This work was supported by the American Heart Association Scientist Development Grant and the Foundation for Anesthesia Education and Research (MLJ). We would like to thank Talaignair N. Venkatraman PhD for his assistance with magnetic resonance imaging.

伦理声明：此协议已获得杜克大学实验动物管理和使用委员会，遵循一切为道德地使用动物的指导方针。 设备1。准备<ol><li>高压灭菌器在手术之前的外科手术工具。 </li><li>用70％乙醇消毒的立体定向装置。 </li><li>打开水浴，保持水温在42℃。 </li><li>溶于生理盐水IV型-S梭菌胶原酶在0.075ü每0.4微升的浓度。 </li></ol> 2，胶原酶注射模型<ol><li>权衡鼠标。 </li><li>在30％O 2/70％氮气 5％异氟醚麻醉鼠标感应室。后，当鼠标呼吸已经放缓至1每秒约2分钟的麻醉信号。 </li><li>插管气管与30毫米的20 G静脉导管。 </li><li>导管连接到啮齿动物呼吸机和机械通气肺与1.6％异氟醚30％ O 2/70％N 2和在每分钟105次呼吸用0.75毫升输送潮气量的比率.. </li><li>刮头皮与电子剃须刀。一旦小鼠被麻醉和插管，将其移动到一个不同的工作站，剃须，然后返回到手术台上。 </li><li>固定头在立体定位框架，并与两个冠状面和矢状缝作为参考点平头。 </li><li>适用眼药膏眼睛。 </li><li>插入直肠温度探头。使用车底循环水床维持肛温在37.0±0.2°C。 </li><li>擦拭用聚乙烯吡咯酮碘手术区随后用70％乙醇，并重复3次。 </li><li>做1厘米的中线切开头皮，用无菌棉签暴露前囟门横向擦拭骨膜。 </li><li>钻1毫米直径的钻孔2.2毫米离开纬度ERAL到前囟与水冷式钻头。 </li><li>旋转胶原酶小瓶5次，然后用25号针头（附设于立体定位框架）与0.5μl的胶原酶溶液洗0.5微升注射器5倍（发布0.5微升的胶原酶溶液在注射器最后一次洗涤后）。 </li><li>使针尖与钻孔然后驱逐0.1微升的注射器和擦拭针头斜面与剃刀丢弃。 </li><li>用显微操作，推进针3毫米深，以皮质和离开一动不动，持续30秒。 </li><li>注入0.4微升超过90秒。 </li><li>异氟醚降低至1％，并留针不动5分钟。 </li><li>退出针头缓慢。 </li><li>应用1 - 2滴0.25％布比卡因和皮下缝合皮肤。 </li><li>关闭异氟醚蒸发器和立体定位框架中移除鼠标。 </li><li>让鼠标恢复自主呼吸，随后拔管。 </li><li>返回鼠标到一个干净的笼子里，并允许自由进入食物和水。 </li></ol> 3，自体血液注射模型<ol><li>按照步骤2.1 - 2.11的胶原酶注入模型。 </li><li>绘制50微升的无菌生理盐水成30 G 50微升注射器。 </li><li>连接微升​​注射器用70厘米PE10管。 </li><li>驱逐所有的生理盐水从微升注射器插入PE10管完全去空气管。 </li><li>拉微升注射器活塞从1毫米到使气泡在PE10管微升注射器装置的远端开口，以避免在以后的程序盐水和血的混合物。 </li><li>擦拭鼠标的前端中央尾动脉区域用70％乙醇，并用剃刀切割的动脉在0.5至1厘米至尾尖。 </li><li>收集40微升的血液从切入PE10管微升注射器设备的尾巴。注意：肝素没有在针，套管，或鼠标使用。 </li><li>附上微升注射器到麟蹄CTION泵。 </li><li>一个27号针头的金属插管部分连接到PE10管的端部，并固定针到立体定位框架上的微操作。 </li><li>驱逐2微升的血掉的27号针头擦拭针头斜面与剃刀丢弃。 </li><li>使针尖与钻孔和插入针3毫米深皮质。 </li><li>注入35微升的自体血液以每分钟2微升的速度。 </li><li>减少异氟烷，以1％和离开针不动10分钟。 </li><li>退出针在30秒内。 </li><li>应用1 - 2滴0.25％布比卡因和皮下缝合皮肤。 </li><li>关闭异氟醚蒸发器和立体定位框架中移除鼠标。 </li><li>让鼠标恢复自主呼吸，随后拔管。 </li><li>返回鼠标到一个干净的笼子里，并允许自由获取食物和水。 </li></ol> 4，假手术<ol><li>遵循同样的程序，胶原酶注射液n模型，除了没有注射针插入后。 </li></ol> 5，手术后护理<ol><li>注入0.5毫升生理盐水皮下注射的在动物的脖子后面的手术过程的晚上。 </li><li>提供软化的食物与水和凝胶食品在小塑料杯中放置在笼子的地板上。每天更换食物7天。 </li><li>每天检查体重下降，伤口愈合和不舒服的迹象7天。 </li><li>如果大于7天的时间间隔恢复是必需的，拆线可根据光吸入麻醉（约1％的异氟醚中的30％O 2/70％N2）进行，如果必要的。 </li></ol>

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Lei, Sheng, Wang, Lascola, Warner, and Laskowitz have no conflicts of interest to declare. James received grant funding by American Heart Association, National Institutes of Health, and Cephalogics.

尽管新兴的临床前研究和由此产生的大量临床试验有前途的治疗15-18，也有表现，以改善脑出血预后无药物干预，并照顾在很大程度上仍然支持。可通过高通量技术，如转录组和蛋白质组的工作产生的可能的治疗的列表。虽然这些技术继续推进潜在的治疗靶点我们的知识，前进的希望的目标向后平移可通过使用临床相关临床前模型19-22最好的检验。因为它们允许快速吞吐选定的候?...

脑出血（ICH）是脑血管病的30日内1折磨的患者中，约有40-50％死于一种比较常见的形式。不幸的是，很少有改善的死亡率在过去的20年2。卫生3和全国学院从美国心脏协会4指引报告强调了发展非物质文化遗产的临床相关模型，以延长病理生理的认识和发展目标，新的治疗方法的重要性。 几种模式的存在是为了模仿人类非物质文化遗产5。由于脑出血病理生理的成熟的认识，它已成为明显的是，有多种型号可用于研究疾病的不同方面。以前使用的模型包括小鼠淀粉样血管病6，脑实质微球插入和通胀7，并直接动脉血浸润8,9。从淀粉样血管病脑叶出血进行了建模与使用转基因小鼠，代表了独特的非物质文化遗产亚型。微球模型模拟急性占位效应的血肿形成，但无法捕捉到大脑的血液存在细胞反应。最后，直接的动脉血液渗受试者大脑从股动脉的动脉压力。因此，该模型模拟动脉压和血液的存在，但不受到大脑由小血管破裂微血管损伤。此外，该模型具有固有的高变异性。有趣的是，自发性高血压大鼠10自发发展非物质文化遗产因为他们的年龄。脑出血后发展研究这些动物可能模​​仿这种疾病的主要合并症诱发人类非物质文化遗产之一的存在。虽然这些其他车型存在，纹状体内注射胶原酶梭菌11或instrastiatal注射液的中utologous全血12顷 ，目前，用于临床前研究的ICH最常见的两种模型。 脑出血模型的选择应根据实验的问题，包括品种选择，诱导形成血肿的方法的目标进行。举例来说，猪是大动物与比较大脑白质大脑体积相对于小鼠。因此，猪模型适合于研究脑白质病理生理以下非物质文化遗产。相比之下，啮齿动物的大脑在很大程度上是灰质，但转基因系统，使有用的啮齿动物脑出血后，评估损伤和恢复的分子机制。每个模型都有其固有的优势和劣势（ 表1），这应前实验慎重考虑。 以下协议证明在小鼠体内的自体血和胶原酶注射模型。这些模型已分别被翻译从最初在大鼠模型13,14和允许使用广泛使用转基因技术探索脑出血后与细胞死亡相关的分子机制。都代表人类非物质文化遗产明显不同损伤的机制，并且都具有明显不同的预期结果中的行为和组织措施方面。因此，某些假设可能会借给自己一个模型比其他，但很多想法可能需要验证在两个模型。 表1比较胶原酶和自体血注射脑出血模型的特点。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td style="width:197px;"></td><td style="width:80px;">注射胶原酶</td><td style="width:66px;">注血</td></tr><tr><td style="width:197px;">易于使用</td><td style="width:80px;"> + + + </td><td style="width:66px;"> + </td></tr><tr&gt; <td style="width:197px;">再生性</td><td style="width:80px;"> + </td><td style="width:66px;"> + </td></tr><tr><td style="width:197px;">出血的大小控制</td><td style="width:80px;"> + </td><td style="width:66px;"> + + + </td></tr><tr><td style="width:197px;">血液返流</td><td style="width:80px;"> + </td><td style="width:66px;"> + </td></tr><tr><td style="width:197px;">模拟人类疾病</td><td style="width:80px;"> + </td><td style="width:66px;"> - </td></tr><tr><td style="width:197px;">简单</td><td style="width:80px;"> + </td><td style="width:66px;"> + </td></tr><tr><td style="width:197px;">使用多个物种</td><td style="width:80px;"> + </td><td style="width:66px;"> + </td></tr></tbody></table>

<table border="0" cellpadding="0" cellspacing="0" width="416">
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			<td height="43" style="height:43px;width:152px;">Name</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Stereotactic frame</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51603</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Probe holder with corner clamp</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51631</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Mini grinder</td>
			<td style="width:88px;">Power Glide</td>
			<td style="width:88px;">Model 60100002</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">0.5 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co.</td>
			<td style="width:88px;">86259</td>
			<td style="width:88px;">25 gauge needle</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">50 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7637-01</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">26G Hamilton needle</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7804-03</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Syringe pump</td>
			<td style="width:88px;">KD Scientific</td>
			<td style="width:88px;">Model 100</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Heat therapy water pump</td>
			<td style="width:88px;">Gaymar Industries, Inc.</td>
			<td style="width:88px;">Model# TP650</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Circulating waterbed</td>
			<td style="width:88px;">CMS Tool &#38; Die, Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Rodent ventilator</td>
			<td style="width:88px;">Harvard Apparatus</td>
			<td style="width:88px;">Model 683</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Isoflurane vaporizer</td>
			<td style="width:88px;">Drager</td>
			<td style="width:88px;">Vapor 19.1</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Air flowmeter</td>
			<td style="width:88px;">Cole Parmer</td>
			<td style="width:88px;">Model PMR1-010295</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Induction chamber</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
			<td style="width:88px;">Self made</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Otoscope</td>
			<td style="width:88px;">Welch Allyn</td>
			<td style="width:88px;">22820</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">intravenous catheter</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">381534</td>
			<td style="width:88px;">20-gauge, 1.16 inch Insyte-W</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Isoflurane</td>
			<td style="width:88px;">Baxter Healthcare Corporation</td>
			<td style="width:88px;">NDC10019-360-69</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Collagenase Type IV-S</td>
			<td style="width:88px;">Sigma</td>
			<td style="width:88px;">C1889</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE20</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427406</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE10</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427401</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">30G 1 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305128</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">27G 1 1/4 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305136</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Surgical scissors</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">21-539</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Forceps</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">17-307</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Needle holder</td>
			<td style="width:88px;">Boboz</td>
			<td style="width:88px;">RS-7840</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Monofilament suture</td>
			<td style="width:88px;">Ethicon</td>
			<td style="width:88px;">8698</td>
			<td style="width:88px;">Size 5-0</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Indicating controller</td>
			<td style="width:88px;">YSI</td>
			<td style="width:88px;">73ATD</td>
			<td style="width:88px;"></td>
		</tr>
	</tbody>
</table>

intrastriatal injection of autologous blood or clostridial collagenase as murine models of intracerebral hemorrhage

脑出血（ICH）是脑血管疾病的一种常见的形式，并与显著发病率和死亡率相关联。缺乏针对止血和血栓清除大型临床试验有效的治疗和失败表明，有必要对非物质文化遗产的机制，进一步推动调查。这项研究可能通过临床前模型提供的框架下进行。两种小鼠模型中普遍使用包括纹状体（基底节区）注射要么自体全血或梭菌胶原酶。因为，每一个模型代表与非物质文化遗产明显不同的病理生理特点，使用一个特定的模式，可能基于什么方面的病是需要研究来选择。例如，自体血注入最准确地代表了大脑的反应的实质内血液的存在，并可能最为密切复制脑叶出血。梭菌胶原酶注射最准确地代表的S商场血管破裂和出血深部血肿的演变特征。因此，每个模型的结果在不同的血肿形成，神经炎症反应，减轻脑水肿的发展，以及神经行为的结果。一名自称是治疗干预的鲁棒性可以使用这两种模式是最好的评估。在这个协议中，诱导非物质文化遗产的利用这两种模式，立即手术示范损伤和早期术后护理技术论证。这两款机型导致重复性的伤害，血肿体积和神经行为缺陷。由于人类非物质文化遗产的异质性，需要多个临床前模型，彻底探讨的病理生理机制，并测试潜在的治疗策略。

由于血肿形成（ 图1）的差异，同侧转动显示醒来自体血注入小鼠体内后，立刻在2 -胶原酶注射后4小时，如血肿扩大发生（ 图2）。同侧转动的情况下应提高关注没有显著的伤害。在第一篇文章伤天，老鼠在这两种模式应该表现出显著神经功能缺损（ 图3）。在注射后24小时，患侧半球显示稳定的血肿量（ 图4）;进一步地，在注射后24小时，脑含水...

Watch this Scientific Journal Video about Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage at JoVE.com

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

自体血液或梭菌胶原酶脑内注射为脑出血的小鼠模型

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

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14.6K 次观看.  Duke University. Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase. 