Name: assessment of blood-brain barrier permeability by intravenous infusion of fitc-labeled albumin in a mouse model of neurodegenerative disease
Uploaded: 2017-11-08T14:00:00
Description: Watch this Scientific Journal Video about Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease at JoVE.com

この作品が"Fondazione Neuromed"でサポートされている、健康参加する「Ricerca グラーヴェ」イタリア省の資金

動物のすべてのプロシージャは IRCCS Neuromed 動物介護審査会によって、承認された &#34; 会館 Istituto スペリオーレ ディ Sanità &#34; (許可番号: 1163/2015-PR) され、2010 年の EU 指令のガイドラインに従って/63/EU 動物実験。
 1 頸静脈に挿入される FITC-アルブミン溶液の調製 注: すべての実験が実施をマニフェスト (週齢 11) HD R6/2 マウス、時代、性別一致の野生...

<ol>
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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=Blood-Brain+Barrier+Leakage+in+Patients+with+Early+Alzheimer+Disease.">Blood-Brain Barrier Leakage in Patients with Early Alzheimer Disease.</a> Radiology. 282 (2), 615 (2017).</li><li>Fernandez-Lopez, D., 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=Blood-brain+barrier+permeability+is+increased+after+acute+adult+stroke+but+not+neonatal+stroke+in+the+rat.">Blood-brain barrier permeability is increased after acute adult stroke but not neonatal stroke in the rat.</a> J Neurosci. 32 (28), 9588-9600 (2012).</li><li>Yang, Y., Rosenberg, G. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+pancreatic+islets+into+the+kidney+capsule+of+diabetic+mice.">Transplantation of pancreatic islets into the kidney capsule of diabetic mice.</a> J Vis Exp. (9), e404 (2007).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J Vis Exp. (65), (2012).</li><li>McCloy, R. 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=Partial+inhibition+of+Cdk1+in+G+2+phase+overrides+the+SAC+and+decouples+mitotic+events.">Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events.</a> Cell Cycle. 13 (9), 1400-1412 (2014).</li><li>Burgess, 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=Loss+of+human+Greatwall+results+in+G2+arrest+and+multiple+mitotic+defects+due+to+deregulation+of+the+cyclin+B-Cdc2/PP2A+balance.">Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance.</a> Proc Natl Acad Sci U S A. 107 (28), 12564-12569 (2010).</li><li>Krueger, M., Hartig, W., Reichenbach, A., Bechmann, I., Michalski, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+breakdown+after+embolic+stroke+in+rats+occurs+without+ultrastructural+evidence+for+disrupting+tight+junctions.">Blood-brain barrier breakdown after embolic stroke in rats occurs without ultrastructural evidence for disrupting tight junctions.</a> PLoS One. 8 (2), e56419 (2013).</li><li>Hirano, A., Kawanami, T., Llena, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+of+the+blood-brain+barrier+in+disease.">Electron microscopy of the blood-brain barrier in disease.</a> Microsc Res Tech. 27 (6), 543-556 (1994).</li></ol>

我々 はここで説明する手法は、主に脳疾患条件下で BBB の漏れを検出するため便利です。BBB の機能不全が多様な神経学的疾患4の共通の特徴として注目されています。我々 は以前 HD6のようなまれな神経変性疾患のマウスモデルで BBB 整合性の初期の異常を説明するのにこのアプローチを使用します。
このメソッドは、相対的な簡易性?...

An erratum was issued for: <a href="https://www.jove.com/video/56389/assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc">Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease</a>. One of the author&#39;s names was corrected from:
Maglione Vittorio
to:
Vittorio Maglione

An erratum was issued for: <a href="https://www.jove.com/video/56389/assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc">Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease</a>. One of the author's names was corrected.

Erratum: Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

中枢神経系 (CNS) 内の恒常性は、適切なコミュニケーションと神経細胞の機能のための前提条件です。中枢神経系柔密によって封鎖される周辺から、内皮細胞血液脳関門 (BBB)、末梢血流と脳との間のインターフェイスを表し、これらの 2 つの地区の1 間のクロストークで極めて重要な役割を果たしています。 ,2。BBB は複雑な専門マイクロ血管内皮細胞) 細胞複合体 - タイトな接合 (TJs) - によって互いにリンク、囲まれて周を主体とする立体構造物の動的ニューロン終末とアストロ サイトの足プロセス1,2。
生理学的な条件の下でそのまま BBB の非常に低透磁率により、脳の栄養素および他の分子の輸送の厳しい規制とにより中枢神経系の変化からユニークな保護、潜在的な末梢神経活動に影響を与える可能性があります血液の組成は、1,2,3を侮辱します。
BBB の整合性および高められた透磁率の中断長い神経の多くの重要な機能を構成するため知られている、神経変性障害4ただしハンティントンの病気 (HD)5,6, を含む、かどうかこのような機能不全が原因の現象や病気の過程で繁殖イベントはまだ不明です。BBB 破壊のタイミングもとらえどころのないまま、しかし、中断の BBB の整合性が、後半イベント早期のステップ6ではなく、病気の進行で,7を表さないことを示します出現の私達のグループと他の証拠,8、長期的な影響を持っている可能性があります。
これを念頭において、早熟役に立つ病気の進行と脳損傷を予測し、正常に代替しよりターゲットを絞った介入できるを開発する戦略を開発するために神経変性の BBB の内訳を明らかにすることが重要です。このような混乱の臨床結果を軽減します。したがって、BBB 減損の信頼性の高い画像は、脳疾患の診療・研究の主要な重要なのです。
本稿では高分子量フルオレセイン イソチオ シアン酸ラベル-アルブミン (FITC-アルブミン) を使用して、HD マウス モデルの BBB 透過性の評価の成功と単純な手順をについて説明します。通常、脳実質にバリアを越えることはできません FITC-アルブミンの漏出を BBB 漏れの指標として測定しました。この手法は、ラットと cerebrovasculature 障害9,10によって特徴付けられる他の病理学の条件に容易に適応可能であります。

<table>
	<tbody>
		<tr>
			<td>Albumin-fluorescin isothiocyanate conjiugate</td>
			<td>SIGMA</td>
			<td>A9771-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>pAb anti-Laminin</td>
			<td>Novus Biologicals</td>
			<td>NB300-144</td>
			<td></td>
		</tr>
		<tr>
			<td>CY3 anti-rabbit made in goat</td>
			<td>MILLIPORE</td>
			<td>AP132</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPERFROST PLUS</td>
			<td>Thermo Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Slips 24 X 50 mm</td>
			<td>Thermo Scientific (DIAPATH)</td>
			<td>61050</td>
			<td></td>
		</tr>
		<tr>
			<td>Kilik Optimal Cutting Temperature (OCT) compound</td>
			<td>Bio Optica</td>
			<td>05-9801</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Mounting Media</td>
			<td>VECTOR</td>
			<td>H-1500</td>
			<td>Mounting media with DAPI</td>
		</tr>
		<tr>
			<td>iNSu/Light Insulin Disposible Syringe</td>
			<td>RAYS Health &#38;Safety</td>
			<td>INS1ML26G13</td>
			<td></td>
		</tr>
		<tr>
			<td>30G 1/2&#34;</td>
			<td>BD Microlance</td>
			<td>304000</td>
			<td>Needle for Insulin disposible Syringe</td>
		</tr>
		<tr>
			<td>Scalpel Handle</td>
			<td>F.S.T.</td>
			<td>91003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>#22 Disposable Scalpel blads</td>
			<td>F.S.T.</td>
			<td>10022-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Iris scissors 10.5 cm</td>
			<td>F.S.T.</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Forceps #5745 45&#176; 0.10 x 0.06 mm</td>
			<td>F.S.T.</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps 10 cm</td>
			<td>F.S.T.</td>
			<td>11051-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Forceps #5 0.1 X 0.06 mm</td>
			<td>F.S.T.</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical patch</td>
			<td>Medicalis</td>
			<td>34788</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile disposable towel drape</td>
			<td>Dispotech</td>
			<td>TVO50VE</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscopic Microscope</td>
			<td>NIKON</td>
			<td>SMZ 745 T</td>
			<td></td>
		</tr>
		<tr>
			<td>Optic Illuminator LED light (C-FLED2)</td>
			<td>NIKON</td>
			<td>1003167</td>
			<td>Optic Illuminator for Stereoscopic Micrscope</td>
		</tr>
		<tr>
			<td>Eclipse Ni-U Microscope</td>
			<td>Nikon</td>
			<td>932162</td>
			<td>Epifluorescence Microscope</td>
		</tr>
		<tr>
			<td>Microscope digital Camera</td>
			<td>Nikon</td>
			<td>DS-Ri2</td>
			<td>Microscope camera</td>
		</tr>
		<tr>
			<td>Intenslight</td>
			<td>Nikon</td>
			<td>C-HGFI</td>
			<td>Microscope lamp</td>
		</tr>
		<tr>
			<td>NIS-Elements 64 bit</td>
			<td>Nikon</td>
			<td>AR 4.40.00</td>
			<td>Analysis Software</td>
		</tr>
		<tr>
			<td>Electric Razor</td>
			<td>Gemei</td>
			<td>GM-3007</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of blood-brain barrier permeability by intravenous infusion of fitc-labeled albumin in a mouse model of neurodegenerative disease

血液脳関門 (BBB) 整合性の破壊は、異なる神経と神経変性疾患の一般的な機能です。開発と BBB の変化を正確に検出する信頼性の高いプロシージャの検証が重要であるし、便利なツールを表す摂動の BBB の恒常性と脳疾患の病態との間の相互作用は、さらなる調査を必要とするが潜在的疾患を予測するためターゲットを絞った治療戦略を進行と開発。
ここでは、BBB の BBB 透過性の欠陥は明らかに早熟で検出可能なハンチントン病のマウスの前臨床モデルで発生するそのような変性条件で漏れを評価するための簡単で効率的な手順を提案します。病気。具体的には、高分子量かにラベル (FITC)-アルブミン、後者は障害時にのみ、BBB を交差することがあるがマウスの頸静脈と血管や実質地区におけるその分布に鋭く融合その後、蛍光顕微鏡によって決定されます。
脳実質の緑色蛍光アルブミンの蓄積異常 BBB 透過性の指標として機能し、J の画像処理ソフトウェアを使用して量的に表わされるとき緑の蛍光強度として報告されます。

FITC-アルブミン血流から緑色の蛍光トレーサーの BBB は脳 parenchymawhen に血管外漏出に頸静脈結果の適切な注入は、6を妥協しました。生理学的な条件の下で注入蛍光アルブミンの分布は脳の血管の内側に制限と周囲の血管領域や脳実質内に信号が検出できなかった (図 1 a、顕微鏡写真で、トップ)。逆に、BBB 神経病理学的状況下?...

Watch this Scientific Journal Video about Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease at JoVE.com

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

本研究では神経変性条件下で血液-脳関門の破壊を評価するための簡単で効率的な手順を提案する.我々 の目的を達成するために我々 は高分子量かにラベル (FITC) を注入-マウス頸静脈にアルブミン静脈および蛍光顕微鏡による脳実質にその漏洩を評価します。

神経変性疾患のマウスモデルで FITC 標識アルブミンの点滴静注による血液脳関門透過性の評価

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

The overall goal of this procedure is to assess the brain-blood barrier permeability in a mouse model of degenerative disorder by intrajugular injection of FITC-labeled albumin. This method can help answer the key questions in the study of brain homeostasis. It can be particularly useful to investigate any possible link between blood-brain barrier damage and neurodegeneration.The main advantage of this technique is that it is fast and easy to perform and can be applied, also, to larger animal models and to other neuro-pathological settings. Demonstrating the surgical procedure of intrajugular injection of FITC-albumen will be Luca Capocci. Salvatore Castaldo will demonstrate the histological sectioning of a brains.And Enrico Amico will demonstrate the procedure for micrograph acquisition. Begin by preparing FITC-albumen solution by dissolving FITC-albumen powder in phosphate buffered saline at 10 milligrams per milliliter. Lay out the autoclaved surgical instruments.Clean the surgical bench with 70%ethanol and place a surgical sterile disposable drape. Then set up the operating stereo microscope. After anesthetizing the mouse with an intraperitoneal injection of an appropriate does of ketamine xylazine solution, monitor animal sedation by giving a gentle toe pinch and ensuring the absence of the withdrawal reflex.Place the anesthetized mouse in a face-up supine position and fix the four legs and the tail on the surgical workbench with adhesive tape. After the mouse is secured, carefully shave the appropriate surgical margin on the neck and disinfect it with povidone-iodine solution in 70%ethanol. Dry the exposed shaved surface with cotton swabs.Next, after making a 1.5 centimeter long longitudinal incision in the mid-clavicular line, starting midway on the thorax and extending to just below the neck, carefully stretch apart connective tissue with forceps under microscopic observation to expose the external jugular vein. Ensure sufficient space on the right and on the left of the jugular vein to facilitate the insertion of the 30 and a half gauge needle for the infusion of the FITC-albumen solution. Inject 100 microliters of 10 milligrams per milliliter FITC-albumen in the right jugular vein over three minutes.This is the most critical step during the procedure. Incorrect insertion of the needle can cause irreversible damage of the vein wall and compromise the entire experiment. Fifteen minutes after injection, after euthanizing the mouse by decapitation, rapidly remove the brain from the skull and immerse it in a volume of pre-chilled isopentane, that is ten times that of the brain, for 15 minutes.Move the isopentane frozen brain into a pre-chilled tube and store it in a 80 degrees Celsius freezer until histological sectioning. Embed frozen brain in optimal cutting temperature compound prior to cryostat sectioning. Cut the brain into 20 micron thick sections with a cryostat and mount them onto microscope glass slides.Perform the FITC-albumin laminin co-staining by using a specific anti-laminin antibody, as previously described. Cover slip slides with mounting medium containing DAPI and leave the slide to dry overnight at four degrees Celsius in the dark. The next day, observe the fluorescence staining under a fluorescence microscope at 20 times magnification, equipped with both FITC and Cy3 filters.Turn on the fluorescence lamp about 10 minutes before use. Turn on the microscope, the connected computer and the digital camera. Run the image analysis software.Use the appropriate objective for optimal signal collection and spatial resolution and select appropriate filters. Acquire a minimum of four, 24-bit color exposures per brain slice. Analyze the fluorescence intensity for each single channel, using the freely available ImageJ software.The following representative fluorescence micrographs of brain cryosections show differential distribution of green fluorescent albumin under both physiological and pathological conditions. The red staining, shown here, represents the vascular marker, laminin, under physiological conditions and under conditions of blood-brain barrier compromise. These merged images show co-localization of green fluorescent albumin and laminin, along with DAPI staining of nuclei in the intact and compromised blood-brain barriers.This graph displays the quantification of the green fluorescence emitted by FITC-albumin, which is reported as the green fluorescence intensity in both healthy and compromised conditions. While attempting this procedure, it's important to remember to freshly prepare each single reagent and predispose the surgical bench. After watching this video, you may have a good understanding of how to assess the permeability of the blood-brain barrier in an animal model of neurodegenerative disease, by measuring the affixation of the green fluorescent albumin into brain parenchyma.

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神経変性疾患のマウスモデルのための嗅覚アッセイ

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

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

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

生体内で血液脳関門透過性アッセイを用いたマウス蛍光トレーサーに分類

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

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

虚血性脳卒中のモデルラットの血液脳関門の破壊のIn Vivo評価

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

病原体や医薬品との相互作用、脳によって障壁の交差を勉強する人間の血-脳インターフェイス モデル

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

ラットモデルにおける集中超音波を用いた位置特異的血液脳関門開口へのベンチトップアプローチ

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease

パーキンソン病のラットモデルにおけるレボドパ誘発性ジスキネジアの誘導と評価

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements ...

ショウジョウバエの便秘アッセイ

Measuring Constipation in a Drosophila Model of Parkinson's Disease

著者スポットライト:パーキンソン病における非運動症状の探求 

パーキンソン病のショウ ジョウバエ モデルにおける便秘の測定

Non-motor symptoms in Parkinson's disease (PD) are common, difficult to treat, and significantly impair quality of life. One prevalent non-motor symptom ...