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עבודה זו נתמכה על ידי קרן נובו נורדיסק, המכון הלאומי של הפרעות נוירולוגיות ושבץ, NINDS/NIH (M.N.). A.L.R.X. ו- S.H-R הם מקבלי מלגת פוסט-דוקטורט, מלגת דוקטורט מטעם קרן לונדבק, בהתאמה.

כל ההליכים בוצעו בהתאם האירופית דירקטיבה 2010/63/האיחוד האירופי לחקר בעלי חיים, אושרו על ידי המועצה לניסויים חיה תחת דני שר הסביבה ומזון (2015-15-0201-00535).
1. נוהל תעלות
<ol><li>בצינורית הכנה הערה: להימנע מלגעת את הצינורית עם כפפות שאינו סטרילי.<ol>
<li>לשבור את קצה מחט 30G שיניים באמצעות בע...

<ol>
	<li>Xie, 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=Sleep+Drives+Metabolite+Clearance+from+the+Adult+Brain.">Sleep Drives Metabolite Clearance from the Adult Brain.</a> Science. , 373-377 (2013).</li><li>Iliff, J. 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=A+paravascular+pathway+facilitates+CSF+flow+through+the+brain+parenchyma+and+the+clearance+of+interstitial+solutes,+including+amyloid+&#946;.">A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid &#946;.</a> Sci. Transl. Med. 4, 147ra111 (2012).</li><li>Jessen, N. A., Munk, A. S. F., Lundgaard, I., Nedergaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Glymphatic+System:+A+Beginner's+Guide.">The Glymphatic System: A Beginner's Guide.</a> Neurochem. Res. 40, 2583-2599 (2015).</li><li>Louveau, 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=Structural+and+functional+features+of+central+nervous+system+lymphatic+vessels.">Structural and functional features of central nervous system lymphatic vessels.</a> Nature. , (2015).</li><li>Aspelund, 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+dural+lymphatic+vascular+system+that+drains+brain+interstitial+fluid+and+macromolecules.">A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules.</a> J. Exp. Med. 212, 991-999 (2015).</li><li>Kress, B. T., 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=Impairment+of+paravascular+clearance+pathways+in+the+aging+brain.">Impairment of paravascular clearance pathways in the aging brain.</a> Ann. Neurol. 76, 845-861 (2014).</li><li>Plog, B. 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=Biomarkers+of+Traumatic+Injury+Are+Transported+from+Brain+to+Blood+via+the+Glymphatic+System.">Biomarkers of Traumatic Injury Are Transported from Brain to Blood via the Glymphatic System.</a> J. Neurosci. 35, 518-526 (2015).</li><li>Jiang, Q., 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=Impairment+of+glymphatic+system+after+diabetes.">Impairment of glymphatic system after diabetes.</a> J. Cereb. Blood Flow Metab. , (2016).</li><li>Peng, W., 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=Suppression+of+glymphatic+fluid+transport+in+a+mouse+model+of+Alzheimer's+disease.">Suppression of glymphatic fluid transport in a mouse model of Alzheimer's disease.</a> Neurobiol. Dis. 93, 215-225 (2016).</li><li>Ore&#353;kovi&#263;, D., Klarica, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+of+cerebrospinal+fluid:+Nearly+a+hundred+years+of+interpretations+and+misinterpretations.">The formation of cerebrospinal fluid: Nearly a hundred years of interpretations and misinterpretations.</a> Brain Res. Rev. 64, 241-262 (2010).</li><li>Dusart, I., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+Cell+Death+and+the+Inflammatory+Reaction+After+Dorsal+Hemisection+of+the+Rat+Spinal+Cord.">Secondary Cell Death and the Inflammatory Reaction After Dorsal Hemisection of the Rat Spinal Cord.</a> Eur. J. Neurosci. 6, 712-724 (1994).</li><li>Eide, K., Eidsvaag, V. A., Nagelhus, E. A., Hansson, H. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+astrogliosis+and+increased+perivascular+aquaporin-4+in+idiopathic+intracranial+hypertension.">Cortical astrogliosis and increased perivascular aquaporin-4 in idiopathic intracranial hypertension.</a> Brain Res. , (2016).</li><li>Pullen, R. G., DePasquale, M., Cserr, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+flow+of+cerebrospinal+fluid+into+brain+in+response+to+acute+hyperosmolality.">Bulk flow of cerebrospinal fluid into brain in response to acute hyperosmolality.</a> Am. J. Physiol. 253, F538-F545 (1987).</li><li>Ichimura, T., Fraser, P. A., Cserr, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+extracellular+tracers+in+perivascular+spaces+of+the+rat+brain.">Distribution of extracellular tracers in perivascular spaces of the rat brain.</a> Brain Res. 545, 103-113 (1991).</li><li>Iliff, J. 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=Brain-wide+pathway+for+waste+clearance+captured+by+contrast-enhanced+MRI.">Brain-wide pathway for waste clearance captured by contrast-enhanced MRI.</a> J. Clin. Invest. 123, 1299-1309 (2013).</li><li>Ratner, V., 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=Optimal-mass-transfer-based+estimation+of+glymphatic+transport+in+living+brain.">Optimal-mass-transfer-based estimation of glymphatic transport in living brain.</a> Proc. SPIE--the Int. Soc. Opt. Eng. 9413, (2015).</li><li>Lee, H., 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=The+Effect+of+Body+Posture+on+Brain+Glymphatic+Transport.">The Effect of Body Posture on Brain Glymphatic Transport.</a> J. Neurosci. 35, 11034-11044 (2015).</li><li>Nouri, S., Sharif, M. R., Sahba, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+ferric+chloride+on+superficial+bleeding.">The effect of ferric chloride on superficial bleeding.</a> Trauma Mon. 20, e18042 (2015).</li></ol>

הציגו לנו פרוטוקול המתאר הליך מפורט של תעלות מגנה cisterna (CMc), אשר מציעה שיטה פשוטה כדי לספק מולקולות שכותרתו תא CSF. CMc מאפשר את החזיית העוקבים CSF דינמיקה, גם אין ויוו וגם ex-vivo, באמצעות שיטות הדמיה שונות או היסטולוגיה.
אחד היתרונות העיקריים של הטכניקה CMc טמון שלה גישה ישי?...

נוזל מוחי שדרתי (CSF) מתרחץ מערכת העצבים המרכזית (CNS) ברחבי המערכת חדרית ולאורך את החללים תת-עכבישי, לחלל מוגדר מבחינה אנטומית של רצף עם החדרים, המקיף את המוח ואת חוט השדרה. אחד התפקידים העיקריים של CSF נועד לספק נתיב לאישור של מטבוליטים, מומסים בגבול מ parenchyma המוח. סיווג נעשית באמצעות glymphatic שנתגלתה לאחרונה מערכת1, המוח אנלוגית למערכת הלימפה ההיקפית. במסמך זה, אנו מתארים ולדון cisterna מגנה תעלות (CMc), שיטה פולשנית למסירה ישירה של מולקולות אל תוך שדרתי. CMc היא שיטת המפתח ללמוד את הפונקציה glymphatic. יתר על כן, ניתן גם להחיל CMc עבור חקר הדינמיקה CSF, משלוח מהיר, המוח ברוחב של הלא-דם מוח מחסום (BBB) מולקולות חדיר אל parenchyma המוח, לאורך החלל perivascular.
CMc מנצלת עקרונות פיזיולוגיים של CSF הדינמיקה התנועה דרך מערכת העצבים כדי לספק מעקב שכותרתו מולקולות או סמים לתוך מרחב מלא נוזל מוחי-שדרתי של המלון cisterna (ס מ). מולקולות מוזרקים דרך בצינורית מושתל לתוך כיסוי קרום קרום atlanto-העורף שהמולקולות ס"מ. ואז מתבצעות על ידי נוזל מוחי-שדרתי בצובר מזרימה parenchyma המוח דרך החלל paravascular1. הסוכן נותב או הניגוד מוזרק דרך CMc עוקב אחר תנועת נוזל מוחי-שדרתי, אשר מאפשר את ההערכה של CSF תנועה, זרם glymphatic על ידי לכימות רמות העוצמה של מולקולות שכותרתו מזין parenchyma המוח. CMc הוא תואם עם טכניקות הדמיה שונות, כולל epifluorescence, 2-פוטון מיקרוסקופיה של דימות תהודה מגנטית (MRI). בנוסף, ההערכה ניתן לבצע שני ויוו או ex-vivo. חשוב לציין, CMc מאפשר הפריט החזותי של מערכת glymphatic בהרדמה או במהלך השינה הטבעית, כמו גם בבעלי חיים ערה, נעה בחופשיות.
הטכניקה CMc יכול להיות מנוצל כדי לחקור היבטים שונים של דינמיקה של נוזלים ב- CSF, אבל הוכיחה להיות שימושי במיוחד ללמוד את המערכת glymphatic. Glymphatic פעילות כוננים הזרימה הולכת חום של CSF מן המרחב periarterial דרך תעלות מים aquaporin-4 (AQP-4), אשר הם בסככות הקרום של endfeet וסקולרית גלישת astrocytic. הזרם הולכת חום מאפשרת למחלף של CSF, נוזל בין-תאי (הקרן) בתוך parenchyma המוח. CSF/הקרן המכיל פסולת מטבולית, מומסים בגבול ואז הוסר parenchyma המוח דרך2,שטח perivenous3. בסופו של דבר, CSF/הקרן מגיע הפריפריה דרך כלי הלימפה קרום תיאר לאחרונה4,5. מערכת glymphatic הוכח חיונית עבור פינוי של מטבוליטים פסולת מזיקים כגון עמילואיד-β2. בהמשך, glymphatic סיווג נמצא לקוי הזדקנות6, לאחר פגיעה מוחית טראומטית7, ו במודלים חייתיים של סוכרת8 ו מחלת אלצהיימר9. ראוי לציין, glymphatic פעילות היא מדינה התלויים, מציג פעילות גבוה משמעותית במהלך שינה או הרדמה בהשוואה ערות1. אכן, אפרוחי ומורדמת להפגין את הפעילות glymphatic הגבוהה ביותר. לפיכך, כימות ניסיוני של פעילות glymphatic חיוני כשלמדתי תפקידה מחלה ובריאות.
מספר מחקרים עסקו CSF dynamics ולהחלפה שלה עם נוזל בין-תאי (הקרן) ב- parenchyma המוח. עם זאת, השיטות באמצעותו מועברות שכותרתו מולקולות הן פולשני למדי, מפעילה נזק מוחי parenchyma ושינויים בלחץ תוך גולגולתי (ICP) (ראה סקירת10). כמה דוגמאות הן ע או זריקות intraparenchymal אשר כרוכות גולגולת או קידוח של צמח קוצני חור בגולגולת. הליכים אלה הוכחו לשנות ICP, ובכך לשבש את פעולת הפונקציה glymphatic2. כמו כן, שיטות פולשניות כאלה זירוז astrogliosis ולהגדיל את immunoreactivity AQP-4 בשטח parenchyma פגום המוח,11,שלה הסביבה12. כמו האסטרוציטים ו- AQP-4 הם רכיבי מפתח של מערכת glymphatic, CMc היא השיטה של בחירה ללימודים שלה. היתרונות הגדולים של CMc בהשוואה הליכים פולשניים יותר הם קיום שלם הגולגולת והמוח parenchyma, הימנעות ICP שינויים astrogliosis, בהתאמה. לפיכך, CMc בשילוב עם כלי הדמיה שונים פותח עבור מגוון רחב של אפשרויות ללמוד לא רק את מערכת glymphatic, אלא גם את הדינמיקה ואת מנגנוני זרימה הומאוסטזיס, כמו גם מודלים חייתיים של מחלות נוירולוגיות.
ההליך תעלות (CMc) מגנה cisterna מאפשר גישה קלה וישירה נוזל מוחי שדרתי (CSF). על ידי הזרקת מולקולות שונות (למשל פלורסנט המשדרים, סוכנים בניגוד MRI) הנסיין ניתן לעקוב אחר תנועתם בתוך תא CSF, להעריך את הפעילות של מערכת glymphatic. הפרוטוקול הבא מתאר שני CMc חריפה של, לזריקות מיד לאחר ניתוח ולאחר כרונית השרשת הצינורית, שבו החיה והשיחזור הליך כירורגי זריקה מאוחר יותר. ההבדל החשוב ביותר בין השתלה אקוטי וכרוני הוא ההשתלה כרונית מאפשרת לצורך המחקר של פעילות glymphatic בעכברים ער.

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		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;"> 
			DAPI (diamidino-2-phenylindole) Solution (1 mg/mL)</td>
			<td style="width:245px;">Thermo Fisher Scientific</td>
			<td style="width:257px;">62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Dextran, Fluorescein, 3000 MW, Anionic</td>
			<td style="width:245px;">Thermo Fisher Scientific</td>
			<td style="width:257px;">D3305</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E-Z Anesthesia EZ-7000 Classic System</td>
			<td style="width:245px;">E-Z Systems</td>
			<td>EZ-7000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Attane Isofluran 1000 mg/g</td>
			<td style="width:245px;">ScanVet</td>
			<td>55226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Euthanimal 200mg/mL (sodium pentobarbital)</td>
			<td style="width:245px;">ScanVet</td>
			<td style="width:257px;">545349</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Ketaminol Vet 100 mg/mL (ketamine)</td>
			<td>Intervet International BV</td>
			<td style="width:257px;">511519</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:485px;">Rompin Vet 20 mg/mL (xylazin)</td>
			<td style="width:245px;">KVP Pharma + Veterin&#228;r Produkte GmbH</td>
			<td style="width:257px;">148999</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Xylocain 20 mg/mL (lidocain)</td>
			<td style="width:245px;">AstraZeneca</td>
			<td style="width:257px;">158543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Marcain 2.5 mg/mL (bupivacain)</td>
			<td style="width:245px;">AstraZeneca</td>
			<td style="width:257px;">123918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Bupaq Vet 0.3 mg/mL (buprenorphine)</td>
			<td style="width:245px;">Richter Pharma AG</td>
			<td style="width:257px;">185159&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

cannula implantation into the cisterna magna of rodents

Cisterna מגנה תעלות (CMc) היא תהליך פשוט המאפשר גישה ישירה נוזל מוחי שדרתי (CSF) ללא נזק פעיל הגולגולת או parenchyma המוח. בחולדות מרדימים, החשיפה של קרום הדורה ע י ניתוח עמום של שרירי הצוואר מאפשר החדרת הצינורית לתוך המלון cisterna (ס מ). הצינורית, חיבר או על ידי מחט משופע בסדר או בורוסיליקט נימי, מחוברת דרך צינור פוליאתילן (PE) מזרק. באמצעות מזרק משאבה, מולקולות יכול אז להיות מוזרק במחירים מבוקרים ישירות לתוך ס מ, אשר היא רציפה עם תת-עכבישי החלל. מן החלל תת-עכבישי, אפשר לאתר CSF פלקסים על ידי זרימת הולכת חום לחלל perivascular סביב רבים חודר, שבו מתרחשת ממס exchange עם נוזל interstitial (ISF). CMc ניתן לבצע זריקות חריפה מיד לאחר הניתוח, או השרשה כרונית, עם הזרקת מאוחר יותר מרדימים או ער, לנוע בחופשיות מכרסמים. כימות של התפלגות מעקב ב- parenchyma המוח יכול להתבצע על ידי epifluorescence, 2-פוטון מיקרוסקופ, דימות תהודה מגנטית (MRI), בהתאם המאפיינים הכימי פיזיקלי של מולקולות מוזרק. לפיכך, CMc בשילוב עם טכניקות הדמיה שונות מציע כלי רב עוצמה עבור הערכה של מערכת glymphatic ו CSF dynamics ובפונקציה. יתר על כן, CMc יכול להיות מנוצל כמו צינור של משלוח מהיר, המוח-רחב של איתות מולקולות ותערובות מטבולית זאת יכולה אחרת לעבור את מחסום הדם מוח (BBB).

על קיבוע של עכברים או חולדות בתוך מסגרת stereotaxic, שרירי הצוואר באזור קרסט העורפית בלשון בוטה גזור כדי לחשוף את מגנה cisterna (ס מ). מבנה משולש ס מ יזוהה בקלות בין החלק סימטרית של הצרבלום לשד (איור 1Aג-1). הצינורית מוכנס 1-2 מ מ לתוך ס מ על ידי בעדינות פירסינג ק?...

Watch this Scientific Journal Video about Cannula Implantation into the Cisterna Magna of Rodents at JoVE.com

Cannula Implantation into the Cisterna Magna of Rodents

השרשה בצינורית לתוך המלון Cisterna של מכרסמים

כאן נתאר פרוטוקול לבצע cisterna מגנה תעלות (CMc), דרך פולשנית כדי לספק קליעים נותבים, מצעים, מולקולות איתות לתוך נוזל מוחי שדרתי (CSF). CMc בשילוב עם שיטות הדמיה שונות, מאפשרת המערכת glymphatic CSF dynamics הערכה, וכן משלוח המוח ברוחב של תרכובות שונות.

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

The overall goal of this surgical procedure is to enable direct delivery of tracers, substrates and signaling molecules into the cerebrospinal fluid. This method can help to answer key questions in the neuroscience filed regarding CSF and ISF dynamics in the brain. This method allows the delivery of different molecules, tracers, active metabolic substrates into the CSF, cerebrospinal fluid, compartment without damaging the skull or the brain parenchyma.The implications of this technique extend towards neurological diseases because it allows the evaluation of CSF-ISF fluxes in the brain and also the clearance of metabolites and toxic proteins from the brain parenchyma. So this method provides insight into the dynamics of the CSF-ISF fluxes and additionally we can use it as a fast, brain-wide delivery route for signaling molecules and molecular substrates that could not otherwise pass the blood-brain barrier. To prepare the cannula, break of the beveled metal tip of a 30 gauge dental needle using a needle holder.Then, insert the beveled metal tip into a 30 centimeter length of PE-10 tubing filled with aCSF. Next, use a one milliliter syringe fitted with a 30 gauge needle to flush the cannula with artificial cerebrospinal fluid. The cannula is then ready for use in the surgery.After anesthetizing the mouse, position the animal over a heating pad on a stereotaxic apparatus. Check for a lack of pedal reflex with a toe pinch, and wait for the animal's respiration to become slow and steady. Next, shave the head and neck.Then, wipe away the fur and sterilize the exposed skin. First, scrub the skin with alcohol. Then make two applications of 0.5 per cent chlorohexidine or two per cent iodine solution.For chronic cannulations, also administer analgesics at the cannulation site. Now, fix the mouse in the stereotaxic frame, making use of either the intraneural arch or the zygomatic arch. Then, tilt the head slightly so that it forms an angle of 120 degrees to the body.Next, apply ophthalmic ointment and reapply it as needed during the surgery. Find the occipital crest, which is the part of the skull protruding immediately above the neck muscles. Lift the overlying skin using a pair of tweezers and cut an almond-shaped piece of skin of approximately one centimeter along the midline.Then, using the occipital crest as a reference point, pull apart the superficial connective tissue to expose the neck muscles below. Use cotton swabs or eye spears to control any resultant bleeding. To proceed, separate the muscles at the midline by carefully running the tweezers down the middle of the incision site in the anterior to posterior axis.Then, with a pair of curved forceps in each hand, join the tips in the middle near the bottom of the skull and pull the muscles aside to expose the cisterna magna. The cisterna magna looks like a tiny inverted triangle, outlined by the cerebellum above and medulla below. Using a surgical eye spear or cotton swab, wipe the dural membrane covering the CM.Next, with curved tweezers in the dominant hand, grasp the cannula near the tube-covered needle.Then, place the middle finger of the other hand on the ear bar to create a stable rest for the cannula. Now, at the center of the CM, puncture the dura at 45 degrees relative to the mouse head. Only insert the needle to where the bevel is under the dura to avoid penetrating into the cerebellum or medulla.If necessary, clean off any leaked CSF with a spear or swab. Then, apply a few drops of cyanoacrylate onto the dural membrane surrounding the cannula and add a drop of glue accelerator to cure the glue immediately. Next, combine dental cement and cyanoacrylate glue.Then, cover the entire incision site and quickly apply a drop of glue accelerator to cure. For injection in mice with acute cisterna magna cannulation, proceed directly to injection of CSF tracers with the aid of an injection pump. For chronic cannulation, cut the tubing down to two or three centimeters and seal it with a surgical weld to retain the inter-cranial pressure levels.Then, administer carprofen. Now, house the mouse alone and keep the cage over a heating pad until the mouse has regained sternal recumbency. To prepare the cannula, break off the beveled metal tip of a 30 gauge dental needle using a needle holder.Then, insert the beveled metal tip into a 30 centimeter length of PE-10 tubing filled with aCSF. Using a 30 gauge needle, flush the tubing. Attach the 30 gauge needle to a 100 microliter syringe filled with distilled water and then attach this to a syring pump.Using the pump, withdraw enough air to create a one centimeter bubble in the cannula. Then, dip the needle into the CSF tracer solution and withdraw 12 microliters into the cannula. Cut the tubing attached to the cannula up to three to four centimeters length and quickly connect the CSF tracer-filled tubing with the implanted cannula with the aid of fine forceps.To inject CSF tracers into the CM of anesthetized animals, use a syringe pump to deliver tracers at one microliter per minute for five to ten minutes. Then allow the tracers to circulate for 30 minutes without moving the cannula. Gently restrain the animal and cut off approximately one centimeter of the tubing from the implanted cannula.Then, quickly connect the two cannulas, using the aid of a fine forceps and a pair of curved tweezers if necessary. Now, inject one microliter of tracer per minute over seven to twelve minutes, using a syring pump. After the injection, let the tracer circulate for 30 minutes while taking the necessary measures to ensure that the cannula remains undisturbed and the tubing remains attached.Then proceed with harvesting the brain. Mouse brains were injected with CSF tracers into the CM as described. A macroscopic dorsal view shows the distribution of the tracers in the subarachnoid cisterns of the cerebellopontine region in the olfactory bulb and in the paravascular space along the middle cerebral arteries.In the ventral portion of the brain, a macroscopic view shows the CSF tracer distribution along the Circle of Willis. Histological sections of CM-injected brains further reveal the paravascular distribution of tracers within the brain parenchyma. Mice injected under anesthesia or while asleep had much more tracer distribution than mice injected while awake and freely moving in their home cage.After watching this video, you should gain a good knowledge of how to perform cisterna magna cannulation that will allow you to deliver chemicals or molecules of interest into the cerebrospinal fluid compartment without any operative damage to the skull or to the brain parenchyma. Once mastered, this technique can be performed in between 20 to 30 minutes if performed properly. While doing this procedure, it is important to apply the correct amount of pressure when puncturing the dura mater to avoid major CSF leakage that could interfere with the intercranial pressure.After its development, this cisterna magna cannulation has paved the way for studies in the CSF-ISF clearance in the brain via the so-called lymphatic system that nowadays is widely used in different animal models of neurological diseases.

cannula-implantation-into-the-cisterna-magna-of-rodents

Research

JoVE Journal

Neuroscience

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

בידוד באף גזע תאים חוש הריח של מכרסמים או בני אדם

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

מערכת אוטומטי לחלוטין תכליתי מאוד עבור בדיקת ריבוי פונקציות קוגניטיביות ורישום פעילות עצבית במכרסמים

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

שיטה להרוויח Craniotomy על גולגולת הגחון של מכרסמים ילוד

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Microelectrode מודרך השרשה של אלקטרודות לתוך גרעין Subthalamic של חולדות לטווח ארוך עמוק גירוי המוח

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Operant Protocols for Assessing the Cost-benefit Analysis During Reinforced Decision Making by Rodents

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their consequences. This process is integral to the normal human behavior and has been shown to be disrupted by neurological and psychiatric disorders such as addiction, schizophrenia, and depression. Rodents have long been used to uncover the neurobiology of human cognition. To this end, several behavioral tasks have been developed; however, most are non-automated and are labor-intensive. The recent development of the open-source microcontroller has enabled researchers to automate operant-based tasks for assessing a variety of cognitive tasks, standardizing the stimulus presentation, improving the data recording and consequently, improving the research output. Here, we describe an automated delay-based reinforcement-guided decision-making task, using an operant T-maze controlled by custom-written software programs. Using these decision-making tasks, we show the changes in the local field potential activities in the anterior cingulate cortex of a rat whilst it performs a delay-based cost-and-benefit decision-making task.

A cost-benefit analysis is a weighing-scale approach that the brain performs during the course of decision making. Here, we propose a protocol to train rats on an operant-based decision-making paradigm where rats choose higher rewards at the expense of waiting for 15 s to receive them.

פרוטוקולים operant להערכת ניתוח עלות-תועלת במהלך קבלת החלטות מחוזק על ידי מכרסמים

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

הכוח של מרווח Interstimulus להערכה של זמני עיבוד בחולדות

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

הזרקת דם ישירה כפולה לתוך מגנה צ'יסטרנה כודל של דימום Subarachnoid

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

Direct Cannula Implantation in the Cisterna Magna of Pigs

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces and has been implicated in the clearance of neurotoxic peptides such as amyloid-beta. Impaired glymphatic function exacerbates disease pathology in animal models of neurodegenerative diseases, such as Alzheimer's, which highlights the importance of understanding this clearance system. The glymphatic system is often studied by cisterna magna cannulations (CMc), where tracers are delivered directly into the cerebrospinal fluid (CSF). Most studies, however, have been carried out in rodents. Here, we demonstrate an adaptation of the CMc technique in pigs. Using CMc in pigs, the glymphatic system can be studied at a high optical resolution in gyrencephalic brains and in doing so bridges the knowledge gap between rodent and human glymphatics.

This article presents a step-by-step protocol for the direct cannula implantation in the cisterna magna of pigs.

השתלת צינורית ישירה ב"ציסטרנה מגנה של חזירים"

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces ...

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. This standard approach can be challenging for targets in the caudal brainstem and upper cervical cord due to specific anatomical challenges as these sites are remote from skull landmarks, leading to imprecision. Here we outline an alternative stereotaxic approach via the cisterna magna that has been used to target discrete regions of interest in the caudal brainstem and upper cervical cord. The cisterna magna extends from the occipital bone to the atlas (i.e., the second vertebral bone), is filled with cerebrospinal fluid, and is covered by dura mater. This approach provides a reproducible route of access to select central nervous system (CNS) structures that are otherwise hard to reach due to anatomical barriers. Furthermore, it allows for direct visualization of brainstem landmarks in close proximity to the target sites, increasing accuracy when delivering small injection volumes to restricted regions of interest in the caudal brainstem and upper cervical cord. Finally, this approach provides an opportunity to avoid the cerebellum, which can be important for motor and sensorimotor studies.

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.

גישה כירורגית סטריאוטקסית למיקרו-איניג'קט גזע המוח הקאודלי וחוט השדרה הצווארי העליון באמצעות ציסטרנה מגנה בעכברים

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. ...

Surgical Implantation of the Micro-Drive on the Mouse Skull

After preparing the mouse for surgery and exposing the skull, place the bone screws in the extreme lateral, rostral or caudal portions of the skull where the bone is thickest and the bone screws are sufficiently far away from the micro drive implant. Use a scalpel blade or drill bit to score the skull near the bone screw hole locations, providing a rough surface for the liquid cyanoacrylate to bind. Then thread each bone screw into place, careful not to pierce the underlying dura.Using a sterile 30-gauge needle, apply liquid cyanoacrylate around each bone screw, thickening the skull where the bone screws have been attached. Ensure that no cyanoacrylate enters the exposed dura above the recording sites.