Name: mouse microsurgery infusion technique for targeted substance delivery into the cns via the internal carotid artery
Uploaded: 2017-01-31T14:00:00
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?...

<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. 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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. 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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。 <p class="jove_...

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。

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			<td height="21" style="height:21px;width:367px;">Anesthesia instrument</td>
			<td style="width:149px;">Vetequip</td>
			<td align="right" style="width:119px;">901806</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Surgical scissors</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">14558-09</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Surgical forceps straight tip</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">00108-11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Surgical forceps angled tip</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">00109-11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Spring scissors</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">15000-08</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Nylon suture</td>
			<td style="width:149px;">Braintree Scientific</td>
			<td style="width:119px;">SUT-S 104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary tubing (Micro-Renathane 0.010&#8221; x 0.005&#8221; per ft.)&#160;</td>
			<td style="width:149px;">Braintree Scientific</td>
			<td style="width:119px;">MRE01050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Closing suture</td>
			<td style="width:149px;">VWR</td>
			<td style="width:119px;">95057-036</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Isoflurane</td>
			<td style="width:149px;">Piramal</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">2,3,5-Triphenyltetrazolium chloride</td>
			<td style="width:149px;">FisherScientific</td>
			<td style="width:119px;">50-121-8005</td>
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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?...

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 ...

The overall goal of this surgical technique is to deliver exogenous substances into the mouse brain without penetrating the cranium. This method can help answer key questions in neuroscience regarding the action of drugs, pathogens, and exosomes in the brain. The main advantage of this technique is that it causes minimal trauma to the animal, preserves the integrity of the neuronal network, and achieves high concentrations of the substance in the brain.Begin by administering isoflurane mixed with oxygen to anesthetize the mouse in an induction chamber. After induction of anesthesia, transfer the animal to the surgical surface under a stereo microscope, and place the nose cone to maintain a surgical plane of anesthesia. Ensure that the respiratory rate is around one to two respirations per second without gasping.In addition, ensure that the animal does not react to whisker stimulation or a toe pinch. Use a sterile swab to apply a drop of ophthalmic lubricant on each eye to prevent dryness. After shaving the surgical site with a razor, prep the area with 70%ethanol and chlorhexidine.While looking through the stereo microscope, use surgical scissors and forceps to create a midline incision in the neck from above the breastbone to below the jaw. Use forceps to carefully separate fatty and connective tissue to expose the trachea. Then place a soft, round object of about zero point five centimeters in diameter under the back of the neck to extend the neck and further expose the area.Next, separate the tissue around the trachea using hooks or a tissue retractor. On the left side of the trachea, carefully tweeze apart the connective tissue to expose the left common carotid artery, or CCA. Using forceps, carefully remove all connective tissue to expose the CCA bifurcation in the beginning of both the external and internal carotid arteries.Once the ECA and ICA are exposed, use forceps to place a segment of one-centimeter nylon suture under the external carotid artery. Then place a second one-centimeter segment. Then tie a permanent knot at the highest point possible of the ECA.At the lowest point possible of the ECA, immediately above the CCA bifurcation, tie a looser, removable knot. Then use vessel clips to close the CCA at the lowest possible point, and to close the ICA. Properly closing the internal carotid artery before making a small cut in the external carotid artery will prevent bleeding.Now, using microdissection spring scissors, make a two-millimeter cut in the ECA between the two knots. It is important to keep the cuts small, as a large cut will cause leaking of the substance to be infused. Assemble the infusion system by attaching six inches of capillary tubing to a tuberculin syringe containing 250 microliters of the substance to be infused.Then gently insert the tip of the capillary tubing into the incision in the ECA. Keep advancing the tip until it reaches midway between the bifurcation and the clip on the CCA. Tie down the lower ECA knot.Verify that the knot is loose enough to allow capillary fluidity, and tight enough to prevent leaking. Then, remove the clip from the ICA. Gently apply pressure to the syringe piston, allowing substance infusion at approximately 10 microliters per second.Following infusion of the test substance, place the clip back on the ICA. Gently remove the capillary tubing. Then, tie down the lower ECA knot completely.Remove the clip from the ICA and the CCA. Then remove the retractor or tissue hooks and pillow. Clean the area to be sutured with sterile saline, and then close the incision using nylon sutures.After administering an approved anti-inflammatory agent and analgesic to alleviate post-operative discomfort, place the animal in a cage on a heating pad for at least one hour, and monitor recovery. This histogram demonstrates HIV detection by real-time PCRs seven days after viral infusion via the left internal carotid artery. As expected, levels of the virus are higher in the left hemisphere.This immunofluorescence image shows a mouse brain section depicting GFP tag CD63, an exosome marker, after extracellular vesicle infusion via the internal carotid artery. The CD63 expressing extracellular vesicles, shown in green, are associated with the mouse brain microvessel. Once mastered, this technique can be done in 20 minutes if it is performed properly.After its development, this technique paved the way for researchers in the field of neurodegenerative diseases to explore drug, pathogens, and exosome interactions in mice.

Research

JoVE Journal

Neuroscience

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

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超音波ガイド下のマウスの前脳へのマイクロインジェクション子宮内で E9.5で

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

神経科学

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使用した複数のCNS準州への効率的な遺伝子送達子宮内でエレクトロポレーション

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

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The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

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Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

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Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...

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