这项研究部分支持的NIH资助RO1NS053407，JH张。我们希望，感谢达蒙先生Klebe作出的宝贵贡献。

所有的程序进行，符合美国国立卫生研究院指南实验动物护理和使用的 Loma Linda大学的动物管理和使用委员会的批准。 1。术前准备工作无菌操作技术，被推荐用于所有的外科手术。立体定向设备消毒和手术前准备无菌手术工具。在所有的动物处理佩戴个人防护装备（PPE）。在手术过程中使用的加热垫，以保持动物的生理体温。 <ol><li>称量8-12周龄小鼠使用三束动物规模的。 </li><li>合作注入氯胺酮（100毫克/千克）和甲苯噻嗪（10毫克/千克）腹腔注射的麻醉7-10分钟才能生效（显示器足够的镇静）。 </li><li>将鼠标放置在一个热毯上，剃​​的头皮。 </li><li>眼膏的双眼。 </li><li>固定气道，轻轻地移动舌头侧面，并小心地保护鼠标的头部立体定向设备上。注意:头必须确保立体定向帧的基础水平。 </li><li>消毒手术区域用聚维酮碘，并用70％乙醇冲洗。重复交替应用的聚维酮碘和70％乙醇为共三次。棉签喷头可以用于此目的。 </li></ol> 2。注血模型<ol><li>一个1厘米长的中线切开头皮＃10手术刀。 </li><li>使用棉签涂抹，以清除覆盖在颅骨的软组织，以暴露的垂直交叉点的冠状面和矢状缝（前囟门）。 </li><li> Hamilton注射器（250微升）安装到注射泵上，和立体定向指示针（26表压）超过前囟。 </li><li>接下来，调整立体机械臂，定位针0.2毫米前和2毫米横向右侧。在这些坐标上使一个小的颅骨钻孔，使用变速1毫米的钻头钻。 </li><li>挂起动物的尾巴，并用70％乙醇消毒其下表面。 </li><li>尾动脉穿刺中央，用无菌针（ 如 26号）和收集动脉血毛细管的unheparinized管。 </li><li>转移的血很快从毛细管的Hamilton注射器的玻璃针管，然后将柱塞。 </li><li>现在30μl以上动脉的血液中含有Hamilton注射器的注射泵上重新连接和插入针（其斜角边缘朝向矢状缝）通过钻孔，，只是直到其锥不再可见。 </li><li>从这个角度提前针3毫米腹侧和2微升/分钟的速率注入5μl的自体血。 </li><li>结束后的第一注射预先针0.7毫米进一步深入。 </li><li>等待5分钟，然后注入25微升的血液进入右侧纹状体。 </li><li>完成后的第二次注射，在额外的10分钟的位置，离开针之前撤回在1毫米/分钟的速率。 </li><li>钻孔用骨蜡封，缝合皮肤。 </li><li>术后镇痛，注入0.05毫克/千克，丁丙诺啡皮下预温液（生理盐水）。 </li></ol> 3。注射胶原酶型号<ol><li>经术前准备后，重复步骤1-4所描述的血液注入模型。 </li><li>填补Hamilton注射器（10微升）与细菌（梭菌）胶原酶VII-S溶解在0.5微升的生理盐水0.075U。避免形成气泡。 </li><li>重新连接到注射泵，微量注射器插入针（26号），通过钻孔，直到它的伞是不再可见。 </li><li>推进针3.7毫米，腹部和胶原酶注入0.075U我置身2μl/ min的速率在右侧纹状体。 </li><li>注射完成后，在额外的10分钟的位置，离开针之前撤回在1毫米/分钟的速率。 </li><li>钻孔用骨蜡封，缝合皮肤。 </li><li>注入0.05毫克/千克的丁丙诺啡皮下预先温热的手术后的流体。 </li></ol> 4。假手术<ol><li>经术前准备后，重复步骤1-4所描述的血液注入模型。 </li><li>腹侧通过钻孔将针头（26号）3.7毫米。针应保持在10分钟的位置之前被在1毫米/分钟的速率排出。 </li><li>钻孔用骨蜡封，缝合皮肤。 </li><li>注入0.05毫克/千克的丁丙诺啡皮下预先温热的手术后的流体。 </li></ol> 5。代表性的成果实验性脑内出血唤起形态为以及在啮齿类动物的行为变化。这些变化可以进行评估，以确保有足够的执行过程，或调查的潜在治疗效果。生成区（ 例如基底节）有针对性的脑出血，是最重要的可重现的方法，并且可以对毛或组织学染色的脑切片（ 图1-2）验证。损伤的基底节感觉赤字，它可以通过各种行为评估量化结果。的拐角转弯测试结果表明，实验右路ICH后，小鼠转身显着更经常从受损的对侧（左）侧同侧和离开，比假手术组动物在手术后的24小时和72小时（ 图3 A）。此外，能够充分减值（左）前肢放置的表面上后，触须刺激，通过前肢放置试验评价。在手术后24小时和72小时，小鼠子遭离弃右路ICH均显着高于假手术动物存款减少。测量脑水肿经常采用量化的实验性脑出血后的脑损伤程度。导致脑组织含水量在同侧皮质和基底神经节在24显著增加（ 图4 A）和72小时（ 图4）手术后的脑注射自体血（30μL）或细菌胶原酶（0.075 U）（而假）。的行为测试（ 图3）和脑水肿的程度（ 图4）的结果表明在给定体积的血液和注射胶原酶模型之间没有区别。 <img alt="图1" src="/files/ftp_upload/4289/4289fig1.jpg" /> 图1。建模ICH（A）的冠状脑切片0.2毫米前的前囟门的简化原理图说明了公关的作用。oposed自体输血或注射胶原酶的位置。侧脑室标记LV。 CPu区代表尾状核，纹状体的一部分，并GP标识苍白球。两者，以及纹状体苍白球属于子皮质核的基团，也被称为基底节​​。 （B）代表的冠状脑切片0.2毫米前囟后纹状体内注射自体全血，在24小时的显微照片。 <img alt="图2" src="/files/ftp_upload/4289/4289fig2.jpg" /> 图2。组织学表现的血肿。代表苏木精伊红（H＆E）染色小鼠脑冠状冰冻切片（10微米），说明血肿大小在24小时后纹状体内注射胶原酶的细菌（0.075 U）。 LH =左侧大脑半球，RH =右半球。 &lt;<html> IMG ALT ="图3"SRC ="/ files/ftp_upload/4289/4289fig3.jpg"/&gt; 图3。在小鼠实验性脑出血后神经功能评估。，脑内注射自体血（30μL）或细菌胶原酶（0.075 U）引起的重现性神经功能障碍。 （A）小鼠实验性脑出血后表现出明显更合适的匝数比假手术组动物在手术后24小时和72小时。 （B）的前爪配售能力的左侧肢体受损脑出血后，在手术后24小时和72小时。值表示为平均值±标准差，采用Kruskal-Wallis单向方差分析排名，其次是学生Newman-Keuls法进行分析。 AP值&lt;0.05为差异有统计学意义，N = 6-12元组，P &lt;0.05相比，假的。 <a href="http://www.jove.com/files/ftp_upload/4289/4289fig3large.jpg" target="_blank">点击这里查看大图</a> 。 IGURE 4"SRC ="/ files/ftp_upload/4289/4289fig4.jpg"/&gt; 图4。在小鼠实验性脑出血后的脑组织含水量。评价自体血脑内注射（30μL）或的细菌胶原酶（0.075 U）导致显着增加脑组织含水量在同侧皮质和基底神经节在24（A）和72小时（B）在ICH-感应。值表示为平均值±标准差，单程方差分析，Tukey事后测试与分析。 AP值&lt;0.05为差异有统计学意义，N = 6-10元组，P &lt;0.05相比，假的。 <a href="http://www.jove.com/files/ftp_upload/4289/4289fig4large.jpg" target="_blank">点击这里查看大图</a> 。

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没有利益冲突的声明。

动物模型的脑出血（ICH）做出了巨大贡献深入的了解疾病的病理生理学，以及常用的开发和评估新的治疗策略在临床前的设置。实质内注射自体血或细菌胶原酶的产生ICH在啮齿类动物中的行之有效的方法。这两种方法在大鼠最初开发，但是，由于转基因和基因敲除菌株的迅速增加的可用性，小鼠成为必不可少的，以进一步阐明出血性脑损伤的机制8。 在人类中，基底节区脑出血占约50％的出血性中风，患者存活最初的事件经常发生，有害的神经功能障碍。因此，在啮齿类动物实验性脑出血，累及基底节，唤起动物的对侧肢体感觉赤字。对迄今为止，已经开发了几个行为评估表征这些损伤的小鼠和大鼠9,10。 在本研究中，我们描述和评价的改性双注入模型，自体全血6，以及一个ICH注入模型的细菌胶原酶7，两个定位在小鼠的基底节（文集纹状体）。我们评估经由转角转弯和前肢放置试验9,11行为缺陷，并在手术后24小时和72小时（ 图3）在两个模型观察感觉损伤增加。在这些时间点，ICH组之间没有显着差异，然而，以前的研究认为，长期损伤的进展胶原酶注射后，长期结果的研究更合适的模型，。脑水肿（脑含水量）使用的wet-weight/dry-weight的方法，如之前报道12,13。我们的研究结果显示，血肿周围脑水肿显着增加诱导脑出血后24小时和72小时（ 图4）。小鼠实验性脑出血或假手术幸存下来，直到一天的牺牲（死亡= 0％）。 两个脑出血模型，采用了立体定向辅助手术，以确保精确和可重复的有针对性的大脑区域的血液或胶原酶注射。达到这个目的需要一个小的开颅手术（1毫米钻孔）。它是必不可少的，以避免由钻头穿孔硬脑膜，由于这种不准确性会加剧在回流的血液或胶原酶在注射过程中的损伤和结果。 最初，作为一个单一的脑内注射14开发的血液注入模型中，但它常常产生了不一致的结果由于回流的血液沿针道15。为了尽量减少这种并发症，双注射方法，在其中一个小的量的血液被注入的正上方的目标的脑区，随后由第二次注射的血液进入基底节6。凝固的血的第一次注射，防止沿针道的逆流。此模型模仿一个迅速发展的血肿，但不引起实际的脑血管破裂。自体血注入模型的一个主要优点是，没有混杂因素，如外源蛋白，用于诱发脑出血。另外，细菌胶原酶模型的模仿自发性脑出血，开发了几个小时，展出约30％的脑出血患者5。细菌胶原酶是一种蛋白酶，溶解毛细血管周围的脑细胞外基质，削弱他们，引起容器破裂和随之而来的血液外渗16。这种模式一般用于调查血肿扩大的机制以及发展前景香港专业教育学院的治疗方法，影响平衡。然而，细菌胶原酶，可以放大的炎症反应，并呈现在高剂量的神经毒性作用3。此外，大量的出血，脑内注射胶原酶后，可能会产生 - 人类非物质文化遗产病理缺血性脑损伤。 有趣的是，雌性小鼠进行实验性脑出血神经功能缺损表现出显着更快的恢复比雄性动物8。类似的研究结果已在缺血性中风模型，因此，雄性啮齿动物更普遍采用的研究中风的病理和治疗评价17。 在这些实验中，我们利用腹膜内注射的麻醉药氯胺酮（100毫克/千克）和甲苯噻嗪（10毫克/公斤）两个ICH车型;然而，以前的研究已经报道了在麻醉啮齿动物的急性高血糖发病率，20内开始分钟在ketamine和甲苯噻嗪注射液18。此外，氯胺酮，N-甲基-D-天冬氨酸（NMDA）受体拮抗剂，可能会减少NMDA受体依赖性的兴奋性毒性，因此，改善脑损伤模型中的结果。挥发性麻醉剂交替使用，如异氟醚，在ICH临床前研究和注射剂的独特优势，包括快速改变的麻醉深度和短的恢复时间19。气体麻醉的主要缺点是需要精心制作的设备（蒸发器，流量计，掩模呼吸回路），以及人类气体暴露的可能性。此外，异氟醚已被报道，以减少出血性卒中后在小鼠20细胞凋亡。需要适应的最佳麻醉取决于长度的手术，动物种或菌株，和感兴趣的结果测量。 我们在描述和演示了两个脑出血模型，有尹居良优势和弱点，而代表特定的ICH性能的。必须考虑每个模型的典型特征，用于临床前ICH调查时。

<table border="1"><tbody><tr><td>材料</td><td>公司</td><td>目录编号</td><td>评论</td></tr><tr><td>立体定向头架</td><td> Stoelting有限公司</td><td> 51600 </td><td></td></tr><tr><td> Nanomite注射泵</td><td>哈佛设备</td><td> PY2 70-2217 </td><td></td></tr><tr><td> Hamilton注射器</td><td>汉密尔顿公司</td><td> 1725RN（250微升） 1701 RN（10微升） </td><td> 26号针头250微升和10微升注射器。 </td></tr><tr><td>微钻头</td><td>精细科学工具</td><td> 18000-17 </td><td></td></tr><tr><td>微钻头毛刺</td><td>精细科学工具</td><td> 19007-09 </td><td>直径为0.9mm </td></tr><tr><td>胶原酶VII-S </td><td> Sigma-Aldrich公司</td><td> C2399 </td><td></td></tr><tr><td> Microhematocrit毛细管</td><td> Fisher Scientific则</td><td> 22-362-574 </td><td> unheparinized </td></tr><tr><td>骨蜡</td><td>爱惜</td><td> W31 </td><td></td></tr><tr><td>缝合</td><td>爱惜</td><td> 1676G </td><td></td></tr><tr><td>氯胺酮</td><td> JHP制药</td><td> 42023-115-10 </td><td> Ketalar </td></tr><tr><td>二甲苯胺噻嗪</td><td> LLOYD实验室</td><td> 139-236 </td><td> AnaSed </td></tr></tbody></table>

modeling intracerebral hemorrhage in mice: injection of autologous blood or bacterial collagenase

自发性脑出血（ICH）定义了一个潜在的危及生命的神经系统毛病，占10％至15％的所有行程相关的住院和没有有效的治疗是迄今为止1,2。由于人类非物质文化遗产的异质性，不同的临床前模型中需要深入探讨未来的治疗策略3。实验性脑出血通常是在啮齿类动物中引起的脑实质内注射或自体血或细菌胶原酶4。选择合适的模型是根据出血的病理生理机制的诱导和伤害的进展。注血模型模拟了迅速进展的出血。另外，细菌胶原酶酶会破坏脑毛细血管基底膜，造成一个活跃的出血，一般发展几个小时。从可量化的结果血肿周围组织水肿和神经功能障碍米两种模式。在本研究中，我们描述和评价的改性双注入模型，自体全血6，以及一个ICH注入模型的细菌胶原酶7基底节雄性CD-1小鼠（纹状体区），这两者的目标。我们评估了诱导脑出血后神经功能的赤字和脑水肿24和72小时。脑内注射自体血液（30微升）或细菌胶原酶（0.075U）造成重现性神经功能缺损的小鼠，在24显着增加脑水肿和手术后72小时（对&lt;0.05）。总之，这两种模式产生一致的出血性梗死和代表进行临床前的非物质文化遗产研究的基本方法。

Watch this Scientific Journal Video about Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase at JoVE.com

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

脑出血（ICH）的临床相关的动物模型，需要扩大我们的出血性中风的知识，并研究新的治疗策略。在这项研究中，我们描述和评价两种的ICH模式，实行单方面或自体全血或细菌胶原酶注射到小鼠基底神经节（纹状体）。

在小鼠的脑出血模型:注射自体血液或细菌胶原酶

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

modeling-intracerebral-hemorrhage-mice-injection-autologous-blood-or

Nanyang Technological University

Research

JoVE Journal

Medicine

24.2K 次观看.  Loma Linda University School of Medicine. 脑出血（ICH）的临床相关的动物模型，需要扩大我们的出血性中风的知识，并研究新的治疗策略。在这项研究中，我们描述和评价两种的ICH模式，实行单方面或自体全血或细菌胶原酶注射到小鼠基底神经节（纹状体）。

视频: Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

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

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

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

Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral and cellular mucosal immune system1,2. 
 Microbiota is shielded from the immune system via a strong mucosal barrier. Infections and antibiotics are known to alter both the normal 
	gastrointestinal tract barrier and the composition of resident bacteria, which may result in possible immune abnormalities3.
HIV causes a breach in the gastrointestinal barrier with progressive failure of mucosal immunity and leakage into the systemic circulation of bacterial bioproducts, such as lipopolysaccharide and 
	bacterial DNA fragments, which contribute to systemic immune activation4-7. Microbial translocation is implicated in HIV/AIDS immunopathogenesis and response to therapy 4,8.
We aimed to characterise the composition of bacteria translocating in peripheral blood of HIV-infected patients. To pursue our aim we set up a PCR reaction for the panbacteric 16S ribosomial gene 
	followed by a sequencing analysis.
Briefly, whole blood from both HIV-infected and healthy subjects is used. Given that healthy individuals present normal intestinal homeostasis no translocation of microflora is expected in 
	these patients. Following whole blood collection by venipuncture and plasma separation, DNA is extracted from plasma and used to perform a broad range PCR reaction for the panbacteric 
	16S ribosomial gene9. Following PCR product purification, cloning and sequencing analyses are performed.

Our experiment will show how to perform a sequencing analysis of bacterial species translocating in peripheral blood of HIV positive patients.

测序外周血中细菌菌群:我们的经验与艾滋病毒感染者

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral ...

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

交付的治疗药物通过侧脑室（ICV）和静脉（IV）注射液在小鼠

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

局部或全身药物干预后评估的变化小鼠中挥发性全身麻醉药敏感性

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

视网膜血流小鼠活体视频显微镜测量

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

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

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective treatment and failure of large clinical trials aimed at hemostasis and clot removal demonstrate the need for further mechanism-driven investigation of ICH. This research may be performed through the framework provided by preclinical models. Two murine models in popular use include intrastriatal (basal ganglia) injection of either autologous whole blood or clostridial collagenase. Since, each model represents distinctly different pathophysiological features related to ICH, use of a particular model may be selected based on what aspect of the disease is to be studied. For example, autologous blood injection most accurately represents the brain&#39;s response to the presence of intraparenchymal blood, and may most closely replicate lobar hemorrhage. Clostridial collagenase injection most accurately represents the small vessel rupture and hematoma evolution characteristic of deep hemorrhages. Thus, each model results in different hematoma formation, neuroinflammatory response, cerebral edema development, and neurobehavioral outcomes. Robustness of a purported therapeutic intervention can be best assessed using both models. In this protocol, induction of ICH using both models, immediate post-operative demonstration of injury, and early post-operative care techniques are demonstrated. Both models result in reproducible injuries, hematoma volumes, and neurobehavioral deficits. Because of the heterogeneity of human ICH, multiple preclinical models are needed to thoroughly explore pathophysiologic mechanisms and test potential therapeutic strategies.

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 ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

在小鼠模型自发转移性肾细胞癌（MRCC）肾切除

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

超声引导下经皮内注射小鼠

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

造型中风小鼠:在远端大脑中动脉永久性混凝

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.