות ללא תסמינים. </li><li style=";text-align:right;direction:rtl"> השתמש השפעה אקראית על מנת לשקף את השונות של חזרות לעכבר נתון. בדוק הומוסקדסטיות על ידי בחינת השאריות ואם יש צורך, להשתמש בפונקציות השונות למודל המבנה השונות של שגיאות בקבוצה בקנה אחד עם פיניירו ו1 בייטס7. קבע 0.05 כסף המשמעות של פ </li></ol>

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylated+alpha-synuclein+can+be+detected+in+blood+plasma+and+is+potentially+a+useful+biomarker+for+Parkinson's+disease.">Phosphorylated alpha-synuclein can be detected in blood plasma and is potentially a useful biomarker for Parkinson's disease.</a> FASEB J. 25, 4127-4137 (2011).</li><li>El-Agnaf, O. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+oligomeric+forms+of+alpha-synuclein+protein+in+human+plasma+as+a+potential+biomarker+for+Parkinson's+disease.">Detection of oligomeric forms of alpha-synuclein protein in human plasma as a potential biomarker for Parkinson's disease.</a> Faseb J. 20, 419-425 (2006).</li><li>Lee, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-linked+immunosorbent+assays+for+alpha-synuclein+with+species+and+multimeric+state+specificities.">Enzyme-linked immunosorbent assays for alpha-synuclein with species and multimeric state specificities.</a> J Neruosci Meth. 199, 249-257 (2011).</li><li>Unterberger, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+disease-associated+alpha-synuclein+in+the+cerebrospinal+fluid:+a+feasibility+study.">Detection of disease-associated alpha-synuclein in the cerebrospinal fluid: a feasibility study.</a> Clin Neuropathol. 33, 329-334 (2014).</li><li>Betemps, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+spreading+in+M83+mice+brain+revealed+by+detection+of+pathological+alpha-synuclein+by+enhanced+ELISA.">Alpha-synuclein spreading in M83 mice brain revealed by detection of pathological alpha-synuclein by enhanced ELISA.</a> Acta Neuropathol. (Berl). 2, 29 (2014).</li><li>Osman, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+monoclonal+antibody+that+recognizes+a+potential+coeliac-toxic+repetitive+pentapeptide+epitope+in+gliadins).">A monoclonal antibody that recognizes a potential coeliac-toxic repetitive pentapeptide epitope in gliadins).</a> Eur J Gastroenterol Hepatol. 13, 1189-1193 (2001).</li><li>Pinheiro, J. C., Bates, D. M., Chambers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+5.">Ch. 5.</a> Mixed-Effects Models in S and S-PLUS. 5, 206-225 (2000).</li><li>Mougenot, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+monoclonal+antibody,+against+human+alpha-synuclein,+in+a+subpopulation+of+C57BL/6J+mice,+presenting+a+deletion+of+the+alpha-synuclein+locus.">Production of a monoclonal antibody, against human alpha-synuclein, in a subpopulation of C57BL/6J mice, presenting a deletion of the alpha-synuclein locus.</a> J Neruosci Meth. 192, 268-276 (2010).</li><li>Specht, C. G., Schoepfer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+the+alpha-synuclein+locus+in+a+subpopulation+of+C57BL/6J+inbred+mice.">Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice.</a> BMC Neurosci. 2, 11 (2001).</li><li>Lee, B. R., Matsuo, Y., Cashikar, A. G., Kamitani, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Ser129+phosphorylation+of+alpha-synuclein+in+melanoma+cells.">Role of Ser129 phosphorylation of alpha-synuclein in melanoma cells.</a> J Cell Sci. 126, 696-704 (2013).</li><li>Perrin, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope+mapping+and+specificity+of+the+anti-alpha-synuclein+monoclonal+antibody+Syn-1+in+mouse+brain+and+cultured+cell+lines.">Epitope mapping and specificity of the anti-alpha-synuclein monoclonal antibody Syn-1 in mouse brain and cultured cell lines.</a> Neurosci Lett. 349, 133-135 (2003).</li><li>Emmanouilidou, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+alpha-synuclein+secretion+in+mouse+and+human+brain+parenchyma.">Assessment of alpha-synuclein secretion in mouse and human brain parenchyma.</a> PLoS One. 6, e22225 (2011).</li><li>Mougenot, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+prion+strains+in+a+transgenic+mouse+models+overexpressing+human+A53T+mutated+alpha-synuclein.">Transmission of prion strains in a transgenic mouse models overexpressing human A53T mutated alpha-synuclein.</a> J Neuropathol Exp Neurol. 70, 377-385 (2011).</li><li>Foulds, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+longitudinal+study+on+alpha-synuclein+in+blood+plasma+as+a+biomarker+for+Parkinson's+disease.">A longitudinal study on alpha-synuclein in blood plasma as a biomarker for Parkinson's disease.</a> Sci Rep. 3, 2540 (2013).</li></ol>

The authors have no competing interests to disclose.

השימוש בELISA הודגם כדי לזהות באופן ספציפי αS D ישירות מhomogenates מוח עכבר במחלה במודל העכבר המהונדס M83. ואכן, זה יכול בקלות ELISA להבחין עכברי M83 חולים מעכברי M83 בריאים באמצעות homogenates המוח רק כל חיץ מלח הגבוה. השלבים הקריטיים ביותר לתו...

Most current methods for detecting disease-associated &#945;-synuclein (&#945;SD) in experimental models of Parkinson's disease (PD), such as immunohistochemistry or Western blot, are time-consuming and not quantitative. This neurodegenerative disease is characterized by alpha-synuclein aggregation mainly in the form of inclusions containing an aggregated form of the normally soluble presynaptic protein &#945;S1,2 (Lewy bodies and Lewy neurites). Normally only marginally phosphorylated, &#945;S is hyperphosphorylated at its serine 129 residue in these inclusions3 and can be monitored by antibodies specifically directed against Ser129 phosphorylated &#945;S, thus providing a reliable marker of the pathology. 
Recent research suggests that a &#8220;prion-like&#8221; mechanism could be involved in the propagation of &#945;S aggregation within the nervous system of an affected patient4,5. These studies reported the acceleration of a synucleinopathy by inoculating brain extracts containing &#945;SD into a transgenic mouse model (M83) expressing an A53T mutated human &#945;S protein associated with a severe motor impairment occurring as the mice age6. In the same manner, intra-cerebral inoculation of aggregated recombinant &#945;S in the same M83 mouse model confirmed the acceleration of aggregation5. The induction of deposits of phosphorylated &#945;S has also been reported after inoculation of C57Bl/6 wild-type mice with either fibrillar recombinant &#945;S or brain extracts from human DLB patients7,8. Sacino et al.9 recently pointed out that after injection of fibrillar human &#945;S, a widespread and progressive cerebral &#945;S inclusion formation could be induced in M83 mice, but not in E46K transgenic mice or non-transgenic mice in which induced &#945;S inclusions were transient, and mainly restricted to the site of injection. Recent studies on monkeys confirmed propagation of &#945;S aggregates after inoculation of PD-derived extracts in species closer to humans10.
The link between &#945;S alterations and Parkinson&#8217;s disease suggest that &#945;SD is a potential biomarker for Parkinson&#8217;s disease11. A recent study showed the detection of oligomeric soluble aggregates of &#945;-synuclein in human cerebro-spinal fluid (CSF) and plasma as a potential biomarker for Parkinson&#8217;s disease based on a conventional sandwich system ELISA using the same antibody to capture and detect &#945;S12. Based on the same method, multimeric proteins were recognized in biological samples, including the brain, because there are multiple copies of epitopes present in the assembled forms13. Very recently, pathological &#945;S in the CSF of patients with a proven Lewy body pathology was detected using both an ELISA kit with a highly specific antibody against &#945;SD (5G4) and an immunoprecipitation assay14. These methods could differentiate patients with PD/DLB from other types of dementia.
The &#8220;prion-like&#8221; propagation of &#945;S aggregation was further studied in transgenic mouse model M83 using an ELISA approach that was designed to specifically identify &#945;SD15. In this study, we report the detailed ELISA protocol used to quantitatively detect &#945;SD in sick mice (whether or not inoculated with &#945;SD from sick M83 mice) and more especially in the brain regions specifically targeted by the pathological process in this M83 transgenic mouse model4.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col span="2" />
	</colgroup>
	<tbody>
		<tr>
			<td>LB509&#160;</td>
			<td></td>
			<td>Abcam</td>
			<td>&#160;ab27766</td>
			<td>Detection antibody 1/2000</td>
		</tr>
		<tr>
			<td>AS11</td>
			<td></td>
			<td>Produced at Anses</td>
			<td></td>
			<td>Detection antibody 1/1000</td>
		</tr>
		<tr>
			<td>4D6&#160;</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>ab1903</td>
			<td>Detection antibody 1/2000</td>
		</tr>
		<tr>
			<td>PSer129&#160;</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>&#160;ab59264</td>
			<td>Detection antibody 1/3000</td>
		</tr>
		<tr>
			<td>PSer129&#160;EP1536Y</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>ab51253</td>
			<td>Detection antibody 1/1000</td>
		</tr>
		<tr>
			<td>syn514&#160;</td>
			<td></td>
			<td>Abcam&#160;</td>
			<td>&#160;ab24717</td>
			<td>Detection antibody 1/500</td>
		</tr>
		<tr>
			<td>clone 42</td>
			<td></td>
			<td>BD Biosciences&#160;</td>
			<td>610787</td>
			<td>Coating and detection antibody (1/2000)</td>
		</tr>
		<tr>
			<td>8A5&#160;</td>
			<td></td>
			<td>Provided by Dr. Anderson</td>
			<td></td>
			<td>Detection antibody 1/2000</td>
		</tr>
		<tr>
			<td>polyclonal anti-&#945;syn antibody</td>
			<td></td>
			<td>Millipore</td>
			<td>&#160;AB5038P</td>
			<td>Coating&#160; antibody</td>
		</tr>
		<tr>
			<td>Anti-mouse&#160; IgG HRP conjugate</td>
			<td></td>
			<td>Southern Biotech</td>
			<td>1010-05</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Anti-rabbit IgG HRP conjugate</td>
			<td></td>
			<td>Southern Biotech</td>
			<td>4010-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse&#160; IgG HRP conjugate</td>
			<td></td>
			<td>Dianova</td>
			<td>115-035-164</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="4">HS buffer</td>
			<td>Tris-HCl 50 mM&#160;</td>
			<td>Euromedex</td>
			<td>26-128-3094-B</td>
			<td rowspan="4">Adjust at pH 7.5 and keep at 4&#176;C</td>
		</tr>
		<tr>
			<td>NaCl 750 mM&#160;</td>
			<td>Euromedex</td>
			<td>1112-A</td>
		</tr>
		<tr>
			<td>EDTA 5 mM&#160;</td>
			<td>Euromedex</td>
			<td>EU0007-B</td>
		</tr>
		<tr>
			<td>DTT 1 mM&#160;</td>
			<td>Sigma</td>
			<td>43815</td>
		</tr>
		<tr>
			<td rowspan="4">PBS</td>
			<td>Na2HPO4 1 mM</td>
			<td>Euromedex</td>
			<td>1309</td>
			<td rowspan="4">Adjust at pH 7.5</td>
		</tr>
		<tr>
			<td>KH2PO4 1,5 mM</td>
			<td>Euromedex</td>
			<td>2018</td>
		</tr>
		<tr>
			<td>NaCl&#160; 137 mM</td>
			<td>Euromedex</td>
			<td>1112-A</td>
		</tr>
		<tr>
			<td>KCl 2,7 mM</td>
			<td>Euromedex</td>
			<td>P017</td>
		</tr>
		<tr>
			<td>Tween 20&#160;</td>
			<td></td>
			<td>Euromedex</td>
			<td>2001-C</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA&#160;</td>
			<td></td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT 1 mM</td>
			<td></td>
			<td>Sigma</td>
			<td>43815</td>
			<td>stock solution 100 mM, toxic</td>
		</tr>
		<tr>
			<td>1% phosphatase cocktail</td>
			<td></td>
			<td>Pierce</td>
			<td>78428</td>
			<td></td>
		</tr>
		<tr>
			<td>1% protease inhibitor cocktail</td>
			<td></td>
			<td>Roche</td>
			<td>04&#160;693&#160;132&#160;001</td>
			<td>50 X concentrated</td>
		</tr>
		<tr>
			<td>Microplate MaxiSorpTM</td>
			<td></td>
			<td>Thermo Scientific&#160;</td>
			<td>442404</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Tampon carbonate 50 mM pH 9.6&#160;</td>
			<td>&#160;Na2CO3, 10H2O&#160;</td>
			<td>Sigma</td>
			<td>71360</td>
			<td>2.86 g/L</td>
		</tr>
		<tr>
			<td>&#160;NaHCO3&#160;</td>
			<td>Merk</td>
			<td>6329</td>
			<td>3.36 g/L, pH9.6</td>
		</tr>
		<tr>
			<td>Superblock T20 PBS blocking buffer&#160;</td>
			<td></td>
			<td>Pierce</td>
			<td>E6423H</td>
			<td>10 X concentrated</td>
		</tr>
		<tr>
			<td>TMB&#160;</td>
			<td></td>
			<td>Sigma</td>
			<td>T0440</td>
			<td>Used for ELISA</td>
		</tr>
		<tr>
			<td>TMB&#160;</td>
			<td></td>
			<td>Analytik Jena AG</td>
			<td>847-0104200302</td>
			<td>Used for epitope mapping</td>
		</tr>
		<tr>
			<td>HCl 1N&#160;</td>
			<td></td>
			<td>Chimie plus</td>
			<td>40030</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribolyser</td>
			<td></td>
			<td>Thermo</td>
			<td>Fast prep FP120</td>
			<td>keep on ice at this step</td>
		</tr>
		<tr>
			<td>Grinding tubes</td>
			<td></td>
			<td>Biorad</td>
			<td>355-1197</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate washer</td>
			<td></td>
			<td>Tecan</td>
			<td>Columbus Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td></td>
			<td>Biorad</td>
			<td>Model 680</td>
			<td></td>
		</tr>
		<tr>
			<td>Low power magnifier&#160;</td>
			<td></td>
			<td>VWR</td>
			<td>630-1062</td>
			<td>X8 magnification</td>
		</tr>
		<tr>
			<td>Forceps Dumont#7</td>
			<td></td>
			<td>WPI</td>
			<td>14097</td>
			<td>For dissection steps</td>
		</tr>
		<tr>
			<td>Transfer pipette 1ml Samso</td>
			<td></td>
			<td>Samso</td>
			<td>043231</td>
			<td></td>
		</tr>
		<tr>
			<td>1,5 ml tubes</td>
			<td></td>
			<td>Dutscher</td>
			<td>033290</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of disease-associated &#945;-synuclein by enhanced elisa in the brain of transgenic mice overexpressing human a53t mutated &#945;-synuclein

בנוסף לשיטות מבוססות כמו כתם מערבי, יש צורך בשיטות חדשות לכמת במהירות ובקלות α-סינוקלאין הקשורים מחלה (αS D) במודלים ניסיוניים של synucleopathies. קו מהונדס עכבר (M83) αS A53T האנושי ופיתוח הפנוטיפ קליני דרמטי בין שמונה ו -22 חודשים של גיל, מאופיינים בתסמינים כוללים ירידה במשקל, השתטחות, וירידת ערך באופן ספונטני מוטורי קשה להביע מעל, שימש במחקר זה. עבור ניתוחים מולקולריים של αS D (αS הקשורים למחלות) בעכברים אלו, ELISA נועד לכמת במיוחד αS D בעכברים חולים. ניתוח של מערכת העצבים המרכזית במודל עכבר זה הראה הנוכחות של αS D בעיקר באזורים במוח הזנב וחוט השדרה. לא היו הבדלים בהתפלגות αS D בין תנאי ניסוי שונים שמוביל למחלה קלינית, כלומר, בuninoculated ובדרך כלל הזדקנות עכברים הטרנסגניים ובעכברים מחוסן עם תמציות מוח מעכברים חולים. זיהוי הספציפי של immunoreactivity D αS באמצעות נוגדן נגד Ser129 phosphorylated αS ידי ELISA בעצם מתואם עם שהושג על ידי כתם ואימונוהיסטוכימיה מערביים. באופן בלתי צפוי, תוצאות דומות נצפו עם כמה נוגדנים אחרים נגד חלק C-מסוף של αS. ההתפשטות של αS D, המצביעה על המעורבות של מנגנון &quot;כמו הפריון-&quot;, לכן יכולה להיות במעקב בקלות ולכמת במודל עכבר זה באמצעות גישת ELISA.

במחקר זה, ELISAs משמש לזיהוי ספציפי αS הקשורים מחלה (αS D) בhomogenates המוח נערכה בחיץ מלח גבוה מעכברי M83 חולים. באמצעות נוגדן ספציפי הכרת pSer129 αS (p = .0074), ELISA בקלות מבדיל עכברים זקנים, חולים (&gt; בת 8 חודשים) מ( 2-5 חודשים) הצעירים, עכברי M83 בריאים (איור 1). כמה נוגדנים אחרי...

Watch this Scientific Journal Video about Detection of Disease-associated Î±-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated Î±-synuclein at JoVE.com

Detection of Disease-associated &amp;#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &amp;#945;-synuclein

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

איתור של α-סינוקלאין קשורים למחלות על ידי ELISA משופר במוח של עכברים הטרנסגניים ביתר A53T אדם מוטצית α-סינוקלאין

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

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Research

JoVE Journal

Medicine

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

10.6K Views.  ANSES - French Agency for Food, Environmental and Occupational Health & Safety. An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated α-synuclein (αSD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated αS form or the C-terminal part of the protein.

Video: Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

השימוש בפיברובלסטים אנושיים ראשוניים לניטור פנוטיפים מיטוכונדריאלי בתחום מחלת פרקינסון

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

אינדוקציה של קרצינומה של תאי מעבר שלפוחית ​​השתן פולשנית בMUC1 עכברים הטרנסגניים חיסוניים אנושיים שלמים: מודל לפיתוח חיסוני

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

הפעלת יתר רעיל גירוי של המוח כMicroslices<em&gt; במבחנה</em&gt; דגם של שבץ

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows for positive pressure bulk flow to deliver greater quantities of therapeutics to an intracranial target than traditional drug delivery methods. The clinical utility of real time MRI guided CED (rCED) lies in the ability to accurately target, monitor therapy, and identify complications. With training, rCED is efficient and complications may be minimized. The agarose gel model of the brain provides an accessible tool for CED testing, research, and training. Simulated brain rCED allows practice of the mock surgery while also providing visual feedback of the infusion. Analysis of infusion allows for calculation of the distribution fraction (Vd/Vi) allowing the trainee to verify the similarity of the model as compared to human brain tissue. This article describes our agarose gel brain phantom and outlines important metrics during a CED infusion and analysis protocols while addressing common pitfalls faced during CED infusion for the treatment of neurological disease.

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. In order to prepare health care professionals for adoption of CED, accessible training models are needed. We describe the use of agarose gel as such a model of the human brain for testing, research, and training.

משלוח משופר הסעה מודרכת תמונה לתוך מודלים Agarose ג&#39;ל של המוח

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

טראומה מוחית חריפה בעכברים אחריו שני פוטונים הדמיה אורכית

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

נוהל כירורגי זעיר פולשנית של גרימת אוטם שריר הלב בעכברים

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

אינדוקציה של טרשת עורקים מואצת בעכברים: מודל "פגיעת חוט"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

במבחנה and In Vivo זיהוי של Mitophagy ב תאים אנושיים, C. Elegansועכברים

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

חקר In Vivo מטבוליזם הגלוקוז עכברים הניזונה דיאטה עתירת שומן באמצעות בדיקת הגלוקוז אוראלי סובלנות (OGTT) ובדיקה עמידות לאינסולין (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.