עבודה זו מומנה על ידי המרכז לטכנולוגיה ותקשורת עצבית (CNCT), מרכז P41 משאבים הממומן על ידי המכון הלאומי ביו הדמיה Bioengineering (NIBIB, P41 EB002030) ונתמך על ידי המכונים הלאומיים לבריאות (NIH).

<ol>
	<li>Cinar, O., Semiz, O., Can, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microscopic+survey+on+the+efficiency+of+well-known+routine+chemical+fixatives+on+cryosections.">A microscopic survey on the efficiency of well-known routine chemical fixatives on cryosections.</a> Acta. Histochem. 108, 487-496 (2006).</li><li>Fritz, M., Rinaldi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+pressure+measurement+with+the+tail-cuff+method+in+Wistar+and+spontaneously+hypertensive+rats:+Influence+of+adrenergic-+and+nitric+oxide-mediated+vasomotion.">Blood pressure measurement with the tail-cuff method in Wistar and spontaneously hypertensive rats: Influence of adrenergic- and nitric oxide-mediated vasomotion.</a> J. Pharmacol. Toxicol. Methods. 58, 215-221 (2008).</li><li>Ikeda, K., Nara, Y., Yamorii, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+systolic+and+mean+blood+pressure+determination+by+anew+tail+cuff+method+in+spontaneously+hypertensive+rats.">Indirect systolic and mean blood pressure determination by anew tail cuff method in spontaneously hypertensive rats.</a> Laboratory Animals. 25, 26-29 (1991).</li><li>Jacobowitz, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+discrete+fresh+regions+of+rat+brain.">Removal of discrete fresh regions of rat brain.</a> Brain Res. 80, 111-115 (1974).</li><li>Jonkers, B. W., Sterk, J. C., Wouterlood, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcardial+perfusion+fixation+of+the+CNS+by+means+of+a+compressed-air-driven+device.">Transcardial perfusion fixation of the CNS by means of a compressed-air-driven device.</a> Journal of Neurosci. Meth. 12, 141-149 (1984).</li><li>Jung-Hwa, T. a. o. -. C. h. e. n. g., Gallant, J., Brightman, P. E., Dosemeci, M. W., A, ., Reese, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+at+the+synapse+after+delayed+perfusion+fixation+in+different+regions+of+the+mouse+brain.">Structural changes at the synapse after delayed perfusion fixation in different regions of the mouse brain.</a> J. Comp. Neurol. 501, 731-740 (2007).</li><li>Kasukurthi, R., Brenner, M. J., Morre, A. M., Moradzadeh, A., Wilson, Z. R., Santosa, K. B., Mackinnon, S. E., Hunter, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcardial+perfusion+versus+immersion+fixation+for+assessment+of+peripheral+nerve+regeneration.">Transcardial perfusion versus immersion fixation for assessment of peripheral nerve regeneration.</a> J. Neurosci. Meth. 184, 303-309 (2009).</li><li>Lamberts, R., Goldsmith, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation,+fine+structure,+and+immunostaining+for+neuropeptides:+perfusion+versus+immersion+of+the+neuroendocrine+hypothalamus.">Fixation, fine structure, and immunostaining for neuropeptides: perfusion versus immersion of the neuroendocrine hypothalamus.</a> J. Histochem. Cytochem. 34, 389-398 (1986).</li><li>Ramos-Vara, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+Aspects+of+Immunohistochemistry.">Technical Aspects of Immunohistochemistry.</a> Vet. Pathol. 42, 405-426 (2005).</li><li>Shi, Z. R., Itzkowitz, S. H., Kim, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+three+immunoperoxidase+techniques+for+antigen+detection+in+colorectal+carcinoma+tissues.">A comparison of three immunoperoxidase techniques for antigen detection in colorectal carcinoma tissues.</a> J. Histochem. Cytochem. 36, 317-322 (1988).</li><li>Walker, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Vertebrate dissection. , (1980).</li><li>Zwienenberg, M., Gong, Q., Lee, L. L., Berman, R. F., Lyeth, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J.+Neurotrauma.">J. Neurotrauma.</a> Monitoring in the Rat: Comparison of Monitoring in the Ventricle, Brain Parenchyma, and Cisterna Magna. 16, 1095-1102 (1999).</li></ol>

החוקרים אין לי מה לחשוף.

הערות נוספות לניתוח מוצלח: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחר הסרעפת פרוץ, להקדים במהירות היפוקסיה ו hypercapnia תיזום שינויים פיזיולוגיים בלתי הפיכים שעלולים הרודפים את הניתוח שלאחר מכן. </li><li style=";text-align:right;direction:rtl"> כאשר המחט מוחדרת והידק במקום, הלחץ השיורי ידחוף הדם בבסיס המחט (פלאשבק). למעשה, בעלי חיים גדולים יותר עשויים במרץ לגרש דם. זה קריטי, כי דם לפחות להגיע אל הבסיס של המחט, כדי למנוע החדרת בועות אוויר אשר מכשול בפני להשלים זלוף. אם אין דם הוא ציין בבסיס המחט, הוצא את המחט ולמלא עם חיץ על ידי הצמדתו נמל יציאה של המתקן זלוף. לאחר חיץ הופעל באמצעות עצה, המחט אפשר לרחם אל לב. </li><li style=";text-align:right;direction:rtl"> מיקום נכון של המחט בתוך אבי העורקים הוא קריטי, זה לא צריך להגיע עד קשת אבי העורקים. </li><li style=";text-align:right;direction:rtl"> אם perfusions רבים מתקיימים אין צורך לאo הגדרת מההתחלה כל פעם. המשתמש צריך להיות נפח ראוי למאגר וגם מקבע בבקבוק אחד (200mls בעלי חיים / פתרון). הבקבוקים ניתן ומילא אם יש צורך. ההיבט החשוב ביותר שיש להביא בחשבון הוא בפלאשינג את הקו המחבר בין זלוף ההתקנה לבעל החיים (שסתום התפוקה). לאחר השלמת זלוף, קו זה יהיה paraformaldehyde 4% בה. הסר את צינורות מהבקבוק חיץ ולהשתמש מזרק 50 מ&quot;ל מלא מים כדי לשטוף את מקבע. ודא שסתום היציאה ממוקם הגביע בזבוז אוסף לסילוק נאות של paraformaldehyde. חזור על כך שלוש פעמים. מילוי עם המאגר באמצעות אותה שיטה שמוצג 2.3. </li><li style=";text-align:right;direction:rtl"> שיטה זו של קיבעון ניתנת להתאמה מאוד את היכולת לשנות גם חיץ fixatives מקבע אחרים (כגון אלו המשמשים EM) בהתאם לדרישות של הניסוי. </li><li style=";text-align:right;direction:rtl"> המערכת הוא גם משמש לעתים קרובות למיצוי של רקמת חיים. לשם כך1 תהליך פשוט משתמש מסירת חיץ בלבד (בקבוק חיץ תוך עדיין מחוברת המערכת כולה מונחת בתוך דלי קרח בקבוק מקבע הוא כמו תוכנית ההתקנה ריק אבל סגור). זה מבטיח זלוף חיץ קר בלחץ האופטימלי. מכאן שהמערכת יכולה להיות מותאם עירוי של צבעים ניתן לתקן או על ידי הוספת צבע ישירות למאגר (אם הכמות היא לא בעיה) או להשתמש במזרק עם כמות מינימלית של חיץ / פתרון צבע בין למאגר - מקבע צעד. </li><li style=";text-align:right;direction:rtl"> במקרה של יציקת כלי דם, ניטור לחץ צעד חיץ חיוני עם זאת בשל צמיגות וגיבוש אופי הפתרון הליהוק או מזרק 50 מ&quot;ל יש להשתמש למסירה ישירה או משנית מצורף הפנויה למנגנון (שסתום בצינור והחזקה מיכל) עשה להתממשק עם המערכת הנוכחית לשמש. הליהוק של כלי הדם במעבדות שלנו שקדמה עיצוב וייצור של מכשירים זלוף. לכן אנחנו לא יכולים רחובאוכלים כי יש למדוד את הלחץ תהיה מדידה מדויקת באתר המסירה. המספרים אולי צריך להיות מותאם על מנת להשיג ההפצה המוצלחת של נוזלים צמיגה. </li></ol>

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> ציוד </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td></td><td></td><td></td><td> הרדמה: </td></tr><tr><td> קטמין / xylazine תערובת (הרדמה יכולה להשתנות לפי המעבדה / מוסד) </td><td> Ketaset </td><td> NDC 0856-2013-01 </td><td> 10 מ&quot;ל בבקבוקון </td></tr><tr><td></td><td></td><td></td><td> ניתוח: </td></tr><tr><td> קצה המחט, GA 27 x 1.25 &quot; </td><td> אמצעי שירותים </td><td> 25251 </td><td></td></tr><tr><td> גדולים קהים / בוטה מספריים מעוגלות (~~~HEAD=NNS 14.5 ס&quot;מ) </td><td> מדע כלים עדינים </td><td> 14519-14 </td><td></td></tr><tr><td> ישר איריס מספריים </td><td> מדע כלים עדינים </td><td> 14058-11 </td><td></td></tr><tr><td&gt; תקן פינצטה </td><td> מדע כלים עדינים </td><td> 11027-12 </td><td></td></tr><tr><td> זוג יפה (גרפה) פינצטה </td><td> מדע כלים עדינים </td><td> 11050-10 </td><td></td></tr><tr><td> 1 hemostat מלקחיים גדולים - מעוגלים או ישרים (~~~HEAD=NNS 19 ס&quot;מ) </td><td> surgicaltools.com </td><td> 17.21.51 </td><td></td></tr><tr><td> 2 hemostat מלקחיים רגילים - ישר משוננים (14 ס&quot;מ) </td><td> מדע כלים עדינים </td><td> 13013-14 </td><td></td></tr><tr><td> 1 hemostat שונה (עם חור בקוטר 15 הגיש את קצה) </td><td> מדע כלים עדינים </td><td> 13013-14 </td><td></td></tr><tr><td> 15-מחט מד בוטה או זית שקצהו (מחט זלוף) </td><td> Fisnar </td><td> 5601137 </td><td></td></tr><tr><td></td><td></td><td></td><td> זלוף: </td></tr><tr><td> HyPerfusion מערכת או שווה ערך &lt;/ Td&gt; <td></td><td></td><td></td></tr><tr><td> פוספט pH שנאגרו מלוחים, 7.4 </td><td></td><td></td><td></td></tr><tr><td> Paraformaldehyde 4% במאגר M פוספט 0.1, pH 7.4 </td><td></td><td></td><td></td></tr><tr><td> זכוכית שטוחה או מגש פלסטיק, כ 10 &quot;x 10&quot; </td><td></td><td></td><td></td></tr><tr><td> קרח כתוש </td><td></td><td></td><td></td></tr><tr><td> מי האמבט (37 ° C) </td><td></td><td></td><td></td></tr><tr><td> שעון עצר </td><td></td><td></td><td></td></tr><tr><td> 50 מ&quot;ל במזרק </td><td> Medline תעשיות </td><td> NPMJD50LZ </td><td></td></tr><tr><td></td><td></td><td></td><td> דיסקציה: </td></tr><tr><td> אבאIR של תקן מספריים חדים ישרים / קהה (~~~HEAD=NNS 12 ס&quot;מ) </td><td> מדע כלים עדינים </td><td> 14054-13 </td><td></td></tr><tr><td> Rongeurs מעוגלים או ישרים בינוני (14-16 ס&quot;מ) או הסרת עצם הגולגולת צבת </td><td> מדע כלים עדינים </td><td> 16020-14 </td><td></td></tr><tr><td> ישר איריס מספריים (~~~HEAD=NNS 9 ס&quot;מ) </td><td> מדע כלים עדינים </td><td> 14058-11 </td><td></td></tr><tr><td> מיקרו מרית (כפול 2 &quot;קצוות שטוחים, אחד מעוגל, אחת קוני ל 1/8&quot;) </td><td> מדע כלים עדינים </td><td> 10091-12 </td><td></td></tr><tr><td></td><td></td><td></td><td> לאחר קיבוע &amp; Storage: </td></tr><tr><td> 50 מ&quot;ל בקבוקון זכוכית </td><td></td><td></td><td></td></tr><tr><td> 40 מ&quot;ל HEPES שנאגרו הנקס פתרון (HBHS) עם אזיד הנתרן (90mg / L) </td><td></td><td></td><td></td></tr></tbody&gt; </table>

whole animal perfusion fixation for rodents

המטרה של קיבעון היא במהירות אחידה לשמר רקמות במצב כמו חיים. תוך שימת רקמות ישירות בעבודות מקבע היטב חתיכות קטנות של רקמות, דגימות גדולות יותר כמו המוח ללא שינוי מהווה בעיה עבור קיבעון טבילה משום מקבע לא מגיע בכל אזורי הרקמה באותו קצב 5,7. לעתים קרובות, שינויים בתגובה היפוקסיה להתחיל לפני רקמה ניתן לשמר 12. היתרון של המרוססת מקבע ישירות דרך מערכת הדם היא כימית יכול להגיע במהירות לכל פינה של האורגניזם באמצעות רשת כלי דם טבעי. על מנת לנצל את מחזור הדם בצורה היעילה ביותר, יש להקפיד להתאים לחצים פיזיולוגיים 3. חשוב לציין כי לחצים פיזיולוגיים תלויים המינים בשימוש. טכניקות קיבוע זלוף משתנים בהתאם רקמות להיות קבוע וכיצד רקמות יעובדו קיבעון הבאה. זה וידאואנו מתארים בעלות נמוכה, בהליך מהיר, קיבעון מבוקר ואחיד באמצעות paraformaldehyde 4% perfused דרך מערכת כלי הדם: דרך הלב של חולדה כדי לקבל את השמירה הטובה ביותר של המוח עבור אימונוהיסטוכימיה. היתרון העיקרי של שיטה זו (לעומת כוח הכבידה-fed מערכות) היא מערכת הדם הוא מנוצל בצורה היעילה ביותר.

Watch this Scientific Journal Video about Whole Animal Perfusion Fixation for Rodents at JoVE.com

Whole Animal Perfusion Fixation for Rodents

כאן אנו מתארים בעלות נמוכה, בהליך מהיר, קיבעון מבוקר ואחיד באמצעות paraformaldehyde 4% perfused דרך מערכת כלי הדם: דרך הלב של חולדה כדי לקבל את השמירה הטובה ביותר של המוח.

בעלי חיים שלמה זלוף קיבוע של מכרסמים

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

whole-animal-perfusion-fixation-for-rodents

Nanyang Technological University

Research

JoVE Journal

Neuroscience

381.4K Views.  University of Michigan. כאן אנו מתארים בעלות נמוכה, בהליך מהיר, קיבעון מבוקר ואחיד באמצעות paraformaldehyde 4% perfused דרך מערכת כלי הדם: דרך הלב של חולדה כדי לקבל את השמירה הטובה ביותר של המוח.

Video: Whole Animal Perfusion Fixation for Rodents

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

ספיגה של 2 כלי השיט / תת לחץ דם: מודל עכברוש של איסכמיה המוח הגלובלית

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

Medicine

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.
Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

אפיון היסטולוגית שלם של Microdevices מוח מושתלים-ורקמה סובבת

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

In Vivo מחסום הדם - מוח חדירות Assay בעכברים באמצעות Fluorescently עם התווית המשדרים

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

הרומן פסיבית ניקוי שיטות לייצור מהיר של שקיפות אופטית כל רקמה CNS

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

שיטה המבוססות על מיקרו-CT אפיון נגעים ואיתור אלקטרודות במוח בעלי חיים קטנים

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific genes. Multiple means of assessing non-coding DNA and various chromatin states have proven useful in predicting the presence of enhancer sequences in the genome, but validating the activity of these sequences and finding the organs and developmental stages they are active in is a labor-intensive process. Recent advances in adeno-associated virus (AAV) vectors have enabled the widespread delivery of transgenes to mouse tissues, enabling in vivo enhancer testing without necessitating a transgenic animal. This protocol shows how a reporter construct that expresses EGFP under the control of a minimal promoter, which does not drive significant expression on its own, can be used to study the activity patterns of candidate enhancer sequences in the mouse brain. An AAV-packaged reporter construct is delivered to the mouse brain and incubated for 1-4 weeks, after which the animal is sacrificed, and brain sections are observed under a microscope. EGFP appears in cells in which the tested enhancer is sufficient to initiate gene expression, pinpointing the location and developmental stage in which the enhancer is active in the brain. Standard cloning methods, low-cost AAV packaging, and expanding AAV serotypes and methods for in vivo delivery and standard imaging readout make this an accessible approach for the study of how gene expression is regulated in the brain.

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

This protocol describes a novel rAAV-based transient enhancer-reporter assay. This assay can be used to induce enhancer-driven expression in vivo in the mouse brain.

פריסת AAV של מבני ביטוי מבוססי משפר in vivo במוח עכבר

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific ...

Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small size of mouse organs, ex vivo perfusion of bone, bladder, skin, prostate, and reproductive organs is challenging or not feasible. Here, we report for the first time an in situ lower body perfusion circuit in mice that includes the above tissues, but bypasses the main clearance organs (kidney, liver, and spleen). The circuit is established by cannulating the abdominal aorta and inferior vena cava above the iliac artery and vein and cauterizing peripheral blood vessels. Perfusion is performed via a peristaltic pump with perfusate flow maintained for up to 2 h. In situ staining with fluorescent lectin and Hoechst solution confirmed that the microvasculature was successfully perfused. This mouse model can be a very useful tool for studying pathological processes as well as mechanisms of drug delivery, migration/metastasis of circulating tumor cells into/from the tumor, and interactions of immune system with perfused organs and tissues.

A protocol is described for in situ perfusion of the mouse lower body, including the bladder, the prostate, sex organs, bone, muscle and foot skin.

הקמת מעגל באתרו של איברי הבטן התחתונה וגפיים האחוריות בעכברים

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small ...

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

חיסון חופשי של מוחות עכבר

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the ...

A Gravity-Fed Transcardial Perfusion Method for Histologic Analysis of the Mouse Central Nervous System

The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To maintain the morphology of these delicate tissues, it is routine to administer a chemical fixative such as paraformaldehyde via cannulation of the heart in anesthetized animals (transcardial perfusion). Transcardial perfusion of the mouse heart has traditionally relied on the use of peristaltic pumps or air pressure to deliver both the saline and fixative solutions necessary for this process. As an easily accessible alternative to these methods, this work demonstrates the use of a gravity-fed method of perfusate delivery that uses materials available in most hardware stores. 
To validate this new perfusion method, this work demonstrates all the subsequent steps necessary for the sensitive detection of phosphorylated &#945;-synuclein in both the brain and spinal cord. Included in these steps are the dissection of the fixed brain and spinal cord tissues, rapid freezing/embedding and cryosectioning of the tissues, and immunofluorescent staining. As this method results in whole-body delivery of the fixative, it may also be used to prepare other non-neuronal tissues for histologic analysis.

A convenient gravity-fed perfusion method for histological analysis of the mouse central nervous system is presented. Immunofluorescent detection of phosphorylated &#945;-synuclein is demonstrated in a mouse model of Parkinson's disease. This work also comprehensively describes the transcardial perfusion, dissection, tissue freezing/embedding, and frozen sectioning steps.

שיטת פרפוזיה טרנסקרדיאלית הניזונה מכוח הכבידה לניתוח היסטולוגי של מערכת העצבים המרכזית של העכבר

The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To ...

<meta charset="utf8"></head><body>פרוטוקול לניתוח עמוד שדרה דנדריטי באמצעות Neurolucida 360

Dendritic Spine Quantification Using an Automatic Three-Dimensional Neuron Reconstruction Software

Synaptic connections allow for the exchange and processing of information between neurons. The post-synaptic site of excitatory synapses is often formed on dendritic spines. Dendritic spines are structures of great interest in research centered around synaptic plasticity, neurodevelopment, and neurological and psychiatric disorders. Dendritic spines undergo structural modifications during their lifespan, with properties such as total spine number, dendritic spine size, and morphologically defined subtype altering in response to different processes. Delineating the molecular mechanisms regulating these structural alterations of dendritic spines relies on morphological measurement. This mandates accurate and reproducible dendritic spine analysis to provide experimental evidence. The present study outlines a detailed protocol for dendritic spine quantification and classification using Neurolucida 360 (automatic three-dimensional neuron reconstruction software). This protocol allows for the determination of key dendritic spine properties such as total spine density, spine head volume, and classification into spine subtypes thus enabling effective analysis of dendritic spine structural phenotypes.

<meta charset="utf8"></head><body>מחבר זרקור: אופטימיזציה של ניתוח עמוד השדרה הדנדריטי להערכה ידנית ואוטומטית מאוזנת בדנדריטים אפיים CA1 של ההיפוקמפוס

Dendritic spines are post-synaptic compartments of most excitatory synapses. Alterations to dendritic spine morphology occur during neurodevelopment, aging, learning, and many neurological and psychiatric disorders, underscoring the importance of reliable dendritic spine analysis. This protocol describes quantifying dendritic spine morphology accurately and reproducibly using automatic three-dimensional neuron reconstruction software.

כימות עמוד השדרה הדנדריטי באמצעות תוכנה אוטומטית לשחזור נוירונים תלת מימדיים

Synaptic connections allow for the exchange and processing of information between neurons. The post-synaptic site of excitatory synapses is often formed ...