Name: assessing neuroprotective effects of glycyrrhizae radix et rhizoma extract using a transient middle cerebral artery occlusion mouse model
Uploaded: 2018-12-09T13:00:00
Description: Watch this Scientific Journal Video about Assessing Neuroprotective Effects of Glycyrrhizae Radix et Rhizoma Extract Using a Transient Middle Cerebral Artery Occlusion Mouse Model at JoVE.com

所有涉及动物的程序都得到了釜山国立大学道德委员会的批准 (批准号码, pu-2016-1087)。图 1显示了这项研究的图形概述。
1. grex 的准备和管理
注: 本研究中使用的 gr 是从一家商业制药公司购买的。
<ol><li>将200克 gr 放入 2, 000 毫升甲醇中, 在室温 (25°c) 下孵育5天。</li>
	<li>使用厚度为 0.26 mm、孔径为5μm 的滤纸对混合物进行过滤, 然后?...

<ol>
	<li>Bejot, Y., Delpont, B., Giroud, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rising+stroke+incidence+in+young+adults:+more+epidemiological+evidence,+more+questions+to+be+answered.">Rising stroke incidence in young adults: more epidemiological evidence, more questions to be answered.</a> Journal of the American Heart Association. 11 (5), (2016).</li><li>Hadadha, M., Vakili, A., Bandegi, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+inhibition+of+hydrogen+sulfide+synthesis+on+ischemic+injury+and+oxidative+stress+biomarkers+in+a+transient+model+of+focal+cerebral+ischemia+in+rats.">Effect of the inhibition of hydrogen sulfide synthesis on ischemic injury and oxidative stress biomarkers in a transient model of focal cerebral ischemia in rats.</a> Journal of Stroke and Cerebrovascular Diseases. 24 (12), 2676-2684 (2015).</li><li>Durukan, A., Tatlisumak, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ischemic+stroke.">Animal models of ischemic stroke.</a> Article in Handbook of Clinical Neurology. 92, 43-66 (2009).</li><li>Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+of+Stroke.">Animal Models of Stroke.</a> Brain and Neurorehabilitation. 4 (1), 1-11 (2011).</li><li>Rizzato, G., Scalabrin, E., Radaelli, M., Capodaglio, G., Piccolo, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+exploration+of+licorice+metabolome.">A new exploration of licorice metabolome.</a> Food Chemistry. 221, 959-968 (2017).</li><li>Zhu, 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=Rapid+determination+of+flavonoids+in+licorice+and+comparison+of+three+licorice+species.">Rapid determination of flavonoids in licorice and comparison of three licorice species.</a> Journal of Separation Science. 39 (3), 473-482 (2016).</li><li>Ota, M., Mikage, M., Cai, S. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbological+study+on+the+medicinal+effects+of+roasted+licorice+and+honey-roasted+licorice.">Herbological study on the medicinal effects of roasted licorice and honey-roasted licorice.</a> Yakushigaku Zasshi. 50 (1), 38-45 (2015).</li><li>Lim, C., 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=Licorice+pretreatment+protects+against+brain+damage+induced+by+middle+cerebral+artery+occlusion+in+mice.">Licorice pretreatment protects against brain damage induced by middle cerebral artery occlusion in mice.</a> Journal of Medicinal Food. 21 (5), 474-480 (2018).</li><li>Koizumi, J. Y., Nakazawa, T., Ooneda, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+of+ischemic+brain+edema.">Experimental studies of ischemic brain edema.</a> Nosotchu. 8 (1), 1-8 (1986).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoxylin+and+eosin+staining+of+tissue+and+cell+sections.">Hematoxylin and eosin staining of tissue and cell sections.</a> Cold Spring Harbor Protocols. 2008, (2008).</li><li>Zhu, Y., Liu, F., Zou, X., Torbey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+unbiased+estimation+of+neuronal+number+in+the+rat+hippocampus+with+different+staining+methods.">Comparison of unbiased estimation of neuronal number in the rat hippocampus with different staining methods.</a> Journal of Neuroscience Methods. 254, 73-79 (2005).</li><li>Alberts, M. J., Ovbiagele, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+strategies+for+ischemic+stroke+prevention:+role+of+multimodal+combination+therapies.">Current strategies for ischemic stroke prevention: role of multimodal combination therapies.</a> Journal of Neurology. 254 (10), 1414-1426 (2007).</li><li>Pinto, A., Tuttolomondo, A., Di Raimondo, D., Fernandez, P., Licata, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrovascular+risk+factors+and+clinical+classification+of+strokes.">Cerebrovascular risk factors and clinical classification of strokes.</a> Seminars in Vascular Medicine. 4 (3), 287-303 (2004).</li><li>Barlow, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+the+brain+regions+associated+with+acute+spasticity+in+patients+diagnosed+with+an+ischemic+stroke.">Identifying the brain regions associated with acute spasticity in patients diagnosed with an ischemic stroke.</a> Somatosensory and Motor Research. 33 (2), 1-8 (2016).</li><li>Roth, S., Liesz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroke+research+at+the+crossroads+-+where+are+we+heading.">Stroke research at the crossroads - where are we heading.</a> Swiss Medical Weekly. 146, 14329 (2016).</li><li>Feuerstein, G. Z., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+stroke.">Animal models of stroke.</a> Molecular Medicine Today. 6 (3), 133-135 (2000).</li><li>Herson, P. S., Traystman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+stroke:+translational+potential+at+present+and+in+2050.">Animal models of stroke: translational potential at present and in 2050.</a> Future Neurology. 9 (5), 541-551 (2014).</li><li>Kumar, A., Gupta Aakriti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+animal+models+of+stroke:+an+update.">A review on animal models of stroke: an update.</a> Brain Research Bulletin. 122, 35-44 (2016).</li><li>O'Collins, V. E., Donnan, G. A., Howells, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+animal+models+of+stroke.">History of animal models of stroke.</a> International Journal of Stroke. 6 (1), 77-78 (2011).</li><li>Ji, S., 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=Bioactive+constituents+of+Glycyrrhiza+uralensis+(licorice):+discovery+of+the+effective+components+of+a+traditional+herbal+medicine.">Bioactive constituents of Glycyrrhiza uralensis (licorice): discovery of the effective components of a traditional herbal medicine.</a> Journal of Natural Products. 79 (2), 281-292 (2016).</li><li>Yang, R., Wang, L. Q., Yuan, B. C., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pharmacological+activities+of+licorice.">The pharmacological activities of licorice.</a> Planta Medica. 81 (18), 1654-1669 (2015).</li><li>Yang, R., Yuan, B. C., Ma, Y. S., Zhou, S., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anti-inflammatory+activity+of+licorice,+a+widely+used+Chinese+herb.">The anti-inflammatory activity of licorice, a widely used Chinese herb.</a> Pharmaceutical Biology. 55 (1), 5-18 (2017).</li></ol>

随着慢性高血压、糖尿病、高脂血症等代谢疾病的发病率不断上升, 脑卒中的主要危险因素, 脑卒中的预防和治疗已成为医学研究的一个重要领域12.13. 中风后语言和运动方面的缺陷与脑组织14的损伤程度密切相关, 导致患者及其家属的生活质量差.重要的是要使用一个适当的中风动物模型, 其中涉及相同的病理变化发生?...

脑血流缺血再灌注引起的脑损伤导致神经细胞死亡和脑组织丢失。随着肥胖、高血压和糖尿病等代谢疾病的传播, 脑血管疾病的发病率不断上升, 这种类型的脑损伤继续增加.全世界老年中风患者的绝对人数急剧增加, 这些患者的医疗费用是一个主要的社会负担, 这些患者往往患有长期残疾。因此, 应尽可能减轻继发性残疾, 以减轻经济负担1,2。
脑梗死最常用的啮齿类动物模型是大脑中动脉闭塞 (mcao) 模型, 其中 mca 被硅涂层手术缝合长丝封闭, 以阻断血液流动, 导致缺血性中风3,4. 使用灯丝作为闭塞材料, 可以通过控制腔内灯丝插入的持续时间来控制闭塞时间和持久性。
以往的研究表明, 即使使用相同的啮齿类动物 mcao 模型, 脑梗死的总量也因实验而异, 导致研究的重现性较低。为了提高重现性, 我们优化了实验中使用的细丝薄荷的厚度。脑缺血期和诱发梗死的初步研究结果表明, 缺血期超过 60分钟, 可以观察和量化受损脑组织的体积区域。
甘草, 又名甘草, 由甘草和葛根的干根和根状茎组成. 它已被用于中国和韩国传统医学的各种用途, 包括作为食品添加剂和药用5,6,7。
在先前的一项研究8中, gr 甲醇提取物 (grex) 预处理显示出 mcao 小鼠的抗凋亡作用, 包括显著防止 b 细胞淋巴瘤 2 (bcl-2) 和 bcl 超大 (bcl-xl) 蛋白表达的降低。本研究通过评价传统 mcao 小鼠模型在确定 grex 在梗死后处理中的效率, 有效地降低了 mcao 所致脑损伤的梗死体积, 从而提高了该模型的重现性。

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:383px;">Glycyrrhizae Radix et Rhizoma</td>
			<td style="width:307px;">Gwangmyoung Pharmaceuticals Co., Korea</td>
			<td style="width:344px;"></td>
			<td style="width:383px;">Glycyrrhizae Radix et Rhizoma</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Qualitative filter paper</td>
			<td>Advantec</td>
			<td style="width:344px;">Filter paper No. 2</td>
			<td>Qualitative filter paper</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D8418-250ML</td>
			<td>Dimethyl sulfoxide (DMSO)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Syringe filter (0.45 &#181;m)</td>
			<td>Sigma</td>
			<td>CLS431220</td>
			<td>Syringe filter (0.45 &#181;m)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Stereo Microscope</td>
			<td>Leica</td>
			<td>M50</td>
			<td>Stereo Microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Stereo Microscope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td>Stereo Microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laser Doppler</td>
			<td>Moor Instrument</td>
			<td>moorVMS-LDF</td>
			<td>Laser Doppler</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anesthesia Tabletop Bracket with N2O&#38;O2 Flowmeter System</td>
			<td>Harvard Appratus</td>
			<td>34-1352</td>
			<td>Anesthesia Tabletop Bracket with N2O&#38;O2 Flowmeter System</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Homeothermic Monitoring System</td>
			<td>Harvard Appratus</td>
			<td>55-7020</td>
			<td>Homeothermic Monitoring System</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Digital Camera</td>
			<td>Canon</td>
			<td>Eos-M2</td>
			<td>Digital Camera</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cryostat</td>
			<td>Leica</td>
			<td>CM3050S</td>
			<td>Cryostat</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscope</td>
			<td>Carl Zeiss</td>
			<td>Zeiss Axio</td>
			<td>Microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Data Analysis</td>
			<td>Systat Software Inc.</td>
			<td>SigmaPlot version 12</td>
			<td>Data Analysis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Data Analysis</td>
			<td>NIH Image</td>
			<td>ImageJ</td>
			<td>Data Analysis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mouse diet</td>
			<td>Doo Yeol Biotech</td>
			<td>Standard rodent chow</td>
			<td>Mouse diet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane</td>
			<td>JOONGWAE</td>
			<td>A02104781</td>
			<td>Isoflurane</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane</td>
			<td>TROIKAA</td>
			<td>ISOTROY 100</td>
			<td>Isoflurane</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Silk suture (4-0 Black silk)&#160;</td>
			<td>AILEE</td>
			<td>SK47510</td>
			<td>Silk suture (4-0 Black silk)&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Silk suture (3-0 White silk)&#160;</td>
			<td>Baekjae</td>
			<td>57</td>
			<td>Silk suture (3-0 White silk)&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nylon suture (8-0 monofilament)&#160;</td>
			<td>AILEE</td>
			<td>NB825</td>
			<td>Nylon suture (8-0 monofilament)&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2,3,5-triphenyltetrazolium chloride (TTC)</td>
			<td>Sigma</td>
			<td>T8877-25G</td>
			<td>2,3,5-triphenyltetrazolium chloride (TTC)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Formalin (Formaldehyde solution)</td>
			<td>JUNSEI</td>
			<td>69360-1263 20KG</td>
			<td>Formalin (Formaldehyde solution)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hematoxylin (Harris Hematoxylin)</td>
			<td>YD Diagnostics</td>
			<td>EasyStain</td>
			<td>Hematoxylin (Harris Hematoxylin)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Eosin (1% Eosin Y Solution)</td>
			<td>MUTO PURE CHEMICALS</td>
			<td>3200-2</td>
			<td>Eosin (1% Eosin Y Solution)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cresyl violet (acetate)</td>
			<td>Sigma</td>
			<td>C5042-10G</td>
			<td>Cresyl violet (acetate)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraformaldehyde&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:344px;">P6148-1KG</td>
			<td>Paraformaldehyde&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sucrose</td>
			<td>JUNSEI</td>
			<td>31365-0350 1KG</td>
			<td>Sucrose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Optimum cutting temperature (OCT) compound</td>
			<td>Scigen</td>
			<td>4583</td>
			<td>Optimum cutting temperature (OCT) compound</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Disecting Knife</td>
			<td>Fine Science Tools</td>
			<td>10055-12</td>
			<td>Disecting Knife</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">#4 Forcep</td>
			<td>Fine Science Tools</td>
			<td>11241-30</td>
			<td>#4 Forcep</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">#5 Forcep</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td>#5 Forcep</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">#6 Forcep</td>
			<td>Fine Science Tools</td>
			<td>11260-20</td>
			<td>#6 Forcep</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">#7 Fine Forcep</td>
			<td>Fine Science Tools</td>
			<td>11274-20</td>
			<td>#7 Fine Forcep</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Surgical Scissors</td>
			<td>Fine Science Tools</td>
			<td>14001-12</td>
			<td>Surgical Scissors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Extra Fine Bonn Scissors</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td>Extra Fine Bonn Scissors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Moria Pascheff-Wolff Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15371-92</td>
			<td>Moria Pascheff-Wolff Spring Scissors</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Vessel Dilating Forcep</td>
			<td>Fine Science Tools</td>
			<td>18153-11</td>
			<td>Vessel Dilating Forcep</td>
		</tr>
	</tbody>
</table>

assessing neuroprotective effects of glycyrrhizae radix et rhizoma extract using a transient middle cerebral artery occlusion mouse model

脑卒中后脑血流再灌注后的缺血导致神经细胞死亡和脑组织丢失。脑卒中最常用的动物模型是大脑中动脉闭塞 (mcao) 模型。以前的研究报告了不同的梗死大小, 即使在类似的 mcao 条件下使用相同的实验动物物种。因此, 我们开发了一种改进的实验方法来解决这种差异。小鼠接受 mcao 使用长丝作为遮挡材料, 以模拟人类中风条件和灯丝厚度进行优化, 以建立更可重现的梗死体积。脑卒中诱导后用甘草提取物处理的小鼠总梗死体积明显下降, 存活细胞数量相对于未治疗的对照组有所增加。该改进的实验方案成功地和可重现地证明了 grex 对缺血性脑卒中的有益作用。

在假操作的正常组, 没有观察到脑梗塞, 而在对照组, 观察到相对广泛的损伤区域。在 mcao 模型组给小鼠的 300 mg/kg grex 中, 损伤面积显著减少 (图 2)。
用 h & amp; e 或 cresyl 紫染色缺血性脑切片, 研究其组织学改变。h & amp; e 染色提供有关细胞10的结构信息和特定功能信息, 而 cresyl 紫色染色用...

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Assessing Neuroprotective Effects of Glycyrrhizae Radix et Rhizoma Extract Using a Transient Middle Cerebral Artery Occlusion Mouse Model

在本研究中, 我们通过建立大脑中动脉闭塞 (mcao) 小鼠模型, 修改了现有的实验方法, 以获得更多的重现性结果。口服甘草和根状茎 (gr) 甲醇提取物 (grex), 脑卒中诱导后, 与未治疗对照组相比, 总梗死量显著降低。

应用短暂的中脑动脉闭塞小鼠模型评价甘草提取物的神经保护作用

Ischemia followed by reperfusion of cerebral blood flow after a stroke leads to the death of nerve cells and loss of brain tissue. The most commonly used ...

该方法有助于回答神经科学和传统口腔医学研究领域有关与模型相关的神经保护天然化合物筛选的关键问题。该技术的主要优点是，感染体积的较高可生产性符合缺血时间剂量。这种方法的视觉演示至关重要，因为使用无菌显微镜将非常薄的机具插入原始中脑动脉是典型的。演示这个程序的将是Se-EunLee，一个在我的实验室学习的小学生。要准备乙二烷和Rhizome提取物，或GRex，首先添加200克的乙二烷和Rhizome，或GR，到两升甲醇，在室温下孵育五天。在孵化结束时，通过0.26毫米厚的滤纸和5微米孔径对溶液进行应变。将GR残留物加入一升新鲜甲醇中，然后再次过滤溶液。将两种滤纸混合，并通过一张新的滤纸进行应变。然后在冷冻干燥前将萃取物浓缩在真空中，以产生GRex。为准备GRex进行管理，将冷冻干燥的产品溶解在二甲基硫化物中，用0.9%的生理盐水稀释混合物。然后通过0.45微米注射器过滤器过滤溶液， 将DMSO的最终浓度调整到5%以下 建立小鼠中脑动脉闭塞模型（MCAO），确认麻醉的6周大22至25克雄性C57BL/6小鼠对尾部刺激缺乏反应，将鼠标放在36.5摄氏度的体温保持毯上，与温度计相连。通过剃须和脱毛霜去除小鼠胸部和颈部的所有头发后，将鼠标放在立体显微镜下，并在颈部中心进行两厘米的皮肤切口。小心地将左普通胡萝卜动脉、外皮动脉和内胡萝卜素动脉的分支与周围的结缔组织分离，并使用4.0丝状缝合线将外型胡萝卜状动脉和具有半钩结的常用胡萝卜动脉缝合，以暂时阻断流入内胡萝卜状动脉的血流。插入0.1至0.12毫米厚11毫米厚的硅胶涂层8.0单丝4.0丝线，通过内侧胡萝卜动脉到左中脑动脉的起源，并使用激光多普勒流量计测量相对脑血流动脉的减少。将插入的灯丝固定到血管上两个小时，同时将脑动脉闭塞。在闭塞期结束时，小心地提取灯丝，以恢复血流22小时的再灌注。并使用3.0丝线缝合的皮肤在五个地方用两个半打结。然后将鼠标返回到其笼子进行麻醉恢复。再灌注结束后一小时，仅口服量，每公斤体重GRex300毫克。MCAO发病24小时后，在每只实验动物的头部中皮上切开一个切口，用倾斜钳子打破骨骼，同时剥去杜拉母体。小心地将大脑从头骨中分离出来，并使用小鼠大脑矩阵将切除的组织切成一毫米厚的部分。根据标准协议，在 2%TTC 解决方案中染色部分 17 分钟。在用数码相机成像样品之前，将部分固定为 10% 的甲醛至少 2 小时。然后使用 ImageJ 分析和量化每个部分的脑梗塞区域。在适当的实验时间点后，MCAO，用10毫升PBS将每个安乐死实验动物转接，然后用10毫升4%的甲醛进行转接。隔离大脑后，正如刚刚证明的，将收获的器官浸入10毫升的30%蔗糖中过夜。第二天早上，将脑组织样本嵌入最佳切温化合物中。将组织切成15微米厚的细胞统计部分。将部分安装到玻璃幻灯片上，将部分在 80%乙醇中淹没 1 分钟。对于 H&E 染色，用赤氧林溶液标记样品 5 分钟，然后用 1% 酸醇滴两次。接下来，将幻灯片浸入饱和碳酸锂溶液中 30 秒，然后用自来水洗涤 30 秒，使用 Eosin 溶液进行 30 秒的反染色。用自来水洗涤去除多余的Eosin溶液，并在95然后用100%乙醇中一分钟上升浸入脱水。然后，对滑梯进行通风，用二甲苯清除至少 10 分钟，然后用安装介质安装盖玻片。对于氯霉素紫罗兰染色，将80%乙醇处理的幻灯片放在滑轨加热器上至少一小时，然后浸入50%乙醇中，用氯仿稀释过夜。第二天早上，在40摄氏度的干烤箱中用0.1%的紫光染色10分钟，随后在95%乙醇中脱水30分钟，两次5分钟100%乙醇浸泡。接下来，清除幻灯片两个五分钟的二甲苯浸入，然后空气干燥。然后使用安装介质安装幻灯片，通过光显微镜进行可视化。在假操作的正常群体中，没有观察到脑梗塞;而在控制中，未经治疗的MCAO动物，观察到相对广泛的受损区域。在每公斤300毫克的GRex治疗模型小鼠中，观察到受损组织在统计学上显著减少。H&E和紫红色色组织部分的组织学分析提供了细胞存活率的指标。事实上，H&E和紫红色染色强度显著降低对照，未经处理的MCAO组相对于假操作的正常组。另一方面，经过GRex治疗的动物表现出更大的组织完整性，这意味着神经元细胞死亡比对照组、未经治疗的组和假操作组都少，这表明提取物对缺血再灌注引起的脑损伤具有强大的神经保护作用。在尝试此程序时，重要的是要记住在此期间监测相对小脑血流量。和工艺导体非常小心，以尽量减少对内皮层的损害。

Research

JoVE Journal

Medicine

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

建模在小鼠体内的行程 - 中东与灯丝模型大脑中动脉闭塞

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

科研

医学

Mouse Model of Middle Cerebral Artery Occlusion

大脑中动脉闭塞的小鼠模型

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

腔内中东大脑中动脉闭塞（MCAO）缺血性中风模型在小鼠体内的激光多普勒血流指导

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

在小鼠永久性大脑中动脉结扎术中的应用

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

内皮素-1诱导大脑中动脉闭塞模型缺血性中风激光多普勒血流仪在大鼠指导

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

利用颅窗口，在可视化的大脑中动脉内皮素-1诱导大脑中动脉闭塞

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

优化系统的脑灌注监测在中风模型大鼠的大脑中动脉闭塞腔内

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

执行永久远端大脑中与老年大鼠颈总动脉闭塞研究皮层缺血具有持续残疾

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...