Name: massive pontine hemorrhage by dual injection of autologous blood
Uploaded: 2021-05-29T12:30:00
Description: Watch this Scientific Journal Video about Massive Pontine Hemorrhage by Dual Injection of Autologous Blood at JoVE.com

本研究由中国国家自然科学基金委员会（81471181和81870933）和广州医科大学（0506308）向叶江开放实验室项目，以及中国国家科学基金会（81701471）和广州市卫生委员会科学项目（20191A011083）向邱先生提供资助，中国国家科学基金会（81501009）向吴先生提供资助。

该议定书经广州医科大学第二附属医院动物护理与使用委员会审查批准。老鼠是由南方医科大学动物中心提供的。实验设计见图1。
1. 动物和仪器
<ol><li>使用8周大的雄性斯普拉格-道利大鼠，体重250±10克。</li>
	<li>在环境温度为 25 °C、相对湿度为 65% 和 12/12h 的光暗循环下，在受控环境条件下将大鼠安置至少 7 天。</li>
	<li>无限制地提供食物和水。</li>
	<...

<ol>
	<li>Charidimou, 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=Brain+hemorrhage+recurrence,+small+vessel+disease+type,+and+cerebral+microbleeds:+A+meta-analysis.">Brain hemorrhage recurrence, small vessel disease type, and cerebral microbleeds: A meta-analysis.</a> Neurology. 89 (8), 820-829 (2017).</li><li>Tao, 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=A+novel+brainstem+hemorrhage+model+by+autologous+blood+infusion+in+Rat:+White+Matter+Injury,+Magnetic+Resonance+Imaging,+and+Neurobehavioral+features.">A novel brainstem hemorrhage model by autologous blood infusion in Rat: White Matter Injury, Magnetic Resonance Imaging, and Neurobehavioral features.</a> Journal of Stroke and Cerebrovascular Diseases. 25 (5), 1102-1109 (2016).</li><li>Ichimura, 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=Surgical+treatment+for+primary+brainstem+hemorrhage+to+improve+postoperative+functional+outcomes.">Surgical treatment for primary brainstem hemorrhage to improve postoperative functional outcomes.</a> World Neurosurgery. 120, 1289-1294 (2018).</li><li>Behrouz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+factors+in+pontine+haemorrhage:+A+systematic+review.">Prognostic factors in pontine haemorrhage: A systematic review.</a> European Stroke Journal. 3 (2), 101-109 (2018).</li><li>Guo, X., 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=Brainstem+iron+overload+and+injury+in+a+rat+model+of+brainstem+hemorrhage.">Brainstem iron overload and injury in a rat model of brainstem hemorrhage.</a> Journal of Stroke and Cerebrovascular Diseases. 29 (8), 104956 (2020).</li><li>Chung, Y., Haines, 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=Experimental+brain+stem+surgery.">Experimental brain stem surgery.</a> Neurosurgery Clinics of North America. 4 (3), 405-414 (1993).</li><li>Lekic, T., Tang, J., Zhang, 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=A+rat+model+of+pontine+hemorrhage.">A rat model of pontine hemorrhage.</a> Acta Neurochirurgica Supplement. 105, 135-137 (2008).</li><li>Lekic, T., 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=Evaluation+of+the+hematoma+consequences,+neurobehavioral+profiles,+and+histopathology+in+a+rat+model+of+pontine+hemorrhage.">Evaluation of the hematoma consequences, neurobehavioral profiles, and histopathology in a rat model of pontine hemorrhage.</a> Journal of Neurosurgery. 118 (2), 465-477 (2013).</li><li>Shrestha, B. 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=Rat+brainstem+hemorrhage+model:+Key+points+to+success+in+modeling.">Rat brainstem hemorrhage model: Key points to success in modeling.</a> World Neurosurgery. 117, 106-116 (2018).</li><li>Luo, M., Tang, X., Zhu, J., Qiu, Z., Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+acute+pontine+infarction+in+rats+by+electrical+stimulation.">Establishment of acute pontine infarction in rats by electrical stimulation.</a> Journal of Visualized Experiments. (162), (2020).</li><li>Wu, L., 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=Keep+warm+and+get+success:+The+role+of+postischemic+temperature+in+the+mouse+middle+cerebral+artery+occlusion+model.">Keep warm and get success: The role of postischemic temperature in the mouse middle cerebral artery occlusion model.</a> Brain Research Bulletin. 101, 12-17 (2014).</li></ol>

在本研究中，我们提供了一个协议，以产生一个巨大的庞然大海大鼠模型。该模型可用于研究大庞丁出血的病理生理机理和预后。
在整个实验中，使用了25只老鼠，其中只有一只死亡。对MRI、毛解剖和 HE 染色的验证表明，该方法的死亡率非常低，成功率很高。要建立大规模的庞然大物出血模型，必须解决两个问题，注射的自体血液往往泄漏到亚拉氏体空间，并沿着针道流回?...

脑内出血占中风患者的五分之一。脑内出血的预后取决于出血的速度、体积和位置。与前脑出血相比，脑干出血具有较高的死亡率和发病率3。大约40%的脑部出血发生在第4个庞氏菌中。庞汀出血的病因学和病理生理学与前脑出血5大出血有很大不同，研究较少。
有两种庞丁出血动物模型。一种是自发出血模型，由注入细菌拼贴酶在庞6，7，8诱发。这种模式的最大优点是出血是自发的。然而，拼贴酶只能诱发少量的庞丁出血。此外，拼贴酶可能会导致大脑的其他伤害。另一种模型是由自体血液的立体定向注射引起的。这种模式的优点是，它很容易掌握与高成功率。从理论上讲，研究人员可以将任何血量注入庞然大雅的任何位置。但是，由于通过针线的回漏，注射量有限。最近，双注法已推广，以减少回漏9。这种方法注射两次自体血，注射间隔为20分钟。双注射法用于诱发轻度 （30 μL） 和中度 （60 μL） 庞廷出血，但不诱发大规模庞然大意出血。在诊所里，大多数预后不佳的庞廷出血患者有大出血（超过10毫升）。
在以前的研究中，我们提供了一个协议，以建立一个庞廷缺血中风模型大鼠10。在这项研究中，我们修改了现有的双注射方法，并提供了一个详细的协议，通过在庞氏两个不同位置的100μL自体血的双重注射，诱导大鼠大量庞然大物出血。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100ml Saline solution</td>
			<td>Guangdong yixiang</td>
			<td>191222201 C1</td>
			<td>Preparing heparin diluent</td>
		</tr>
		<tr>
			<td>100&#956;l Microinjector</td>
			<td>Shanghai Gaoge</td>
			<td></td>
			<td>Injection of autologous blood</td>
		</tr>
		<tr>
			<td>1ml Syringe</td>
			<td>Jiangsu Zhiyu</td>
			<td>20191014</td>
			<td>Withdraw autologous blood from the tail vein</td>
		</tr>
		<tr>
			<td>75% Alcohol</td>
			<td>Shandong Lierkang</td>
			<td></td>
			<td>Disinfection of rat tail</td>
		</tr>
		<tr>
			<td>Adhesive tape</td>
			<td>Shanghai Jinzhong</td>
			<td></td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Animal anesthesia system</td>
			<td>RWD</td>
			<td>R510-31S-6</td>
			<td>Inducing and maintaining anesthesia</td>
		</tr>
		<tr>
			<td>Balance beam</td>
			<td>Jiangsu Saiangsi</td>
			<td></td>
			<td>For neurological deficit scores</td>
		</tr>
		<tr>
			<td>Blades</td>
			<td>Shanghai Feiying</td>
			<td>74-C</td>
			<td>For gross anatomy</td>
		</tr>
		<tr>
			<td>Bone cement</td>
			<td>Shanghai Xinshiji</td>
			<td>20180306</td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Brain tank</td>
			<td>Shenzhen LEIYEA</td>
			<td></td>
			<td>For gross anatomy</td>
		</tr>
		<tr>
			<td>Butorphanol tartrate</td>
			<td>Jiangsu Hengrui</td>
			<td></td>
			<td>For pain management</td>
		</tr>
		<tr>
			<td>Electric cranial drill</td>
			<td>Nanjing&#160; Darwin biotechnology</td>
			<td>20180090018</td>
			<td>Making a burr hole on the skull</td>
		</tr>
		<tr>
			<td>EP tube</td>
			<td>Nantong Surui</td>
			<td></td>
			<td>Transfer autologous blood</td>
		</tr>
		<tr>
			<td>Erythromycin eye cream</td>
			<td>Yunnan pharmacy</td>
			<td></td>
			<td>Eyes protection</td>
		</tr>
		<tr>
			<td>HE dye liquor</td>
			<td>Solarbio</td>
			<td>G1120</td>
			<td>For HE staining</td>
		</tr>
		<tr>
			<td>Heating&#160;pad</td>
			<td>Dangerous Jungle</td>
			<td>JR01</td>
			<td>Keeping warm</td>
		</tr>
		<tr>
			<td>Heparin sodium injection</td>
			<td>Chengdu Haitong Pharmacal Company</td>
			<td>190701</td>
			<td>Preparing heparin diluent</td>
		</tr>
		<tr>
			<td>IndoPhors</td>
			<td>Guoyao of China</td>
			<td></td>
			<td>Sterilization</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD</td>
			<td>20080701</td>
			<td>Inducing and maintaining anesthesia</td>
		</tr>
		<tr>
			<td>Light dark box</td>
			<td>Jiangsu Saiangsi</td>
			<td></td>
			<td>For neurological deficit scores</td>
		</tr>
		<tr>
			<td>Micro-injection pump</td>
			<td>Baoding Leifu</td>
			<td>TFD03-01-C</td>
			<td>Injection of autologous blood</td>
		</tr>
		<tr>
			<td>MRI system</td>
			<td>Philips</td>
			<td></td>
			<td>Confirmation of infarction in vivo</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Shanghai Jinzhong</td>
			<td>J32020</td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Penicilin</td>
			<td>Guoyao of China</td>
			<td></td>
			<td>Infection Prevention</td>
		</tr>
		<tr>
			<td>Q-tips</td>
			<td>Jiangxi Songhe</td>
			<td></td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Scalp heedle</td>
			<td>Jiangxi Hongda</td>
			<td>20200313</td>
			<td>Withdraw autologous blood from the tail vein</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Shanghai Kaiyuan</td>
			<td>170902</td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Shearing scissors</td>
			<td>Shanghai Jinzhong</td>
			<td>Y00040</td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>RWD</td>
			<td>900-00001-00</td>
			<td>for surgical positioning</td>
		</tr>
		<tr>
			<td>Surgical towel</td>
			<td>Xinxiang Huakangweicai</td>
			<td>20070601</td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Suture needle</td>
			<td>Shanghai Jinzhong</td>
			<td></td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Suture scissors</td>
			<td>Shanghai Jinzhong</td>
			<td>J25041</td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Tissue holding forcepts</td>
			<td>Shanghai Jinzhong</td>
			<td>J31080</td>
			<td>Surgicl instruments</td>
		</tr>
	</tbody>
</table>

massive pontine hemorrhage by dual injection of autologous blood

我们提供一个协议，在老鼠中建立一个巨大的庞然大河出血模型。这项研究使用了重约250克的老鼠。一百微升的自体血从尾静脉中取出，立体有毒地注射到庞斯中。注射过程分为两个步骤：首先，将10μL的血液注射到特定位置，前柱位置（AP）-9.0毫米：横向（拉特） 0 毫米：垂直（垂直）-9.2毫米，然后第二次注射位于AP-9.0毫米的残余血液;拉特 0 毫米;垂直 -9.0 毫米，间隔 20 分钟。平衡束测试、肢体放置测试和修改后的 Voestch 神经分数用于评估神经功能。磁共振成像 （MRI） 用于评估体内出血量。这种模型的症状与大量庞汀出血患者一致。

共使用25只动物，3只用于控制，6只用于30μL，6只用于60μL，10只用于100μL血液注射。一只接受100μL注射自体血（1/10）的老鼠在手术后24小时内死亡。
手术后在第1天、第3天、第7天和第14天进行行为测试。手术后不同时间点对照组和注射血组的分数在表2中显示。庞丁出血导致神经缺陷，如角膜反射和盘旋（<strong class...

Watch this Scientific Journal Video about Massive Pontine Hemorrhage by Dual Injection of Autologous Blood at JoVE.com

Massive Pontine Hemorrhage by Dual Injection of Autologous Blood

我们提出了一个协议，通过双注射自体血液，在老鼠中建立一个巨大的庞然大河出血模型。

大量庞廷出血通过双注射自体血

We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred ...

我们提供一个协议，建立在老鼠的大规模庞然大水出血。此模型易于建立、可重复且非常成功。蒸馏注射自体大鼠的方法可用于在任何大脑区域出血。想要尝试这些技术的人应该学习如何使用立体声轴框架，并熟悉庞廷解剖学。首先在鼠标上放置手术窗帘。沿着标记的中线用手术刀切开。使用棉签去除任何潜在的血液。然后在头皮瓣的每一侧放置一块钳子，以暴露头骨。将棉签浸入 0.9% 盐水中，轻轻从头骨中取出结缔组织。使用记号笔将布雷格马的中心点标记为原点。使用微型钻头进行颅骨切除术，小心地进行。完成后，从立体声框架中取出微钻。将 100 微升汉密尔顿注射器放入立体声电子支架中。打开喷射泵开关。单击快速吸入按钮。然后将肝素溶液吸气至100微升，并将其完全排干。将氯西丁和75%酒精手术磨砂从根部到尖端至少涂抹三次，对皮肤进行消毒，软化角质，扩张尾静脉，提高注射成功率。将头皮针灸连接到一毫升注射器上，然后将头皮针灸插入距尾尖三厘米的横向尾静脉，并取出 150 微升血液。从一毫升注射器中取出头皮针灸，将血液输送到管中。快速工作，防止血液凝固。换手术手套选择退出模式。将体积设置为 100 微升，并将速度设定为每分钟 200 微升。单击运行按钮，将 100 微升血液吸进汉密尔顿注射器中。切换到注入模式，并将速度设置为每分钟一微升。推进注射器，直到尖端达到大脑表面以下9.2毫米，并注入前10微升血液。单击运行按钮。停止注射，将注射器留在位20分钟，以防止血液流入亚拉赫诺德空间。收回注射器，直到尖端到达大脑表面以下九毫米处，然后以每分钟一微升的相同速度重新开始注射，直到残余血液完全注射。将注射器留在原位 10 分钟，以避免血液回流。慢慢地从大脑中取出注射器。然后使用骨水泥覆盖颅骨切除孔。当水泥干燥时，用四个零聚酰胺缝合丝缝合伤口。缝三四针后绑2个一一标准手术结。观察老鼠每15分钟，直到它完全从麻醉中恢复过来，然后用加热垫将其送回原来的笼子里。为老鼠提供免费食物和水。共有25只动物用于控制30微升、60微升和100微升血液注射。手术后在第一天、第三天、第七天和第十四天进行行为测试。庞廷出血导致神经缺陷，如角膜反射和盘旋减少。注射100微升血液也诱发肌痛。MRI扫描在手术后24小时进行。在血液注射组中，出血被检测为池塘 Basiler 部分带有 ISO 的低强化边缘，以稍微增压核心。出血量随着注射量的增加而增加。大鼠在手术后24小时14天被牺牲。和两毫米厚的部分。在注射部位周围发现出血，并在池塘底部分布。在100微升血液注射组出血周围有轻微水肿。一些老鼠在手术后三天被牺牲，石蜡部分被制成 HE 染色。结果表明，在出血区富集的血液注射组炎症细胞中，血红蛋白仍含有完整的红血球。尝试此协议时，请务必记住注射位置。这种方法可用于在任何特定的大脑区域诱发出血。

Research

JoVE Journal

Neuroscience

Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

Dual Somatic Recordings from Gonadotropin-Releasing Hormone GnRH Neurons Identified by Green Fluorescent Protein GFP in Hypothalamic Slices

双体录音从促性腺激素释放激素（GnRH）神经元下丘脑薄片鉴定绿色荧光蛋白（GFP）

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating ...

科研

神经科学

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

基因传递到产后大鼠脑非心室质粒注射和电穿孔

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

自体血液的注入模型小鼠自发性脑出血

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

Live Imaging of Drosophila Larval Neuroblasts

Live Imaging of Drosophila Larval Neuroblasts

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

实验脱髓鞘和小鼠脊髓髓鞘再生由溶血卵磷脂的焦点注射

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

小鼠蛛网膜下腔出血模型脑血管痉挛量化的容积法

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Minimally Invasive Endoscopic Intracerebral Hemorrhage Evacuation

微创内窥镜内腹出血疏散

Intracerebral hemorrhage (ICH) is a subtype of stroke with high mortality and poor functional outcomes, largely because there are no evidence-based ...

Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

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, ...

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 ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

双直接注射血液到西斯特纳麦格作为苏巴拉奇内德出血的模型

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...