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

这项工作由中国国家自然科学基金（81573406,81373393,81273506,81221003,81473186和81402907），浙江省自然科学基金（LR15H310001）和浙江省科技创新队队长计划（2011R50014）资助。

所有实验均经浙江大学动物实验委员会道德准则批准实施，完全符合国家卫生研究院“实验动物护理与使用指南”。努力减少任何疼痛或不适，并使用最少数量的动物。 皮质硬膜下切片的OGD <ol><li> 解决方法： <ol><li>制备1000毫升的r-ACSF（124毫摩尔/ L，氯化钠，5毫摩尔/ L的KCl，1.25毫摩尔/ L KH 2 PO 4，2毫摩尔/升MgSO 4，26毫摩尔/ L的NaHCO 3?...

<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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+real-time+monitoring+of+glutathione+redox+status+in+single+pyramidal+neurons+in+organotypic+hippocampal+slices+during+oxygen-glucose+deprivation+and+reperfusion.">Optimized real-time monitoring of glutathione redox status in single pyramidal neurons in organotypic hippocampal slices during oxygen-glucose deprivation and reperfusion.</a> ACS Chem Neurosci. 6 (11), 1838-1848 (2015).</li><li>Medvedeva, Y. V., Ji, S., Yin, H. Z., Weiss, J. 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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在这里我们提出两个实验模型来研究APC对脑缺血的神经保护作用。在脑切片中，APC通过在再灌注发生后用20％CO 2鼓泡的酸性缓冲液中孵育小鼠皮质纹状体切片来实现，而在MCAO模型中，APC是通过在再灌注后向小鼠吸入20％CO 2而实现的。这两种模型反映了APC对脑缺血的神经保护作用。保护与通过缺血后处理实现的保护相当，但是具有更宽的时间窗口。在MCAO模型，再灌注后相比10分钟?...

中风是全球死亡和残疾的主要原因之一。在过去几十年中，为了找到有效的卒中治疗，已经做出了巨大的努力，但是成绩还不尽如人意。后处理是在缺血发作后由亚毒性应激操纵的过程。后处理，包括缺血性，缺氧，低血糖和远程缺血后处理，触发内源性适应机制，并已被证明是有前途的治疗对脑缺血1，2，3，4。然而，缺血后处理可能引起额外的损伤。肢体远程缺血后处理通常需要5的几个周期-在同侧或双侧后肢5，6，7 20分钟闭塞和再灌注。钍因此，这些后处理操作在临床实践中是危险的或不切实际的。为了克服这些缺点，我们开发了APC为小鼠8局灶性脑缺血的疗法。通过吸入20％CO 2诱导，APC以更可行和更安全的方式显着减少缺血性脑损伤。最近我们已经证明，APC延长了再灌注窗口，突出了APC对卒中治疗的重要性9 。 在这里我们提出两个实验模型来研究APC对脑缺血的神经保护作用。第一个是小鼠皮质纹状体切片中的氧 - 葡萄糖剥夺（OGD）模型。通过人造脑脊液（ASCF）将脑切片快速制备和转移到人造环境中，可以维持细胞活力和神经元电路，这样可以在体外研究脑功能10sup&gt;， 11 。在ASCF模仿脑缺血缺氧缺糖，诱导缺血性损伤12，13，14。在OGD之后，将脑切片用常规ASCF（r-ASCF）刷新以提供再灌注，然后使用用20％CO 2鼓泡的酸性ASCF处理APC。与原代培养细胞相比，皮质硬膜下切片维持完整的组织学表征。 为了研究体内脑功能，采用小鼠中脑动脉闭塞（MCAO）模型。通过颈总动脉插入火焰钝的单丝阻止大脑中动脉。作为最广泛使用的中风模型之一，MCAO模型显示临床相关性，单丝的应用使得更容易实现再灌注。简单地通过reperfusio发病后吸入含有20％CO 2含氧量正常的混合气体n，APC对脑梗死体积减少所表明的脑缺血有显着的保护作用。

<table border="0" cellpadding="0" cellspacing="0" width="723">
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		<col />
	</colgroup>
	<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

中风是全球死亡和残疾的主要原因之一，治疗方法有限。作为神经保护的内源性策略，后处理已被证明是有希望的针对脑缺血的治疗方法。然而，复杂的程序和潜在的安全问题限制了其临床应用。为了克服这些缺点，我们开发了酸性后处理（APC）作为实验性局灶性脑缺血的治疗方法。 APC是指温和酸中毒治疗由局部缺血再灌注期间吸入的CO 2。在这里，我们分别提出了两种模型来在体外和体内执行APC。使用小鼠的氧 - 葡萄糖剥夺（OGD）治疗和小鼠的皮质静脉闭塞和中脑动脉闭塞（MCAO）来模拟脑缺血。 APC可以通过将脑切片转移到用20％CO 2鼓泡的酸性缓冲液中来实现r通过吸入20％CO 2的小鼠。 APC显示出对脑缺血的显着保护作用，如组织存活力和脑梗死体积所反映的。

在上述皮质纹状体切片模型中，在再灌注后1小时通过TTC测定定量皮质硬膜下切片存活力。通过将490nm处的吸收归一化到对照切片来计算TTC转化。根据TTC转换，APC在起始时间和持续时间依赖的方式保护OGD诱导的再灌注损伤。详细地，1和3分钟的酸中毒治疗在OGD 15分钟后5分钟显着改善了活力，而5分钟没有（OGD：0.609±0.029,5 / 1：0.758±0.034,5 / 3：0.821±0.041， 5/5：0.672±0.053，数据?...

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

酸性后处理可防止脑缺血。在这里我们提出两个模型来执行APC。分别通过体外氧葡萄糖剥夺后将皮质硬膜下切片转移到酸性缓冲液中，并在体内大脑中动脉闭塞后吸入20％CO 2 。

实验模型研究酸性后处理对脑缺血的神经保护作用

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