Name: experimental models to study the neuroprotection of acidic postconditioning against cerebral ischemia
Uploaded: 2017-07-31T12:30:00
Description: Watch this Scientific Journal Video about Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia at JoVE.com

この研究は、中国国立自然科学財団（81573406,81373393,81273506,81221003,81473186および81402907）、浙江省自然科学財団（LR15H310001）およびS＆Tイノベーションチームの浙江省主導チーム（2011R50014）の資金提供を受けて行われました。

すべての実験は、浙江大学動物実験委員会の倫理指針に従って承認され、実施され、実験動物の管理と使用のための国立衛生研究所ガイドラインに完全に準拠していた。痛みや不快感を最小限に抑えるための努力がなされ、最低限の動物数が使用された。 コルチコステリアスライスのOGD <ol><li> ソリューションの準備： <ol><li>準備した1000mLのR-ACSF（124ミリモ...

<ol>
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Y., 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+novel+neuroprotective+strategy+for+ischemic+stroke:+transient+mild+acidosis+treatment+by+CO2+inhalation+at+reperfusion.">A novel neuroprotective strategy for ischemic stroke: transient mild acidosis treatment by CO2 inhalation at reperfusion.</a> J Cereb Blood Flow Metab. 34 (2), 275-283 (2014).</li><li>Shen, Z., 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=PARK2-dependent+mitophagy+induced+by+acidic+postconditioning+protects+against+focal+cerebral+ischemia+and+extends+the+reperfusion+window.">PARK2-dependent mitophagy induced by acidic postconditioning protects against focal cerebral ischemia and extends the reperfusion window.</a> Autophagy. , (2017).</li><li>Skolnik, J., Takacs, L., Szende, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+oxygen+consumption+of+slices+from+kidney,+brain,+cortex+and+liver+in+hypoxia.">In vitro oxygen consumption of slices from kidney, brain, cortex and liver in hypoxia.</a> Nature. 209 (5020), 305 (1966).</li><li>Lynch, G., Schubert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+in+vitro.+brain+slices+for+multidisciplinary+studies+of+synaptic+function.">The use of in vitro. brain slices for multidisciplinary studies of synaptic function.</a> Annu Rev Neurosci. 3, 1-22 (1980).</li><li>Zheng, S., Zuo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+preconditioning+reduces+purkinje+cell+death+in+an+in+vitro+model+of+rat+cerebellar+ischemia.">Isoflurane preconditioning reduces purkinje cell death in an in vitro model of rat cerebellar ischemia.</a> Neuroscience. 118 (1), 99-106 (2003).</li><li>Yin, B., Barrionuevo, G., Weber, S. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+vulnerability+of+CA1+vs+CA3+pyramidal+neurons+after+ischemia:+possible+relationship+to+sources+of+Zn2++accumulation+and+its+entry+into+and+prolonged+effects+on+mitochondria.">Differential vulnerability of CA1 vs CA3 pyramidal neurons after ischemia: possible relationship to sources of Zn2+ accumulation and its entry into and prolonged effects on mitochondria.</a> J Neurosci. , (2016).</li><li>Pignataro, 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=In+vivo+and+in+vitro+characterization+of+a+novel+neuroprotective+strategy+for+stroke:+ischemic+postconditioning.">In vivo and in vitro characterization of a novel neuroprotective strategy for stroke: ischemic postconditioning.</a> J Cereb Blood Flow Metab. 28 (2), 232-241 (2008).</li><li>Zhang, X., Ding, H. 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ここでは、脳虚血に対するAPCの神経保護を研究するための2つの実験モデルを提示する。脳切片において、APCは、再灌流後に20％CO 2で泡立てた酸性緩衝液中でマウスの皮質層をスライスすることによって達成されるが、MCAOモデルでは、再灌流後に20％CO 2を吸入することによってAPCが達成される。両方のモデルは、脳虚血に対するAPCの神経保護を反映する。保護は、虚血後事後?...

脳卒中は世界中の死亡および障害の主要な原因の1つです。過去数十年間に脳卒中の有効な治療法を見出すために大きな努力がなされてきたが、達成は非常に不十分である。ポストコンディショニングは、虚血性エピソードに続く亜毒性ストレスによって操作されるプロセスである。ポストコンディショニングは、虚血、低酸素、低血糖およびリモート虚血ポストコンディショニングを含め、内因性の適応メカニズムをトリガーし、脳虚血1、2、3、4に対する治療を有望であることが証明されてきました。しかし、虚血後事後調整は、さらなる損傷を導入する可能性がある。同側または両側後肢5、6、7日に20分間閉塞および再灌流-通常5の数サイクルを必要とするリモート虚血ポストコンディショニングを四肢。 Thそのため、これらの事後条件付け操作は臨床的に危険であり実用的ではありません。これらの欠点を克服するために、我々はマウスにおける局所脳虚血の治療薬としてAPCを開発した8 。単に20％CO 2を吸入することによって誘発されるAPCは、虚血性脳損傷をより現実的かつより安全な方法で有意に低減する。最近、我々は、APCが再灌流ウインドウを拡張し、脳卒中治療9に対するAPCの意義を強調していることを証明した。 ここでは、脳虚血に対するAPCの神経保護を研究するための2つの実験モデルを提示する。最初のものは、マウスの皮質層スライスの酸素 - グルコース欠乏（OGD）モデルである。人工脳脊髄液（ASCF）のような人工環境への脳スライスの迅速な調製および伝達は、細胞生存能力およびニューロン回路を維持することができ、インビトロで脳機能を研究することを可能にする10 &lt;11 。 ASCF模倣におけるOGD脳虚血及び虚血性傷害12、13、14を誘導します。 OGDの後、脳スライスを通常のASCF（r-ASCF）で再灌流して再灌流を行い、次いで20％CO 2で泡立てた酸性ASCFを用いてAPCで処置する。皮質層スライスは、初代培養細胞と比較して無傷の組織学的特徴付けを維持する。 インビボで脳機能を研究するために、マウス中大脳動脈閉塞（MCAO）モデルを使用する。中大脳動脈は、総頸動脈を介して火炎平滑モノフィラメントを挿入することによって閉塞される。最も広く使用される脳卒中モデルの1つとして、MCAOモデルは臨床的関連性を示し、モノフィラメントの適用は再灌流をより容易に達成する。再灌流開始後に20％CO 2を含む酸素正常混合ガスを吸入するだけでAPCは、脳梗塞容積の減少によって示される脳虚血に対して有意な保護効果を示した。

<table border="0" cellpadding="0" cellspacing="0" width="723">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">S5886</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium chloride</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">P5405</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium phosphate monobasic</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">P9791</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium sulfate</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">M2643</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium bicarbonate</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">S5761</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium chloride dihydrate</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">C5080</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D-(+)-Glucose</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">G7021</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vibratome</td>
			<td style="width:183px;">Leica</td>
			<td style="width:157px;">VT1000 S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2,3,5-triphenyltetrazolium hydrochloride</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">T8877</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Absolute Ethanol</td>
			<td style="width:183px;">Aladdin Industrial Corporation</td>
			<td style="width:157px;">E111993</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl sulfoxide</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">D8418</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laser Doppler Flowmetry</td>
			<td style="width:183px;">Moor Instruments Ltd</td>
			<td style="width:157px;">Model Moor VMS-LDF2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Diethyl ether anhydrous</td>
			<td style="width:183px;">Sinopharm Chemical Reagent Corporation</td>
			<td style="width:157px;">80059618</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trichloroacetaldehycle hydrate</td>
			<td style="width:183px;">Sinopharm Chemical Reagent Corporation</td>
			<td style="width:157px;">30037517</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10% Formalin</td>
			<td style="width:183px;">Aladdin Industrial Corporation</td>
			<td style="width:157px;">F111936</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24-well plates</td>
			<td style="width:183px;">Jet Biofil</td>
			<td style="width:157px;">TCP-010-024</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

experimental models to study the neuroprotection of acidic postconditioning against cerebral ischemia

脳卒中は限られた治療法を用いて世界中の死亡および障害の主要な原因の1つです。神経保護のための内因性戦略として、事後コンディショニング治療は、脳虚血に対する有望な治療法であることが証明されている。しかしながら、複雑な処置および潜在的な安全性の問題は、それらの臨床適用を制限する。これらの欠点を克服するために、我々は、実験的局所脳虚血の治療法として、酸性後条件付け（APC）を開発した。 APCは、虚血後の再灌流中にCO 2を吸入することによる軽度のアシドーシス治療を指す。ここでは、APC をインビトロおよびインビボで実行するための2つのモデルをそれぞれ示します。マウスの酸素グルコース欠乏（OGD）処置およびマウスの皮質層の閉塞および中大脳動脈閉塞（MCAO）を用いて、脳虚血を模倣した。 APCは、脳スライスを20％CO 2で泡立てた酸性緩衝液に移すことによって簡単に達成することができる20％CO 2をマウスに吸入させる。組織生存率および脳梗塞容積に反映されるように、APCは脳虚血に対して有意な保護効果を示した。

上記の皮質層スライスモデルにおいて、皮質層のスライス生存率を、再灌流1時間後のTTCアッセイによって定量化した。 TTC変換は、490nmでの吸収を対照スライスに正規化することによって計算した。 TTC変換によれば、APCは、発症時間および持続時間依存的様式で、OGD誘発再灌流損傷に対して保護した。詳細には、1分および3分のアシドーシス治療は、15分のOGD後5分で生...

Watch this Scientific Journal Video about Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia at JoVE.com

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

酸性ポストコンディショニングは、脳虚血を防ぐ。ここでは、APCを実行する2つのモデルを示します。それらはインビトロで酸素 - グルコース欠乏後に皮質層スライスを酸性緩衝液に移し、インビボで中大脳動脈閉塞後に20％CO 2を吸入することによってそれぞれ達成される 。

脳虚血に対する酸性ポストコンディショニングの神経保護を研究するための実験モデル

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for ...

The overall goal of acidosis treatment in a cortico striatal slice model in a middle cerebral artery occlusion model is to study the neuroprotective effect of acidic postconditioning against cerebral ischemia. This method can help answer key questions of how acidosis postconditioning may protect ischemic brain injury, and to develop new strategy for stroke therapy. The main advantage of this technique is being able to use widely available experiment models to study the neuroprotection of acidosis postconditioning.Demonstrating the procedure will be Yarong Zheng a graduate student from my lab. Begin by removing the brain from the skull of a decapitated mouse with a thin spatula and dropping it carefully into a beaker containing ice cold regular ACSF equilibrated with five percent carbon dioxide and 95%oxygen. Apply cryoprecipitate glue to the vibratone plate in two strips.Put a piece of three percent agarose on the glue strip furthest from the blade to support the brain. Then use forceps to transfer the brain to filter paper. Cut off the frontal pole and the cerebellum with a blade.Place the brain tissue vertically on the free strip of glue with the brain leaning against the agarose. Add ice cold ACSF to the cutting reservoir to submerge the brain and add ice to the ice holder area. Keep the ACSF in the reservoir bubbled with five percent carbon dioxide and 95%oxygen.After setting the vibratone to cut 400 micron sections, and correctly positioning the brain relative to the cutting blade, press the start-stop button to start automatic cutting. Use a Pasteur pipette with a cut tip to transfer five brain slices one by one and place them into the tissue holder in ice cold regular ACSF bubbled with five percent carbon dioxide and 95%oxygen. After 30 minutes at room temperature place the brain slices in a water bath at 37 degrees Celsius for ten minutes to recover synaptic function.Place glucose free ACSF and acidic ACSF into the 37 degree Celsius water bath and bubble the solutions with five percent carbon dioxide and 95%nitrogen and 20 percent carbon dioxide and 80%oxygen respectively for 30 minutes. Carefully transfer four of the five brain slices into a preheated 37 degree Celsius, glucose free ACSF and incubate for 15 minutes. To study the time window for acidic preconditioning transfer one slice directly from glucose free ACSF to acidic ACSF and transfer the other three slices to regular ACSF for reperfusion.After three minutes transfer the slice in acidic ACSF to regular ACSF. Then after five minutes in regular ACSF transfer one of the slices from regular ACSF to the tissue holder in acidic ACSF for three minutes. Transfer another slice in the same manner 15 minutes after reperfusion.The remaining slice that was placed in glucose free ACSF but not acidic ACSF is set as the oxygen glucose deprivation group. Transfer the slices from the ACSF into the wells of a 24 well plate containing TTC solution. Stretch out the slice in the solution.And then incubate at 37 degrees Celsius in a shallow water bath for 30 minutes. Next transfer the slices into clean, weighed, 1.5 milliliter centrifuge tubes covered in foil and measure the dry weight of the slices. After weighing, extract Formazan by adding a one to one solution of ethanol and dimethyl sulphoxide to the tubes at a volume to weight ratio of 10 to one.Incubate away from light for 24 hours. The next day transfer the ethanol dimethyl sulphoxide solution from the tubes to a 96 well plate and measure the absorbance at 490 nanometers using a plate reader. First confirm proper anesthesia by the absence of a toe pinch reflex.Then place the anesthetized mouse with LDF probe attached to its skull in a supine position and fix using sterile cotton threads. Disinfect the shaved skin of the neck with 75%ethyl alcohol. Then after making a peri median skin incision in the neck blunt dissect the soft tissues with forceps to expose the blood vessels.Add one drop of saline to the exposed tissues to keep them hydrated. Next, use ophthalmic forceps to dissect the common carotid artery from surrounding tissue and the vagus nerve. Be careful not to damage the vagus nerve.Place a micro vessel clamp at the distal end of the common carotid artery. And tie a dead knot with 6-0 silk sutures at the proximal end. Then tie a loose knot proximal to the clamp as a temporary suture.Next, use micro ophthalmic scissors to make a small longitudinal incision between the two knots as close to the dead knot as possible. Now insert a 12 millimeter, tip blunted monofilament through the incision into the artery lumen and advance it a few millimeters. Tighten the loose knot around the tip of the monofilament and then remove the clamp.Then use ophthalmic forceps to advance the filament into the internal carotid artery until the LDF software shows a sharp decline in blood flow. Record the start time of occlusion. Then cut off the LDF probe, and put the mouse in a 30 degree Celsius incubator for the one hour duration of occlusion.55 minutes after the start of occlusion reanesthetize the animal with isoflurane and position the animal as before. Then open the neck incision, and reexpose the common carotid artery. After the occlusion period, use ophthalmic forceps to gently pull out the filament to achieve reperfusion.Then turn the temporary suture into a permanent one by tightening the knot. For acidosis treatment change the gas inhaled by the nose cone to 20%carbon dioxide 20%oxygen, and 60%nitrogen for five minutes. After closing the incision with an interrupted surgical suture place the mouse in a 30 degree Celsius heated cage until the mouse regains consciousness.This histogram shows the results from the TTC assay performed on cortical slices after oxygen glucose deprivation and acidic postconditioning as demonstrated in this video. Three minutes of acidosis treatment was neuroprotective if treatment was initiated immediately or five minutes after oxygen glucose deprivation, but not if the slices were reperfused for 15 minutes. Mice were subjected to 60 minutes of middle cerebral artery occlusion and treated by inhaling 20%carbon dioxide for five minutes at five, 50, or 100 minutes after reperfusion.Infarct volume was quantified by two, three, five triphenyltetrazolium hydrochloride staining 24 hours after reperfusion as indicated by the black dotted lines. This histogram shows the percent infarct volume for each condition. The neuroprotection from acidic postconditioning was robust even when the onset time was delayed to 50 minutes after reperfusion.However, acidosis treatment initiated at 100 minutes did not block the ischemic injury. After watching the video you should have a good understanding of how to study the neuroprotection of acidosis postconditioning on OGD model of brain slices, and on MSO model of mice. While attempting the procedures it is important to remember that the extension and the duration of acidosis is very important to reproduce the protective effects.

Research

JoVE Journal

Neuroscience

An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces

ブレインマシンインタフェースの閉ループ性能を研究する実験プラットフォーム

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine ...

神経科学

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

燐光の酸素依存性の焼入に基づいて脳の血液の酸素化の測定

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

ラット中大脳動脈の血管縫合閉塞による局所脳虚血モデル

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

網膜色素上皮生理学と病態の研究のための実験的モデル

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound (fTCD)

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound fTCD

機能性経頭蓋ドップラー超音波を使って子供の脳の側性化の評価（fTCD）

There are many unanswered questions about cerebral lateralization.  In particular, it remains unclear which aspects of language and nonverbal ability are ...

Strategies for Study of Neuroprotection from Cold-preconditioning

コー​​ルド - プレコンディショニングによる神経保護の研究のための戦略

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

小脳顆粒ニューロンの遺伝子操作<em&gt;体外</em&gt;と<em&gt;インビボ</em&gt;ニューロンの形態と移行を研究するために、

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

脳血管内皮細胞での生体エネルギー機能の評価

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

A Magnetic Resonance Imaging Protocol for Stroke Onset Time Estimation in Permanent Cerebral Ischemia

恒久的な脳虚血における脳卒中発症時間推定のための磁気共鳴イメージ投射プロトコル

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

研究保護および腫瘍細胞の侵襲性をモデルとして脊髄海馬スライス培養

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...