Name: cannula implantation into the cisterna magna of rodents
Uploaded: 2018-05-23T14:00:00
Description: Watch this Scientific Journal Video about Cannula Implantation into the Cisterna Magna of Rodents at JoVE.com

この作品は、NINDS/NIH (m. n.) ノボ ノルディスク財団と国立研究所の神経疾患や脳卒中、によって支えられました。A.L.R.X. と S.H R ポスドク親睦とルンドベック財団から博士課程奨学金の受取人は、それぞれ。

すべてのプロシージャは動物の研究のためヨーロッパの指令 2010年/63/EU に従って行われ、デンマーク環境や食品 (2015-15-0201-00535) 省の下で動物実験委員会によって承認されました。
1. カテーテル留置の手順
<ol><li>カニューレの準備 注: は、非滅菌手袋でカニューレを触れないでください。<ol>
<li>ニードル ホルダーを使用して 30 G 歯科針の斜めの金属チップを断ちます?...

<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>

我々 はチステルナ マグナ カニュレーション (CMc)、CSF のコンパートメントに標識分子を配布する簡単な方法を提供するための詳細な手順を説明するプロトコルを提案しました。CMc では、髄液循環動態、体内と体外では、異なる画像モダリティや組織を使用しての両方の後続の可視化ことができます。
CMc 技術の主な利点の 1 つは、開頭して脳を公開するこ?...

脳脊髄液 (CSF) は、心室システム全体と解剖学的に定義された領域を連続で、脳室と脳や脊髄をとりまく、くも膜下のスペースに沿って、中枢神経系 (CNS) を浴びる。CSF の主な機能の 1 つは代謝と脳実質からの溶質のクリアランスのためのルートを提供するためにです。クリアランスは、最近発見された glymphatic システム1、末梢のリンパ系にアナログ脳を介して促進されます。ここで、我々 説明し、チステルナ マグナ カニュレーション (CMc) 低侵襲法 CSF 中に分子の直接配信します。CMc は、glymphatic 関数を勉強するため重要なメソッドです。さらに、CMc は、髄液循環動態の研究と血管周囲のスペースに沿って、脳実質に非血液脳関門 (BBB) を透過性分子の脳全体に高速配信するためにも適用できます。
CMc 槽 (CM) の脳脊髄液で満たされた空間に標識トレーサー分子や薬を提供する中枢神経系を介して髄液循環運動動態の生理学的原則を悪用します。分子は、cm 分子は paravascular スペース1を介して脳実質に CSF 一括流れによってそれから運ばれる環椎後頭骨硬膜膜屋根に注入カニューレによって挿入されます。CMc によって挿入されたトレーサーまたはコントラストのエージェントにより CSF の動きと glymphatic 流入の評価、脳実質が入力された分子の強度レベルを定量化することで脳脊髄液の動きに追従します。CMc は、落射蛍光、2 光子の顕微鏡検査、磁気共鳴画像 (MRI) などさまざまなイメージング技術と互換性が。また、この評価をすることができます体内または体外で実行します。重要なは、CMc は、麻酔下でまたは自然な睡眠中にだけでなく、目を覚まし、自由に移動する動物の glymphatic システムの可視化のためことができます。
CMc 手法が CSF における流体力学のさまざまな側面を研究することが出来、glymphatic システムを学ぶために特に有用であると証明しました。Glymphatic 活動は終足血管折り返しアストロ サイトの膜に繋留するアクアポリン 4 (アクアポリン 4) 水路を介して動脈領域から脳脊髄液の対流をドライブします。対流の流れにより、髄液と脳実質内の間質液 (ISF) のインターチェンジ。CSF/ISF 代謝廃棄物や溶質を含むが静脈スペース2,3を介して脳実質から削除されます。最終的には、CSF/ISF 最近記述されていた硬膜リンパ管4,5を介して周囲に達する。Glymphatic システムは、アミロイド β2など有害な廃棄物代謝物質のクリアランスのため重要な示されています。さらに、glymphatic のクリアランスは加齢で6、外傷性脳傷害7後、糖尿病8 、9アルツハイマー病の動物モデルにおける障害者です。特に、glymphatic の活動は状態に依存しない、睡眠や覚醒1と比較すると麻酔中に有意に高い活性を示すです。確かに、若い麻酔下の動物は、最高の glymphatic 活動を展示します。したがって、健康と病気におけるその役割を勉強するとき、glymphatic 活動の実験の定量化は重要です。
いくつかの研究は、髄液循環動態と脳実質内に間質液 (ISF) とその交流に対処しています。ただし、標識分子を配信する方法、むしろ侵略的、脳実質の損傷、頭蓋内の圧力 (ICP) の変更をトリガー (を参照してください10を確認)。いくつかの例が脳や頭蓋骨に穴の開頭術やぎざぎざの掘削を伴うクモ膜注射。ICP、glymphatic 関数2をこうして中断することを変更するこれらの手順を示されています。また、このような侵襲的方法アストログリオーシスを誘発して脳実質損傷領域とその周辺11,12アクアポリン 4 免疫反応性を高めます。アストロ サイトとアクアポリン 4 は glymphatic システムの重要な要素である、CMc、その研究で選択の方法です。CMc より侵襲的処置と比較しての主な利点は、そのまま頭蓋骨と脳実質、ICP の変化とアストログリオーシス、それぞれ回避のメンテナンスです。したがって、glymphatic システムだけでなく、ダイナミクスを研究する可能性の広い範囲との恒常性だけでなく、神経疾患の動物モデルにおける流れのメカニズムのさまざまなイメージング ツールと組み合わせて内の CMc を開きます。
チステルナ マグナ カニュレーション (CMc) プロシージャは、脳脊髄液 (CSF) への直接アクセスをできます。異なる分子 (例えば蛍光トレーサー、MRI 造影剤) を注入することにより実験者は CSF コンパートメント内の動きを追跡し、glymphatic システムのアクティビティを評価することがことができます。次のプロトコルでは、注射、手術と動物が後で注入の手術から回復するカニューレの慢性注入直後のため、両方急性 CMc について説明します。急性および慢性の移植の最も重要な違いは、慢性的な注入が覚醒マウス glymphatic 活動の研究のためにできることです。

<table border="0" cellpadding="0" cellspacing="0" width="1153">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">SOPIRA Carpule 30G 0.3 x 12mm</td>
			<td style="width:245px;">Kulzer</td>
			<td style="width:257px;">AA001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Polyethylene Tubing 0.024&#8221; OD x 0.011&#8221; ID</td>
			<td style="width:245px;">Scandidact</td>
			<td>PE10-CL-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30G x &#189;&#8221; 0.3 x 12 mm Luer-Lock</td>
			<td style="width:245px;">Chirana T. Injecta</td>
			<td>CHINS01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:485px;">Chlorhexidine 0.5% (chlorhexidine digluconate)</td>
			<td style="width:245px;">Meda AS</td>
			<td style="width:257px;">no catalogue number, see link in comments</td>
			<td>http://www.meda.dk/behandlingsomrader/desinfektion/desinfektion-af-hud/klorhexidin-sprit-medic-05/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcohol Swab 70% Isopropyl Alcohol 30 x 60mm</td>
			<td>Vitrex Medical A/S</td>
			<td>520213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Viskoese Oejendraeber Ophtha</td>
			<td>Ophtha</td>
			<td>145250</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:485px;">Wooden applicator, Double cotton bud (&#216; appr. 4 - 5 mm, length appr. 12 mm)</td>
			<td style="width:245px;">Heinz Herenz</td>
			<td style="width:257px;">1032018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eye spears</td>
			<td>Medicom</td>
			<td>A18005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ferric chloride 10% solution</td>
			<td style="width:245px;">Algeos</td>
			<td style="width:257px;">NV0382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimtech Science Precision Wipes Tissue Wipers</td>
			<td style="width:245px;">Kimberly Clark Professional</td>
			<td style="width:257px;">05511</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Loctite Super Glue Precision 5g</td>
			<td style="width:245px;">Loctite</td>
			<td style="width:257px;">no catalogue number, see link in comments</td>
			<td>http://www.loctite-consumer.dk/da/produkter/superglue-liquid.html&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insta-Set CA Accelerator</td>
			<td style="width:245px;">Bob Smith Industries</td>
			<td style="width:257px;">BSI-152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dental Cement Powder</td>
			<td style="width:245px;">A-M Systems</td>
			<td style="width:257px;">525000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical weld&#160;</td>
			<td style="width:245px;">Kent Scientific Corporation</td>
			<td style="width:257px;">INS750391</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;">Hamilton syringe GASTIGHT&#174; , 1700 series, 1710TLL, volume 100 &#956;L, PTFE Luer lock</td>
			<td>Hamilton syringes</td>
			<td style="width:257px;">1710TLL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">LEGATO 130 Syringe pump</td>
			<td style="width:245px;">KD Scientific</td>
			<td style="width:257px;">788130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Paraformaldehyde powder, 95%</td>
			<td style="width:245px;">Sigma Aldrich</td>
			<td style="width:257px;">158127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Phosphate buffered saline (PBS; 0.01M; pH 7.4)</td>
			<td style="width:245px;">Sigma Aldrich</td>
			<td style="width:257px;">P3813</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Ovalbumin, Alexa Fluor 647 Conjugate</td>
			<td style="width:245px;">Thermo Fisher Scientific</td>
			<td style="width:257px;">O34784</td>
			<td></td>
		</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

チステルナ マグナ カニュレーション (CMc) は、頭蓋骨や脳実質への手術損傷なし脳脊髄液 (CSF) への直接アクセスを可能にする簡単な手順です。麻酔をかけられた齧歯動物、首の筋肉の鈍的切離による硬膜の露出槽 (CM) にカニューレを挿入ことができます。ベベル針またはホウケイ酸ガラス キャピラリーのいずれかで構成された、カニューレは、注射器にポリエチレン (PE) 管経由で添付されます。シリンジ ポンプを使用すると、分子を挿入できます制御率では、くも膜下腔と連続 CM に直接。くも膜下腔から我々 は間質液 (ISF) と溶質の交流が発生する貫通細動脈の周りの血管周囲スペースに対流に CSF フラックスを追跡できます。CMc または実行できますすぐに術後急性の注射のため慢性的な注入、麻酔で目を覚まし、以降のインジェクションの齧歯動物を自由に移動します。磁気共鳴画像 (MRI)、注入された分子の物理化学的特性によって、2 光子顕微鏡落射蛍光脳実質におけるトレーサー分布の定量が可能です。このように、様々 なイメージング技術と組み合わせて CMc は、glymphatic システムと髄液循環動態とその機能の評価のための強力なツールを提供しています。さらに、シグナル伝達分子と血液脳関門 (BBB) をそれ以外の場合はクロスができませんでした代謝基質の脳全体に高速配信するための導管として CMc を活用することができます。

脳定位固定装置のフレームのラットやマウスの固定時に後頭稜付近の首の筋肉は槽 (CM) を公開する解剖ぶっきらぼう。CM の三角形の構造は、小脳の尾側部分と髄質 (図 1 a-1 C) 容易に認識されます。カニューレは、優しくピアス環椎後頭膜 (図 1) で、1-2 mm を CM に挿入されます。硬膜はタフな構造とカニューレの...

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

齧歯動物の槽注入カニューレ

ここチステルナ マグナ カニュレーション (CMc)、脳脊髄液 (CSF) にトレーサー、基板、シグナル分子を提供する低侵襲の方法を実行するプロトコルについて述べる。別の画像診断装置と組み合わせると、CMc が glymphatic システム、CSF のダイナミクス評価、様々 な化合物の脳全体で配信できます。

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.

げっ歯類やヒトから鼻嗅覚幹細胞を単離する

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 ...

神経科学

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.

マルチ認知機能のテストとげっ歯類の神経活動を記録するための完全に自動化された汎用性の高いシステム

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.

新生児げっ歯類の腹スカルに開頭術を行うには方法

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.

微小電極は、長期的な脳深部刺激のためのラットの視床下核に電極の移植ガイド付き

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.

齧歯動物による補強意思の中に費用便益分析を評価するためのオペラントのプロトコル

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.

刺激間隔の齧歯動物の一時的な処理の評価のための力

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.

くも膜下出血のモデルとしての水槽マグナへの血液の二重直接注射

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.

豚のシスターナマグナにおける直接カニューレ移植

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.

マウスのCisterna Magna を介して 尾側脳幹および上部頸椎脊髄をマイクロインジェクションする定位的外科的アプローチ

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.