Name: mouse microsurgery infusion technique for targeted substance delivery into the cns via the internal carotid artery
Uploaded: 2017-01-31T14:00:00.000+00:00
Duration: 6 min 41 s
Description: Watch this Scientific Journal Video about Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery at JoVE.com

We would like to thank Dr. Lei Chen (Icahn School of Medicine at Mount Sinai, NY) who first established the use of this model in our laboratory, and to Dr. Gretchen Wolff (German Cancer Research Center, Heidelberg, Germany) for disseminating the technique in our laboratory. Supported in part by HL126559, DA039576, MH098891, MH63022, MH072567, DA027569, and NSC 2015/17/B/NZ7/02985.

以下のプロトコルに関係する手順は、マイアミ大学の施設内動物管理使用委員会（IACUC）によって承認されています。また、すべての手順は、実験動物管理・インターナショナルの評価と認定協会（AAALAC）によって承認された施設で行われています。 手術のためのマウスの作製<ol><li>実験室麻酔システムを使用して、酸素と混合イソフルランでマウスを麻酔。 （材料表を参照してください）商用マシンに2 L / minで％と酸素の流れ4-5との間で設定でイソフルランを使用してください。実体顕微鏡下で、手術の表面に動物を移し、ノーズコーンを使用して麻酔を維持（ - 2 L / minで2.5と酸素流量1.5を設定イソフルランを使用します）。 <ol><li>あえぎなし2呼吸/秒 - マウス呼吸数は約1であることを確認してください。また、動物は、ウィスカ刺激反応とペダルrを示さないことを確認eflex（つま先ピンチ）。手術中の呼吸数と労力、少なくとも5分ごとに監視します。齧歯類麻酔監視のために特定の施設内動物管理使用委員会や獣医のガイドラインに従ってください。 </li></ol></li><li>乾燥を防ぐために無菌の綿棒を使用して、各眼に点眼潤滑剤の液滴を適用します。抗炎症や不快感を軽減するために鎮痛剤の投与が推奨されます。 </li><li>動物がその背中の上に横たわると、ノーズコーンを使用して麻酔を維持します。 </li><li>エタノール70％とクロルヘキシジンと面積を3回拭くことによって、動物の首のエリアを清掃してください。 （詳細は下記）カミソリを使用して、動物の手術のエリアを剃ります。 </li></ol>一般的な頸動脈の2解剖（CCA） <ol><li>実体顕微鏡下で全体のマイクロサージェリーの手順を実行します。手術用ハサミとピンセットを使用すると、胸骨上からトン以下に、首に浅い正中切開を行います彼の顎（約3〜4センチ）。 </li><li>鉗子を注意深く別個の脂肪および結合組織を使用して気管を露出させます。 </li><li>さらにエリアを露出し、首を拡張するために、マウスの首の後ろに枕（丸い物体、直径約0.5cm）を配置します。 </li><li>組織リトラクターやフックのいずれかを使用して組織を分離します。 </li><li>気管の動物の左側には、慎重に左CCAを露出させるために結合組織を離れてピンセットで抜きます。 </li><li>鉗子を使用して、慎重にCCAの分岐部を露出させるために、すべての結合組織、および外部と内部の両方の頸動脈の先頭を削除します。 </li></ol>物質の注入のためのCCAの調製<ol><li>鉗子を使用して、外頸動脈（ECA）の下にナイロン縫合糸の二つのセグメント（約1cmずつ）挿入します。 </li><li> ECAの可能な限り最高の時点での恒久的な結び目を置きます。 </li><li> ECAの可能な最低点、すぐにCCA分岐上記の場所で取り外し可能な結び目。この結び目は、上部の結び目に比べて緩いすべきです。 </li><li>可能な限り低い点で、血管クリップを使用して、CCAを閉じます。 </li><li>血管クリップを使用して、内頸動脈（ICA）を閉じます。 </li><li>使用して顕微解剖春のはさみは2ノットの間、ECA（約2mm）の小さなカットを行います。 </li></ol> ICA を介した 4物質注入<ol><li>輸液システムを組み立てます。注入される物質を250μl（薬物、病原体、細胞外ベシクル、とりわけ）を含むツベルクリン注射器にと行っ切開部に静かにキャピラリー先端を挿入します。キャピラリーチューブの6インチ（X 1.2ミリメートル2.5ミリメートル理想的な寸法）を取り付けステップ3.6インチ</li><li>それが分岐し、CCAを閉じるクリップの中間点に達するまで毛細管を下にスライドし続けます。 </li><li>下ECAの結び目を下に接続します。結び目は、毛細管流動を可能にするのに十分な緩みとleakinを防止するのに十分タイトであることを確認しますグラム。 </li><li> ICAからクリップを削除します。 </li><li>ゆっくりと注射器ピストン可能物質の点滴毎秒約10μlに圧力を加えます。 </li></ol> 5.注入後の手順<ol><li>バックICA上のクリップを配置します。 </li><li>静かにキャピラリーチューブを取り外します。 </li><li>完全に下ECAの結び目を下に接続します。 </li><li> ICAとCCAからクリップを削除します。 </li></ol> 6.切開閉会と術後ケア<ol><li>開創または組織フックと枕を削除します。 </li><li>滅菌生理食塩水を使用して、きれいな縫合エリア。 </li><li>ナイロン縫合糸/針とピンセットを用いて切開を閉じます。 </li><li>術後の不快感を軽減するために、抗炎症および鎮痛管理します。 </li><li>少なくとも1時間とモニタ回復のための加熱パッド上に配置されたケージに動物を置きます。 </li></ol>

<ol>
	<li>Chen, L., Swartz, K. R., Toborek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+microport+technique+for+applications+in+cerebrovascular+research.">Vessel microport technique for applications in cerebrovascular research.</a> J Neurosci Res. 87 (7), 1718-1727 (2009).</li><li>Frisella, W. 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=Intracranial+injection+of+recombinant+adeno-associated+virus+improves+cognitive+function+in+a+murine+model+of+mucopolysaccharidosis+type+VII.">Intracranial injection of recombinant adeno-associated virus improves cognitive function in a murine model of mucopolysaccharidosis type VII.</a> Mol Ther. 3 (3), 351-358 (2001).</li><li>Wei, L., Erinjeri, J. P., Rovainen, C. M., Woolsey, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collateral+growth+and+angiogenesis+around+cortical+stroke.">Collateral growth and angiogenesis around cortical stroke.</a> Stroke. 32 (9), 2179-2184 (2001).</li><li>Wu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+delivery+of+methotrexate+to+epidermal+growth+factor+receptor-positive+brain+tumors+by+means+of+cetuximab+(IMC-C225)+dendrimer+bioconjugates.">Targeted delivery of methotrexate to epidermal growth factor receptor-positive brain tumors by means of cetuximab (IMC-C225) dendrimer bioconjugates.</a> Mol Cancer Ther. 5 (1), 52-59 (2006).</li><li>Pignataro, G., Studer, F. E., Wilz, A., Simon, R. P., Boison, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotection+in+ischemic+mouse+brain+induced+by+stem+cell-derived+brain+implants.">Neuroprotection in ischemic mouse brain induced by stem cell-derived brain implants.</a> J Cereb Blood Flow Metab. 27 (5), 919-927 (2007).</li><li>Sugiyama, Y., Kusuhara, H., Suzuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+and+biochemical+analysis+of+carrier-mediated+efflux+of+drugs+through+the+blood-brain+and+blood-cerebrospinal+fluid+barriers:+importance+in+the+drug+delivery+to+the+brain.">Kinetic and biochemical analysis of carrier-mediated efflux of drugs through the blood-brain and blood-cerebrospinal fluid barriers: importance in the drug delivery to the brain.</a> J Control Release. 62 (1-2), 179-186 (1999).</li><li>Pardridge, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+and+gene+delivery+to+the+brain:+the+vascular+route.">Drug and gene delivery to the brain: the vascular route.</a> Neuron. 36 (4), 555-558 (2002).</li><li>Vantyghem, S. A., Postenka, C. O., Chambers, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrous+cycle+influences+organ-specific+metastasis+of+B16F10+melanoma+cells.">Estrous cycle influences organ-specific metastasis of B16F10 melanoma cells.</a> Cancer Res. 63 (16), 4763-4765 (2003).</li><li>Huang, R. 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=Efficient+gene+delivery+targeted+to+the+brain+using+a+transferrin-conjugated+polyethyleneglycol-modified+polyamidoamine+dendrimer.">Efficient gene delivery targeted to the brain using a transferrin-conjugated polyethyleneglycol-modified polyamidoamine dendrimer.</a> FASEB J. 21 (4), 1117-1125 (2007).</li><li>Liu, R., Martuza, R. L., Rabkin, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracarotid+delivery+of+oncolytic+HSV+vector+G47Delta+to+metastatic+breast+cancer+in+the+brain.">Intracarotid delivery of oncolytic HSV vector G47Delta to metastatic breast cancer in the brain.</a> Gene Ther. 12 (8), 647-654 (2005).</li><li>Kumar, P., 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=Transvascular+delivery+of+small+interfering+RNA+to+the+central+nervous+system.">Transvascular delivery of small interfering RNA to the central nervous system.</a> Nature. 448 (7149), 39-43 (2007).</li><li>Wrobel, J. K., Wolff, G., Xiao, R., Power, R. F., Toborek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+Selenium+Supplementation+Modulates+Growth+of+Brain+Metastatic+Tumors+and+Changes+the+Expression+of+Adhesion+Molecules+in+Brain+Microvessels.">Dietary Selenium Supplementation Modulates Growth of Brain Metastatic Tumors and Changes the Expression of Adhesion Molecules in Brain Microvessels.</a> Biol Trace Elem Res. , (2015).</li></ol>

The authors have nothing to disclose.

ここで説明する輸液顕微は、本体1、12を通じて不要な普及を防止する、CNSに様々な生物学的特徴の外因性物質を提供する上で非常に成功することが証明されています。血液脳関門の破壊は、いくつかのCNS関連疾患の病理学的特徴です。したがって、血液脳関門と外因性物質の関係を評価することは非常に重要かつ興味深いものです。 提示され、この手術モデルは、動物に限定外傷を引き起こし、非常に低い死亡率1、12に関連しています。また、手順は、CNSの機能又は脳血流1、12と干渉しません。この手順の最も重要な側面は、のみを許可すべきであるECAでの正しいカットを実行することですキャピラリーチューブを挿入します。広いカットが反対サイト上の他のECAの手術を必要とする、物質の漏れが注入されることになります。このような状況を防ぐために、キャピラリーチューブはカットよりも大きな直径を有しているべきであり、また動脈を通じて進入を容易にするために鋭いエッジを有するべきです。技術的側面について、完全に訓練された人員は、20分以内に、この手術を行うことができます。 この技術の主要な制限は、同じICAを通して一度だけ注入の可能性です。 CNS、使用すると、ここで説明1【選択技術は、BBB内に新しい正と負の相互作用に関する研究を支援するため、ならびに病原体を送達する可能性を有している、以前に記載されている必要があり、マイクロ容器に繰り返し物質送達のため、薬、および脳への生理的な薬。

中枢神経系のin vivoモデル（CNS）疾患は、脳の中に、このような薬物、病原体、またはエキソソームなどの外因性物質の効果的な送達を必要とします。したがって、理想的な配信方法は、動物に最小限の外傷を引き起こす神経ネットワークの整合性を維持し、脳の1の高い物質の濃度を達成する必要があります。 局所物質送達のいくつかの外科的方法は、内シース、脳内、及び脳室内注射またはインプラント2、3、4、5を含む、記載されています。これらのアプローチは、しかし、CNSへの外傷と考えられており、目的の物質の唯一の少量の投与を可能にしています。また、外因性の物質が急速に脳脊髄液6によって除去することができることが示唆されています</s&gt;まで、および上記の技術が使用されるときに脳実質への低い浸透範囲が7観察されています。それらは他の器官8,9による取り込みに起因するCNSに物質を送達するの低い有効性を示すものの、経口、肺、皮下、および静脈内経路などの全身送達方法は、より一般的に、動物モデルにおいて使用されています。したがって、これらの送達経路は、副作用および毒性10,11のリスクを増加させる、投与物質の高められた用量を必要とします。 ここでは、効果的に内頸動脈を介して脳に直接物質を提供するマウス輸液顕微法を、説明します。 CNSへの送達を標的化することに加えて、この技術は、正常な生理学的障壁を迂回し、従ってバイオロジカに非常に関連していません脳への治療薬または病原体の通路に関与リットルを処理します。

<table><tbody><tr><td>Anesthesia instrument</td><td>Vetequip</td><td>901806</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tool</td><td>14558-09</td><td></td></tr><tr><td>Surgical forceps straight tip</td><td>Fine Science Tool</td><td>00108-11</td><td></td></tr><tr><td>Surgical forceps angled tip</td><td>Fine Science Tool</td><td>00109-11</td><td></td></tr><tr><td>Spring scissors</td><td>Fine Science Tool</td><td>15000-08</td><td></td></tr><tr><td>Nylon suture</td><td>Braintree Scientific</td><td>SUT-S 104</td><td></td></tr><tr><td>Capillary tubing (Micro-Renathane 0.010&amp;rdquo; x 0.005&amp;rdquo; per ft.)&amp;nbsp;</td><td>Braintree Scientific</td><td>MRE01050</td><td></td></tr><tr><td>Closing suture</td><td>VWR</td><td>95057-036</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal</td><td></td><td></td></tr><tr><td>2,3,5-Triphenyltetrazolium chloride</td><td>FisherScientific</td><td>50-121-8005</td><td></td></tr></tbody></table>

mouse microsurgery infusion technique for targeted substance delivery into the cns via the internal carotid artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

ここに記載のマウス注入顕微法は、非常に多用途であり、脳転移の形成1、12の代表的なモデルでの腫瘍細胞の送達を含む、直接脳内に異なる物質を送達するために使用されてきました。 この技術はまた、CNS内の異なる病原体の病理学的側面を評価するのに適しています。 HIV感染症のマウスモデルでは、注入手術はCCAに直接ウイルス粒子を注入するために使用しました。我々は、7日目、手術後、CNSは、リアルタイムPCRにより、HIVに対して陽性であることがわかりました。また、同側半球感染は6-10半球（ 図2）よりも高い折り目ました。 ここで説明する注入手術の別の適切なアプローチが提供することです<html> CNSへの細胞外小胞（ECVs）、主にエキソソーム、。 図3は、注入後のマウスの24時間の同側の脳内の緑色蛍光タンパク質（GFP）で標識されたCD63、エキソソームのマーカーの存在を示します。 <img alt="図1" src="/files/ftp_upload/54804/54804fig1.jpg" /> 図1：内頸動脈を介して注入の概略図。インサート（A）および（B）は、それぞれ、上下ノットの局在を示します。 （c）は、キャピラリチューブが挿通されるECAの小さな切り込み（灰色の線）を表します。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54804/54804fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <html> 4804fig2.jpg &quot;/&gt; 図2：HIV の検出リアルタイムPCRによる内頸動脈を介したウイルス注入後。バーは7日、左内頸動脈を介して、注入後のマウスから採取した平均HIV DNAレベルを示します。エラーバーは標準偏差を示します。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54804/54804fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/54804/54804fig3.jpg" /> 図3：内頸動脈を介して ECVs注入後のマウス脳節描いたGFPタグ付きCD63、エキソソームマーカーの免疫蛍光。代表画像は、CD63を発現するECVs（緑）に関連したマウス脳微小血管を示しています。スケールバー：50μmです。= &quot;_空白」を取得&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。

Watch this Scientific Journal Video about Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery at JoVE.com

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

CNSへの標的化物質送達のためのマウス顕微注入法<i&gt;経由で</i&gt;内頸動脈

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

mouse-microsurgery-infusion-technique-for-targeted-substance-delivery

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

10.8K ビュー.  University of Miami. The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

ビデオ: Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

脳転移のマウスモデルを生成するために頸動脈内癌細胞注入

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

がん研究

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an injecting needle produces during its trajectory is a major concern especially in the mouse brain for not only the brain is small but also sometimes multiple injections are needed.  We show here a method to produce glass capillary needles with a 50-&mu;m lumen which significantly reduces the brain damage and allows a precise targeting into the rodent brain. This method allows a delivery of small volumes (from 20 to 100 nl), reduces bleeding risks, and minimizes passive diffusion of drugs into the brain parenchyma. By using different size of capillary glass tubes, or changing the needle lumen, several types of substances and cells can be injected. Microinjections with a glass capillary tube represent a significant improvement in injection techniques and deep brain targeting with minimal collateral damage in small rodents.

In this article, we show a method to make glass capillary needles with a 50-&mu;m lumen. This technique significantly reduces the brain damage, minimizes passive diffusion of drugs and allows a precise targeting into the rodent brain.

薬の配達のためのMicroinjections、ウイルスベクター、または細胞移植で脳深部構造のターゲティング

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

神経科学

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with high precision while minimizing localized trauma and inflammation. Also, the ability to customize the system to accommodate animals of different species and sizes is desirable. This video presentation demonstrates the construction of an infusion system that can be fitted to any particular research animal. The delivery microcannula diameter is approximately 10-fold smaller than most infusion cannulas presently used. This translates into enhanced accuracy and reduced trauma to the brain region under study. The delivery cannula can also be sculpted to fit the contour of the surface of the animal's skull, thereby allowing closure of the scalp incision neatly over the infusion system, precluding the need for a skull-mounted pedestal, reducing risk of infection, and ensuring a greater level of comfort to the animal. The system is assembled in an air-free environment and requires the researcher to fashion glass micropipettes with a heat source. These construction methods require special skills that are best acquired, if not in person, using video instruction.  (This article is based on work first reported in J Neurosci Methods. 2008 Jan 30;167(2):213-20. Epub 2007 Aug 28.).

As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns.

神経刺激剤の徐放のためのMicroinfusionシステムの構築と注入。

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with ...

生物学

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

腔内MCAOのマウスモデル：クレシルバイオレット染色による脳梗塞の評価

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

医学

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

血液脳関門の破壊のラットモデルは、対象となる神経血管治療を許可する

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

がん細胞の内頸動脈注射による脳転移のモデル化

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

マウスの脳に直接薬剤送達のための異粘膜移植手順

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

<meta charset="utf8"></head><body>マウスモデルでの注射後の総頸動脈修復

Facilitating Repeat Intracarotid Injections in Mouse Models by a Novel Injection Site Repair Technique

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there is an urgent need to develop methods for intracarotid injections in mouse models, including methods to repair the carotid artery in mice after injection to allow for subsequent injections. We developed a method of intracarotid injection in a mouse model to deliver therapeutics into the internal carotid artery (ICA) with two alternative procedures. 
During injection, the needle is inserted into the common carotid artery (CCA) after tying a suture around the external carotid artery (ECA) and injected therapeutics are delivered into the ICA. Following injection, the common carotid artery (CCA) can be ligated, which limits the number of intracarotid injections to one. The alternative procedure described in this article includes a modification where intracarotid artery injection is followed by injection site repair of the CCA, which restores blood flow within the CCA and avoids the complication of cerebral ischemia seen in some mouse models. 
We also compared the delivery of bone marrow-derived human mesenchymal stem cells (BM-hMSCs) to intracranial tumors when delivered through intracarotid injection with and without injection site repair following the injection. Delivery of BM-hMSCs does not differ significantly between the methods. Our results demonstrate that injection site repair of the CCA allows for repeat injections through the same artery and does not impair the delivery and distribution of injected material, thus providing a model with greater flexibility that more closely emulates intracarotid injection in humans.

<meta charset="utf8"></head><body>著者スポットライト:腫瘍溶解性ウイルスとマイクロRNAの動脈内送達戦略による膠芽腫治療の進歩

Repair of the intracarotid artery in a mouse model after injection returns blood flow to the artery without negatively impacting the distribution of the injected material. Injection site repair facilitates subsequent injections through the same artery and prevents cerebral ischemia in mouse strains that lack a complete Circle of Willis.

新しい注射部位修復技術によるマウスモデルにおける反復頸動脈内注射の促進

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there ...

CNSへの標的化物質送達のためのマウス顕微注入法

要約

さらに動画を探す

この動画の章

脳転移のマウスモデルを生成するために頸動脈内癌細胞注入

薬の配達のためのMicroinjections、ウイルスベクター、または細胞移植で脳深部構造のターゲティング

神経刺激剤の徐放のためのMicroinfusionシステムの構築と注入。

腔内MCAOのマウスモデル：クレシルバイオレット染色による脳梗塞の評価

血液脳関門の破壊のラットモデルは、対象となる神経血管治療を許可する

がん細胞の内頸動脈注射による脳転移のモデル化

マウスの脳に直接薬剤送達のための異粘膜移植手順

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

新しい注射部位修復技術によるマウスモデルにおける反復頸動脈内注射の促進

新しい注射部位修復技術によるマウスモデルにおける反復頸動脈内注射の促進