Name: sub-acute cerebral microhemorrhages induced by lipopolysaccharide injection in rats
Uploaded: 2018-10-17T14:30:00
Description: Watch this Scientific Journal Video about Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats at JoVE.com

我们感谢健丰老师和首都医科大学的同事在核磁共振成像方面的指导。我们还感谢宜昌第二人民医院神经病学系提供技术支持。

这里描述的所有方法都已获得中国人民解放军陆军总医院动物护理和使用委员会 (ACUC) 的批准。
1. 材料
<ol><li>LPS 注射液的制备<ol>
<li>将25毫升蒸馏水添加到25毫克的 LPS 粉末中, 从鼠伤寒到最终浓度为1毫克/毫升。将注射剂储存在4摄氏度的无菌管中。 注意: LPS 是有毒的。</li>
		<li>在正常0.9% 盐水溶液中制备 2% eb 溶液, 以保持 eb 注射液在工作浓度。</li>
	</ol>
</li>
</ol><p ...

<ol>
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M., 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=Cerebral+microbleeds:+A+guide+to+detection+and+interpretation.">Cerebral microbleeds: A guide to detection and interpretation.</a> Lancet Neurology. 8 (2), 165-174 (2009).</li><li>Hoffmann, 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=High-Field+MRI+reveals+a+drastic+increase+of+hypoxia-induced+microhemorrhages+upon+tissue+reoxygenation+in+the+mouse+brain+with+strong+predominance+in+the+olfactory+bulb.">High-Field MRI reveals a drastic increase of hypoxia-induced microhemorrhages upon tissue reoxygenation in the mouse brain with strong predominance in the olfactory bulb.</a> Plos One. 11 (2), e0148441 (2016).</li><li>Sumbria, R. K., 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=Effects+of+phosphodiesterase+3A+modulation+on+murine+cerebral+microhemorrhages.">Effects of phosphodiesterase 3A modulation on murine cerebral microhemorrhages.</a> Journal of Neuroinflammation. 14 (1), 114 (2017).</li><li>Sumbria, R. K., 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=Aging+exacerbates+development+of+cerebral+microbleeds+in+a+mouse+model.">Aging exacerbates development of cerebral microbleeds in a mouse model.</a> Journal of Neuroinflammation. 15 (1), 69 (2018).</li><li>Mello, B. S. 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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=Changes+in+astroglial+markers+in+a+maternal+immune+activation+model+of+schizophrenia+in+Wistar+rats+are+dependent+on+sex.">Changes in astroglial markers in a maternal immune activation model of schizophrenia in Wistar rats are dependent on sex.</a> Frontiers in Cellular Neuroscience. 9 (489), (2015).</li><li>Dutta, G., Zhang, P., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lipopolysaccharide+Parkinson's+disease+animal+model:+mechanistic+studies+and+drug+discovery.">The Lipopolysaccharide Parkinson's disease animal model: mechanistic studies and drug discovery.</a> Fundamental &amp; Clinical Pharmacology. 22 (5), 453-464 (2008).</li><li>El-Sayed, N. S., Bayan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+role+of+resveratrol+targeting+estradiol+and+neprilysin+pathways+in+lipopolysaccharide+model+of+Alzheimer+disease.">Possible role of resveratrol targeting estradiol and neprilysin pathways in lipopolysaccharide model of Alzheimer disease.</a> Advances in Experimental Medicine and Biology. 822 (822), 107-118 (2015).</li><li>Combrinck, M. I., Perry, V. H., Cunningham, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+infection+evokes+exaggerated+sickness+behaviour+in+pre-clinical+murine+prion+disease.">Peripheral infection evokes exaggerated sickness behaviour in pre-clinical murine prion disease.</a> Neuroscience. 112 (1), 7-11 (2002).</li><li>Pantoni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+small+vessel+disease:+from+pathogenesis+and+clinical+characteristics+to+therapeutic+challenges.">Cerebral small vessel disease: from pathogenesis and clinical characteristics to therapeutic challenges.</a> Lancet Neurology. 9 (7), 689-701 (2010).</li><li>Rosand, 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=Spatial+clustering+of+hemorrhages+in+probable+cerebral+amyloid+angiopathy.">Spatial clustering of hemorrhages in probable cerebral amyloid angiopathy.</a> Annals of Neurology. 58 (3), 459-462 (2005).</li><li>Robinson, 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=Microstructural+and+microglial+changes+after+repetitive+mild+traumatic+brain+injury+in+mice.">Microstructural and microglial changes after repetitive mild traumatic brain injury in mice.</a> Journal of Neuroscience Research. 95 (4), 1025-1035 (2017).</li><li>Kraft, P., 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=Hypercholesterolemia+induced+cerebral+small+vessel+disease.">Hypercholesterolemia induced cerebral small vessel disease.</a> Plos One. 12 (8), e0182822 (2017).</li><li>Schreiber, S., Bueche, C. 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中草药的研究在过去几年中有所增加。然而, 中草药的机制仍然不清楚, 促使科学家建立模拟这种特殊情况的动物模型。例如, 霍夫曼等。开发了缺氧诱导的中草药小鼠模型, 表明中草药是由缺氧和脑血管自动调节12的破坏引起的。路透社等。5在 APP23-transgenic 小鼠中建立了中草药模型, 表明脑淀粉样血管病 (CAA) 在中草药病因中起着重要作用. 费舍尔<em...

经典脑 microhemorrhages (中草药) 是指血液降解产物的微小血管周围沉积物, 如含铁血黄素从脑1中的红细胞。根据鹿特丹扫描研究, 中草药可以发现在近17.8% 的人60–69岁和38.3% 在那些80年2。中草药在老年人中的患病率较高, 中草药的积累与认知和神经精神功能障碍之间的相关性已建立3,4。最近报道了中草药的几种动物模型, 包括 IV 型胶原酶立体定向注射液5、应用转基因6、β n-甲基氨基-l-丙氨酸暴露7、高血压8等诱发的啮齿动物模型,由全身炎症诱发的中草药是最接受的选择之一。费舍尔等。9首次使用的 LPS 来源于鼠伤寒沙门氏菌, 开发出一种急性中草药小鼠模型。随后, 同一组报告了使用相同方法2的亚急性中草药小鼠模型的发展。
LPS 被认为是一个标准化的炎症刺激通过腹腔注射。先前的研究证实, LPS 注射液可能导致神经炎症, 在动物模型2,10中大量的小胶质细胞和星形胶质细胞活化反应。此外, 神经炎症活化激活与中草药数量之间有正相关关系, 已建立2、10。在前人研究的基础上, 通过腹腔注射 LPS, 建立了中草药大鼠模型。
检测技术的进步导致了中草药研究调查的数量增加。最广泛公认的检测中草药的方法包括通过苏木精和伊红 (he) 染色检测红血球, 普鲁士蓝染色9检测铁铁, 通过免疫荧光法检测埃文斯蓝 (EB) 沉积物成像和7.0 特斯拉磁共振成像-易感性加权成像 (MRI-社会工作)10。本研究旨在开发一种中草药的筛选方法。

<table>
	<tbody>
		<tr>
			<td>LPS</td>
			<td>Sigma-Aldrich</td>
			<td>L-2630</td>
			<td>for inflammation induction</td>
		</tr>
		<tr>
			<td>EB</td>
			<td>Sigma-aldrich</td>
			<td>E2129</td>
			<td>for EB leakage detection</td>
		</tr>
		<tr>
			<td>DAPI dying solution</td>
			<td>Servicbio</td>
			<td>G1012</td>
			<td>count medium for IF</td>
		</tr>
		<tr>
			<td>Perl&#8217;s Prussian staining</td>
			<td>Solarbio</td>
			<td>G1424</td>
			<td>Kit for Prussian staining</td>
		</tr>
		<tr>
			<td>HE staining</td>
			<td>Solarbio</td>
			<td>G1120</td>
			<td>Kit for HE staining</td>
		</tr>
		<tr>
			<td>chloral hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>47335U</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>phosphate buffer saline (PBS)</td>
			<td>Solarbio</td>
			<td>P1022</td>
			<td>a kind of buffer solution commonly used in experiment</td>
		</tr>
		<tr>
			<td>0.9% saline solution</td>
			<td>Hainan DonglianChangfu Pharmaceutical Co., Ltd., China</td>
			<td></td>
			<td>solution for perfusion</td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td>a kind of solution commonly used for fixation</td>
		</tr>
		<tr>
			<td>20% sucrose solution</td>
			<td>Solarbio</td>
			<td>G2461</td>
			<td>a kind of solution commonly used for fixation</td>
		</tr>
		<tr>
			<td>30% sucrose solution</td>
			<td>Solarbio</td>
			<td>G2460</td>
			<td>a kind of solution commonly used for fixation</td>
		</tr>
		<tr>
			<td>vet ointment</td>
			<td>Solcoseryl eye gel, Bacel, Switzerland</td>
			<td></td>
			<td>for rat&#39;s eyes protection</td>
		</tr>
	</tbody>
</table>

sub-acute cerebral microhemorrhages induced by lipopolysaccharide injection in rats

脑 microhemorrhages (中草药) 在老年患者中是常见的, 与各种神经精神障碍相关。中草药的病因是复杂的, 神经炎症经常被观察作为共同发生。本文介绍了一种由脂多糖 (LPS) 注射液诱导的亚急性中草药大鼠模型, 以及一种检测中草药的方法. 系统性 LPS 注射液相对简单、经济、经济实惠。LPS 注射液的一个主要优点是它的稳定性导致炎症。通过观察、苏木精和伊红 (he) 染色、Perl 普鲁士染色、埃文斯蓝 (EB) 双重标记和磁共振成像-易感性加权成像 (MRI-中草药) 技术, 可以检测到 LPS 注射液引起的。最后, 本报告还讨论了其他开发中草药动物模型的方法, 包括它们的优点和/或缺点。

中草药可以使用各种方法检测到。最接受的方法包括: (1) 表面中草药的总观测和评估 (如图 1、上部板所示);(2) he 染色检测红细胞 (图2A、上部板) 或普鲁士染色检测红细胞裂解产生的铁 (图 2A、下部板);(3) eb 双重染色检测从 BBB 泄漏中提取的 eb 沉积物 (图 3, 左面板);(4) MRI-中草药 hypointe...

Watch this Scientific Journal Video about Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats at JoVE.com

Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

我们提出了一种诱导和检测杜勒大鼠 LPS 注射液引起的中草药的实验方案, 可用于今后中草药的发病机制研究。

脂多糖注射液致大鼠亚急性脑 Microhemorrhages

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, ...

该方法有助于回答脑微出血领域的关键问题，如脑微出血的病因、分布、检测等。该技术的主要优点是LPS注射稳定诱导炎症。此外，LPS 喷射非常简单、经济且经济高效。虽然这种方法可以提供脑出血诱导的见解，它也可以应用于其他脑部疾病，如抑郁症，精神分裂症，帕金森病，阿尔茨海默病，和普里昂病。以每公斤体重1毫克的剂量对10周大的雄性Sprague Dawley大鼠进行LPS注射，然后将每只大鼠返回到家庭笼子。6 小时 16 小时后重复 LPS 注射。请注意，以每公斤1毫克的剂量注射SD大鼠可能导致5%的死亡率。年轻或年长的大鼠或怀孕雌性大鼠的死亡率可能进一步上升。LPS 注射后将大鼠返回到笼子，并提供食食物和饮料。在末期麻醉下，对每只大鼠进行五到八毫米的深度心脏穿刺。在第三和第四个间空间处，刺破点应为胸骨左边缘五毫米。将针头握到位，用含有埃文斯蓝色管的管子更换空管。等到血液恢复被观察，然后注射埃文斯蓝的剂量0.2毫升每100克的体重到左心室。如果通过免疫荧光成像评估埃文斯蓝漏通过血液/大脑屏障，请将大鼠保持10分钟的超供状态。使用冰冷 200 至 300 毫升 0.9% 盐水溶液进行心脏灌注，以清除脑血管和大脑。切换到冰冷4%甲醛固定。然后，在隔离大脑后，在PBS中浸入20%蔗糖至少6个小时，或直到大脑沉入管底。将溶液更改为 30%蔗糖，再修复 6 小时。最后，使用低温恒温器准备10毫米厚的脑组织部分。用 PBS 清洗幻灯片三次，每次五分钟，然后用 4，6'diamidino-2-苯酚或 DAPI 溶液在室温下孵育幻灯片 15 分钟。在 PBS 中像之前一样清洗幻灯片后，使用 PBS-甘油溶液安装幻灯片。在荧光显微镜上捕捉图像。EB沉积用红色荧光表示，核用蓝色荧光表示。在蒸馏水中清洗滑梯，开始 H&E 染色。然后浸入赤氧树脂溶液8分钟。或者，对于P应普鲁士蓝色染色，在反应溶液中染色，用铁氰化物和盐酸等件混合物10分钟。染色后，用自来水清洗五分钟。在 1%酸醇中区分 30 秒。在自来水中洗涤1分钟后，在0.2%氨水或饱和碳酸锂溶液中染色30秒至1分钟。接下来，用自来水洗五分钟。用 eosin 溶液防污 30 秒至 1 分钟。通过90%酒精脱水，然后95%酒精，绝对酒精每0.5至2分钟。脱水后，用二甲苯清除30秒。安装后，使用亮场荧光显微镜分析 H&E 染色。从血管中释放的红血球是CCM的成分，在H&E染色下以红橙色出现。表面CCMH可以通过红色箭头指示的毛观测来检测。埃文斯蓝色染色揭示了明显的红色荧光，其中埃文斯蓝色分子从受伤的血脑屏障泄漏。H&E染色显示毛细管外发现的红血球，普鲁士染色显示从红血球的解解中得出的铁离子点。在尝试此程序时，重要的是要记住，LPS 诱导脑微出血的剂量大于帕金森病或精神分裂症模型中使用的剂量。因此，动物的环境应保持清洁，以降低死亡率。按照这个程序，可以执行电子显微镜、磁共振成像和免疫荧光成像等方法，以确定脑微出血中血脑屏障外层结构的损伤、体内脑微出血的分布以及胶质细胞活化。

Research

JoVE Journal

Neuroscience

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

焦距脑缺血模型大鼠大脑中动脉的血管内缝合闭塞

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 ...

科研

神经科学

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

在实验室老鼠研究求可卡因电击诱导恢复原状的程序

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

利用双光子显微镜的艾滋病毒引起的神经炎症的小鼠模型脑血管结构的量化

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

由亲炎细胞因子的内脑注射诱导的广度脑皮质脱叶的鼠模型

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

爆炸性神经损伤对压生啮鼠中脑动脉的影响

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

电刺激在大鼠中建立急性庞廷梗死

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

大鼠运动皮层中的激光诱发脑损伤

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Detrusor Underactivity Model in Rats by Conus Medullaris Transection

大鼠逼尿肌活动不足模型通过圆锥髓质横断

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was ...

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

通过脑室内注射血红蛋白模拟新生儿脑室内出血

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and ...