This work was supported by the Australian Research Council Centre of Excellence for Integrative Brain Function (ARC Centre Grant CE140100007), an NHMRC project grant (#1086643). Prof. George Paxinos is supported by a Senior Principal Research Fellow NHMRC grant (#1043626).

משפט ואתיקה: כל הנהלים הקשורים בנושאים בעלי חיים לעקוב אחר ההנחיות של טיפול בבעלי חיים ועדת האתיקה (ACEC) מאוניברסיטת ניו סאות &#39;ויילס (מספר ACEC שאושרה הינה 14 / 94a). 1. הכנה של חוט השדרה עכבר שקוף <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנת פתרון הידרוג&#39;ל קר קרח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנת פתרון paraformaldehyde 16% (PFA) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להוסיף 16 גרם אבקת paraformaldehyde לתוך 70 מ&quot;ל מים מזוקקים מחומם מראש (50-55 מעלות צלזיוס) ומערבבים על בוחש מגנטי מחוממת עד paraformaldehyde נמס. הערה: אל תאפשר הפתרון לחמם מעל 55 מעלות צלזיוס ולהיות מודעים לכך paraformaldehyde הוא רעיל. </li><li style=";text-align:right;direction:rtl"> מעבירים את הפתרון paraformaldehyde כדי גליל ולהוסיף מים מזוקקים עד 100 מ&quot;ל. מצננים את הפתרון paraformaldehyde באמצעות מקרר 4 ° C.. </li></ol></li><li style=";text-align:right;direction:rtl"> ממיסים 125 מ&quot;ג יוזם VA-044 ב 26.25 מ&quot;ל מים מזוקקים ו לקרר את הפתרון בתוך 4° מקרר C. </li><li style=";text-align:right;direction:rtl"> פתרון acrylamide מגניב 5 מ&quot;ל (40%), 1.25 מ&quot;ל פתרון Bis (2%) (זהירות: Bis ופתרונות acrylamide רעילים, עלול לגרום למומים גנטיים או סרטן), 5 מ&quot;ל 10x PBS, 12.5 מ&quot;ל 16% פתרון PFA, ואת פתרון יוזם VA-044 על הקרח. </li><li style=";text-align:right;direction:rtl"> מערבבים את כל הפתרונות שהוצעו על הקרח. הערה: הנפח הכולל הוא 50 מ&quot;ל. </li></ol></li><li style=";text-align:right;direction:rtl"> זלוף וגבייה של קורד עכבר השדרה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לטשטש עכבר עם זריקה intraperitoneal של קטמין (80 מ&quot;ג / ק&quot;ג) ו xyzaline (5 מ&quot;ג / ק&quot;ג) מדולל מלוחים 0.9% נורמלי. המינון לעכבר יכול להיות נמוך מזה של חולדות. </li><li style=";text-align:right;direction:rtl"> על משטח פלסטיק מתאים במנדף, לתקן את הגפיים של העכבר מהגוף (סרט דביק יעיל) פעם העכבר אינו מגיב קמצוץ כדי עורו של כף הרגל. </li><li style=";text-align:right;direction:rtl"> חותכים את העור העכבר מעל החזה ואחר כך בחזה העצם כדי לחשוף את הלב עם מספריים. </li><li style=";text-align:right;direction:rtl"> באמצעות משאבת peristaltic, עם מחט 25 G מצורף thסוף הדואר של צינורות, הכנס את המחט לתוך החדר השמאלי של הלב, לגזור אטריום הזכות ליצור נקודת יציאה בדם ולאחר מכן להתחיל כביסה עם 40 מ&quot;ל 0.9% מלח רגיל בשיעור של כ -10 מ&quot;ל / דקה. </li><li style=";text-align:right;direction:rtl"> כשיושלם, perfuse את העכבר עם 35 פתרון הידרוג&#39;ל קר מ&quot;ל קרח. הערה: perfuse במהירות של 10 מ&quot;ל / דקה, כדי למנוע נפיחות של רקמת העצבים. </li><li style=";text-align:right;direction:rtl"> לחתוך את העור על הגב עם להב ולאחר מכן להסיר את השרירים ליד החוליות. לאחר חיתוך קשתות בחוליות בצד אחד מרפרף אותם לצד השני, לקחת חוט השדרה מתוך עמוד השדרה עם מספריים בסדר. </li></ol></li><li style=";text-align:right;direction:rtl"> ניקוי חוט השדרה עכבר <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את חוט השדרה 15 מ&quot;ל של תמיסת הידרוג&#39;ל לילה ב 4 ° C.. </li><li style=";text-align:right;direction:rtl"> הסר 10 מ&quot;ל של פתרון הידרוג&#39;ל ויוצקים את שאר הפתרון עם מגזרים חוט השדרה אל צינור 5 מ&quot;ל. מלאו את צינור 5 מ&quot;ל עם הפתרון הידרוג&#39;ל עד שהוא לגמרי מלא. </li><li style=";text-align:right;direction:rtl"> למתוח חתיכה קטנה של parafilm על גבי הצינור 5 מ&quot;ל ולאחר מכן לעטוף parafilm סביב הצוואר של הצינור. הערה: ודא המגעים parafilm הפתרון הידרוג&#39;ל ואין בועות בין פתרון הידרוג&#39;ל ואת parafilm. </li><li style=";text-align:right;direction:rtl"> שים את הצינור 5 מ&quot;ל ב 37 &#160; מעלות צלזיוס בתנור למשך הלילה. שימו הפתרון הידרוג&#39;ל עד שזה הופך ג&#39;ל. </li><li style=";text-align:right;direction:rtl"> קח את הצינור 5 מ&quot;ל מהתנור ולהסיר את הג&#39;ל מהצינור עם ברגים (כגון קצה פיפטה גרפו 1 מ&quot;ל). הסר את הג&#39;ל רקמה חוט השדרה על ידי דבק הרקמות הגסות הג&#39;ל ולאחר מכן לקחת את הרקמה הגסה משם (הג&#39;ל ידבק הרקמה ולכן יוסר על ידי הרקמה). </li><li style=";text-align:right;direction:rtl"> לאחר הסרת הג&#39;ל רקמת חוט השדרה, לשטוף את חוט השדרה 4 פעמים עם PBS 1x (pH 7.4) במשך 24 שעות על שייקר. </li><li style=";text-align:right;direction:rtl"> הכן את הפתרון סליקה מעוקב ידי המסת אוריאה 3.85 גרם ו 3.85 גרם N, N, N &#39;, N&#39;-tetrakis (2-Hydroxypropyl) ethylenediam<html> ine ב 5.38 מ&quot;ל מים מזוקקים. הערה: בוחש חם משמש. להוסיף פוליאתילן גליקול 2.31 גרם האתר מונו-p-isooctylphenyl / טריטון X-100 הפתרון פעם ברור מתקרר לטמפרטורת החדר. הערה: 10 גרם של שלושה כימיקלים אלה הם לעכבר אחד. </li><li style=";text-align:right;direction:rtl"> חותכים את חוט השדרה עכבר coronally לתוך 2-3 מקטעים ארוכים מ&quot;מ עם סכין גילוח ולשים אותם לתוך 5 מ&quot;ל של הפתרון סליקה מעוקב לעיל. מניחים על שייקר בתנור ב 37 &#160; ° צלזיוס במשך 3 ימים. </li><li style=";text-align:right;direction:rtl"> שלושה ימים לאחר מכן, לשנות את הפתרון וסליקה אחד טרי. הערה: הפתרון מעל מוכן לפני השימוש. </li><li style=";text-align:right;direction:rtl"> שניים עד שלושה ימים לאחר מכן לאחר רענון פתרון הסליקה מעוקב, לבדוק את השקיפות של הרקמה כנגד נייר עם גופן 8 אותיות. אם הוא שקוף, האותיות ניתן לראות דרך רקמת פינה. הסר את פתרון הסליקה ולהוסיף 4 מיליליטר של PBST (0.1% Triton-X100) כדי לשטוף את הרקמה 4 פעמים ביום (כל 6 שעות). </li></ol></li></ol>ילדה = &quot;jove_title&quot;&gt; 2. immunofluorescence מכתים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דגירת מגזרי חוט שדרת פתרון הנוגדן הראשוני (-סרוטונין אנטי, וגדל ארנב, מדולל 1: 100 ב PBST) במשך 3 ימים על שייקר בתנור 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> הסר את הפתרון נוגדן ולהוסיף 4 מ&quot;ל של PBST (0.1% Triton-X100) לשטוף את פלחי חוט השדרה 4 פעמים ביום (כל 6 שעות) בתנור 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> הסר את הפתרון PBST ולהוסיף הפתרון נוגדנים משני (594 IgG מצומדות עז נגד ארנב, מדולל 1: 100 ב PBST) כדי לדגור הרקמה במשך 3 ימים על שייקר בתנור 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> הסר את פתרון נוגדנים משני ולהוסיף 4 מיליליטר של PBST לשטוף מגזרי חוט השדרה 4 פעמים ביום (כל 6 שעות) על שייקר בתנור 37 מעלות צלזיוס. למחרת הרקמה מוכנה הדמיה. </li></ol> הדמיה 3. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הסר את הפתרון PBST ולהוסיף 4 מ&quot;ל של גליצרול 85% על מנת להפוך את מקדם השבירה של הרקמה אפילו. </li><li&gt; בדקי את הבהירות של הרקמה. לאחר ברור נע בין 1 hr, לשים את הרקמה על גבי זכוכית נושאת גדולה, דקה אשר מתאימה את המסגרת החזקה של מיקרוסקופ פלואורסצנטי רב-צילום. </li><li style=";text-align:right;direction:rtl"> לשים כמה טיפות של גליצרול 85% ליד רקמת חוט השדרה ו coverslip אותו עם כוס coverslip 22 x 50 מ&quot;מ 2. הערה: הכיוון של חוט השדרה מחליט כיצד התמונה נראית. </li><li style=";text-align:right;direction:rtl"> שים את השקופית זכוכית על מסגרת ההחזקה של מיקרוסקופ פלואורסצנטי רב פוטון ולהעביר את הרקמה לתוך מסלול האור. </li><li style=";text-align:right;direction:rtl"> בחר את ננומטר לייזר נאון הליום קו 594 ולהתאים את עוצמת הלייזר לרמה אופטימלית על ידי בדיקת הבהירות של איתות חיובי בתמונה חיה. </li><li style=";text-align:right;direction:rtl"> בחר את אזור הסריקה של רקמת חוט השדרה באמצעות המטרה 20X (במים, NA 0.7) ולהכין ליצור Z- מחסנית, ואת העומק של כל שלב מוגדר 3 מיקרומטר. </li><li style=";text-align:right;direction:rtl"> סרוק את הרקמה מלמעלה לתחתית הערימה-z תחת objec 20Xמגני אוזניים ואז המטרה 63X (בשמן, NA 1.4) (גודל הצעד היה 1 מיקרומטר), בהתאמה. לשחזר קטעי וידאו 3D באמצעות תוכנת 3D 28. </li></ol>

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A., Bhansali, P., Jurgens, C., Guido, W., Mason, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ClearT:+a+detergent-+and+solvent-free+clearing+method+for+neuronal+and+non-neuronal+tissue.">ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue.</a> Development. 140 (6), 1364-1368 (2013).</li><li>Ert&#252;rk, A., Bradke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+of+entire+organs+by+3-dimensional+imaging+of+solvent+cleared+organs+(3DISCO).">High-resolution imaging of entire organs by 3-dimensional imaging of solvent cleared organs (3DISCO).</a> Exp Neurol. 242, 57-64 (2013).</li><li>Kim, S. Y., Chung, K., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+microscopy+mapping+of+connections+in+the+intact+brain.">Light microscopy mapping of connections in the intact brain.</a> Trends Cogn Sci. 17 (12), 596-599 (2013).</li><li>Spence, R. 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=Bringing+CLARITY+to+gray+matter+atrophy.">Bringing CLARITY to gray matter atrophy.</a> NeuroImage. 101, 625-632 (2014).</li><li>Ando, K., 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=Inside+Alzheimer+brain+with+CLARITY:+senile+plaques,+neurofibrillary+tangles+and+axons+in+3-D.">Inside Alzheimer brain with CLARITY: senile plaques, neurofibrillary tangles and axons in 3-D.</a> Acta Neuropathol. 128 (3), 457-459 (2014).</li><li>Zhang, H., Rinaman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+CLARITY+for+visualizing+immunofluorescence+labeling+in+the+developing+rat+brain.">Simplified CLARITY for visualizing immunofluorescence labeling in the developing rat brain.</a> Brain Struct Funct. , (2015).</li><li>Lee, H., Park, J. H., Seo, I., Park, S. H., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+application+of+the+electrophoretic+tissue+clearing+technology,+CLARITY,+to+intact+solid+organs+including+brain,+pancreas,+liver,+kidney,+lung,+and+intestine.">Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine.</a> BMC Dev Biol. 14, 781 (2015).</li><li>Yang, B., 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=Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158 (4), 945-958 (2014).</li><li>Rosset, A., Spadola, L., Ratib, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OsiriX:+an+open-source+software+for+navigating+in+multidimensional+DICOM+images.">OsiriX: an open-source software for navigating in multidimensional DICOM images.</a> J Digit Imaging. 17, 205-216 (2004).</li></ol>

The authors have nothing to disclose.

הפרוטוקול המתואר מראה כיצד סיבי serotonergic תמונה בחוט השדרה עכבר עם CLARITY בשילוב וטכניקות מעוקב. הוא מציג תהליך סליקה מהר לעומת פרוטוקול הסליקה הפסיבי שפותח על ידי Cheung et al. 14 והתומר et al. 15 ומאפשר רקמת חוט השדרה כדי להיות נתמך היטב על ידי הידרוג&#39;ל במהל...

Supraspinal projections are responsible for the modulation of diverse behaviors such as pain perception. One of the projections carrying nociceptive information contains serotoninergic fibers, which originate from the hindbrain raphe and adjacent reticular nuclei1,2. Physiological and pharmacological studies have demonstrated an increased release of serotonin in the dorsal horn of the spinal cord after electrical stimulation of the raphe nuclei in the hindbrain3-5. In the rat and opossum, serotonergic raphespinal fibers have dense terminals, not only in the dorsal horn6-8, but also in the intermediate zone7,9,10, the ventral horn7,11, and even lamina 1012,13. There are no similar studies in the mouse. The present study aimed to map the termination pattern of serotonergic fibers arising from the hindbrain raphe nuclei and their adjacent reticular nuclei in the mouse spinal cord using the recently published CLARITY14 method and its modification - CUBIC15. 
Conventional fluorescence or peroxidase immunohistochemistry of the spinal cord clearly shows the distribution of serotonergic fibers in the gray matter of the spinal cord in 30-40 &#181;m thick cross-sections. However, this approach does not show the continuity of the serotonergic fiber tracts in the white matter and their collaterals in the gray matter. Although the 3D reconstruction of histological sections has advanced our knowledge of fiber tracts, it remains a challenge for histologists and anatomists to follow a single tract due to small distortions in the tissue caused by cutting. To circumvent this obstacle a number of researchers have developed various protocols for making the whole tissue structure transparent, and collecting an image of unaltered tissue in a single video file17-21. So far, the clear, lipid-exchanged, acrylamide-hybridized rigid, imaging/ immunostaining compatible, tissue hydrogel (CLARITY) technique, developed by Deisseroth's group14,15, as well as CUBIC, developed by Susaki et al16 are the most successful. Since the publication of the protocols, many researchers have started using these techniques to investigate various aspects of biological tissues, including, not only the brain22-25, but also the heart, kidneys, intestine, and the lungs26,27. 
By fixing the mouse spinal cord with the hydrogel solution (CLARITY) and clearing with the CUBIC reagents (which is a much faster method than that described by the original CLARITY protocol14,15), a spinal cord tissue block of 2-3 mm long was cleared within two weeks and immunofluorescence staining for serotonin completed in eight days. With just a combination of chemical agents, conventional immunohistochemistry can be used to create an image of individual fiber tracts in a 3D video file in approximately one month.

<table><tbody><tr><td>Photoinitiator VA044</td><td>Wako</td><td>va-044/225-02111</td><td>http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td></tr><tr><td>40% acrylamide solution</td><td>Bio Rad</td><td>161-0140</td><td>http://www.bio-rad.com/en-au/sku/161-0140-40-acrylamide-solution</td></tr><tr><td>2% Bis Solution</td><td>Bio Rad</td><td>161-0142</td><td>http://www.bio-rad.com/en-au/sku/161-0142-2-bis-solution?parentCategoryGUID=5e7a4f31-&lt;br/&gt;879c-4d63-ba0b-82556a0ccf1d</td></tr><tr><td>paraformaldehyde</td><td>Sigma</td><td>158127</td><td>http://www.sigmaaldrich.com/catalog/product/sial/158127?lang=en&amp;amp;region=AU</td></tr><tr><td>urea</td><td>Merck Millipore</td><td>66612</td><td>http://www.merckmillipore.com/AU/en/product/Urea---CAS-57-13-6---Calbiochem,EMD_BIO-66612</td></tr><tr><td>N,N,N&amp;rsquo;,N&amp;rsquo;-tetrakis (2-hydroxypropyl) ethylenediamine</td><td>Merck Millipore</td><td>821940</td><td>http://www.merckmillipore.com/AU/en/product/Ethylenediamine-N,N,N&#039;,N&#039;-tetra-2-propanol,MDA_CHEM-821940</td></tr><tr><td>Triton-X 100</td><td>Merck Millipore</td><td>648462</td><td>http://www.merckmillipore.com/AU/en/product/TRITON&amp;reg;-X-100-Detergent---CAS-9002-93-1---Calbiochem,EMD_BIO-648462</td></tr><tr><td>sucrose</td><td>Sigma</td><td>S0389</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/s0389?lang=en&amp;amp;region=AU</td></tr><tr><td>serotonin antibody</td><td>Merck Millipore</td><td>AB938</td><td>http://www.merckmillipore.com/AU/en/product/Anti-Serotonin-Antibody,MM_NF-AB938</td></tr><tr><td>goat anti rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&amp;reg; 594 conjugate</td><td>Life Technologies</td><td>&amp;nbsp;A-11012</td><td>https://www.lifetechnologies.com/order/genome-database/antibody/Rabbit-IgG-H-L-Secondary-Antibody-Polyclonal/A-11012</td></tr><tr><td>multi-photon microscope</td><td>Leica</td><td>Leica TCS SP5 MP STED</td><td>http://www.leica-microsystems.com/products/confocal-microscopes/details/product/leica-tcs-sp5-mp/</td></tr></tbody></table>

imaging serotonergic fibers in the mouse spinal cord using the clarity/cubic technique

סיבי יורד ארוך אל חוט השדרה חיוני תנועה, תפיסת כאב, והתנהגויות אחרות. דפוס סיום סיבים בחוט השדרה של רוב מערכות סיבים אלה לא נחקר ביסודיות בכל מינים. סיבי serotonergic, אשר פרויקט אל חוט השדרה, נחקרו בחולדות ואופוסומים על סעיפים היסטולוגית המשמעות התפקודית שלהם כבר להסיק על סמך דפוס סיום סיבים שלהם בחוט השדרה. עם התפתחות CLARITY וטכניקות מעוקב, אפשר לחקור מערכת סיבים זה והפצתו בחוט השדרה, אשר צפוי לחשוף תכונות שלא היו ידועים קודם של מסלולים supraspinal serotonergic. כאן, אנו מספקים פרוטוקול מפורט הדמיה הסיבים serotonergic בחוט השדרה העכבר באמצעות CLARITY בשילוב וטכניקות מעוקב. השיטה כרוכה זלוף של עכבר עם פתרון הידרוג&#39;ל והבהרה של הרקמה עם COMBINation סליקת ריאגנטים. רקמת חוט שדרה נוקתה למשך קצת פחות משבועות, ואת מכתים immunofluorescent עוקב נגד סרוטונין הושלם בתוך פחות מעשרה ימים. עם מיקרוסקופ פלואורסצנטי רב פוטון, הרקמה נסרקה תמונת 3D שוחזרה באמצעות תוכנת Osirix.

סעיף זה מציג תוצאות מכתים נוגדן סרוטונין במוח שדרת העכבר השקוף באמצעות שילוב של CLARITY ופרוטוקולים מעוקבים. אנו מראים כי סיבי serotonergic נוכח בכל הרבדים של חוט השדרה עם דומיננטיות בחלק הגחוני של צופר הגחון (איור 1, ראו גם וידאו 1). רקמת הבי...

Watch this Scientific Journal Video about Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique at JoVE.com

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

הדמית סיבי serotonergic בחוט שדרת עכבר שימוש CLARITY / טכניקה מעוקבת

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

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Research

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Neuroscience

10.5K Views.  Neuroscience Research Australia. Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Video: Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

בתחום ההדמיה vivo של חוט השדרה בעזרת עכבר דו פוטון מיקרוסקופית

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.

To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen.

הדמיה כחכחה מערכות ביולוגיות שלמות ברמה תאית על ידי 3DISCO

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

נוהל השתלה קאמרי בעמוד השדרה לאורך<em&gt; In vivo</em&gt; הדמיה של כבל עכבר השדרה

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

הדמיה שני פוטונים של Dynamics הסלולרי בחוט השדרה העכבר

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

סליקה אופטית של מערכת העצבים המרכזית עכבר שימוש CLARITY הפסיבי

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 &#181;m (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY&#160;can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

חיסון של רטינות הר שלם עם שיטת ניקוי רקמת בהירות

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

הדמיה וכימות של דנדריטים עצביים שלמים באמצעות ניקוי רקמות CLARITY

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

שיטת ניקוי רקמות להדמיה עצבית מסולמות מזוסקופיים למיקרוסקופיים

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments.
These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment.
In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

הקמת מודל פגיעה בעמוד השדרה של חבלה בעכבר המבוסס על טכניקה זעיר פולשנית

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...

Imaging the Gut with "CLARITY"

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving acrylamide embedding to delipidate tissue (without sectioning) and to preserve the 3-D tissue structure for immunostaining. The technique is highly relevant in imaging the dynamic gut environment where different cell types interact during homeostasis and disease states. This method optimized for the mouse gut is described here, which helps to trace cell types like epithelia, enteroendocrine, neurons, glia, and the neuronal projections into the epithelia or enteroendocrine cells that mediate microbial sensing or nutrient chemo sensing respectively. The gut tissue (1-1.5 cm) is fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 &#176;C overnight on day 1. On day 2, PFA is discarded, and the tissue is washed thrice with PBS. The tissue is hydrogel embedded to preserve its integrity by incubation in 4% hydrogel (acrylamide) solution in PBS (diluted from 30% ProtoGel) overnight at 4 &#176;C. On day 3, the tissue-hydrogel solution is incubated at 37&#160;&#176;C for 1 h to allow hydrogel polymerization. Tissue is then washed thrice gently with PBS to remove excess hydrogel. The subsequent step of delipidation (clearing) involves tissue incubation in sodium dodecyl sulfate (8% SDS in PBS) at 37 &#176;C for 2 days (days 4 &#38; 5) on a shaker at room temperature (RT). On day 6, the cleared tissue is thoroughly washed with PBS to remove SDS. Tissue can be immunostained by incubation in primary antibodies (diluted in 0.5% normal donkey serum in PBS containing 0.3% Triton X-100), overnight at 4&#176;C, and subsequent incubation in appropriate secondary Alexa Fluor antibodies for 1.5 h at RT, and nuclear staining with DAPI (1: 10000). The tissue is transferred onto a clean glass slide and mounted using VectaShield for confocal imaging.

<meta charset="utf8"></head><body>הדמיית המעיים ב"בהירות"

We describe the protocol for passive CLARITY (PACT) staining of mouse intestine to enable visualization of subepithelial tissues, including neurons, glia and enteroendocrine cells (EEC) without tissue sectioning. The protocol involves hydrogel embedding of formaldehyde-fixed tissue, and subsequent delipidation using an anionic detergent to "clear" the tissue.

הדמיית המעיים ב"בהירות"

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving ...