The author is grateful for the assistance of Mrs. Sharon Spana (University of Sydney) in rodent monitoring, handling and experimentation. The authorwould like to thank Prof. Jonathan Stone and Dr. Dan Johnstone for the assistance in the preparation of this manuscript. PhD funding support has been provided by University of Sydney and Australian Center for Excellence in Vision.

伦理声明:本协议遵循悉尼大学的动物护理准则，AEC＃5657。麻醉是经动物伦理委员会（悉尼大学，AEC＃5657）。 1，设备的准备<ol><li>使用实时皮肤温度跟踪。打开电脑和数据采集硬件。 </li><li>打开温度记录软件，调整设定温度之间30-35℃和采样，以每100毫秒的频率。 </li><li>可选:插入直肠体温计，以保证核心温度保持稳定在37.5℃。 </li></ol> 2.校准手册血压计<ol><li>新生儿连接手臂袖口sphygmomanometer.Use大小2袖口为250-550克的老鼠。适配器可能需要袖带管路连接到血压计。 </li><li>由任一松开空气释放阀或从适应断开袖带管袖带放气要么。确保无压力保持在管中及压力计针搁置在零的椭圆形/矩形内。 </li><li>检查油管，压力计和袖带之间的压力。充气的充气球的温柔泵袖口，直到它读取100毫米汞柱的压力表。确保压力保持不变。慢慢打开空气释放值袖带放气。 </li></ol> 3.动物的制备注意:动物是经过光损伤需要暗适应远程缺血前一天晚上。动物进行光损伤需要暗饲养（12小时光照:黑暗周期（5勒克斯）） <ol><li>在任何醒着还是anesthetised啮齿类动物进行远程缺血。保证动物有一个健康的肌张力。按捏上后肢，以确认有足够的肌肉存在确保这一点。 RIP诱导保护，防止光损伤已在大鼠久坐经测试长达6个月的年龄。 </li><li>对于R Anesthetised准备IP <ol><li>注入大鼠腹膜内注射60毫克/千克氯胺酮和5mg / kg的甲苯噻嗪。通过延伸腿和捏在脚的下侧的皮肤检查麻醉深度。动物有没有反射，如果它深深地anesthetised。应用人工泪液，以免角膜干燥，而在麻醉下。 </li><li>放置大鼠在任一热垫或循环水加热器管保持37.5℃的恒定体温。定位大鼠与下肢&#39;脚垫朝上俯卧位。右侧或左肢体可经历远程缺血。 </li></ol></li><li>对于RIP清醒的准备 注:清醒动物实验需要两个人。一个人抑制了动物和第二个人操作手动血压计。实验者必须有信心来执行的程序，约束会增加受伤的处理程序的风险。接受远程缺血大鼠必须牛逼空调o手动克制。取决于直觉准则手册约束应该从30秒进展到最多5分钟在数星期。胆小的动物，无法适应环境约束手动应排除清醒实验。最后，手动约束很可能引起应力（和潜在介绍的困惑的研究）对动物和假队列（袖带放置无炎症）必须被用来准确地解释的RIP的研究结果。 <ol><li>切一块毛巾到15厘米×30〜50厘米长，然后将短边垂直于老鼠的脊椎，覆盖头部后肢的顶部。 </li><li>掖短边下老鼠的躯干紧紧地开始缠上毛巾的剩余长边的老鼠。固定臂下的包裹动物以仰卧姿势。如果老鼠是根据左臂举行，不受毛巾老鼠的右侧肢体。 </li></ol></li></ol> 4.应用Ø˚F皮肤温度探头<ol><li>延伸是经受缺血和放置在皮肤探头放在脚掌的大鼠的腿。定位皮肤探头最大化温度探头和皮肤之间的接触。牢固地将探头插入脚垫并加盖探头纸带。 </li><li>通过跟踪温度记录软件温度检查皮肤探针放置。确保皮肤温度是30-34之间℃，并保持稳定。跟踪皮肤温度1-2分钟。调整皮肤探头如果温度不稳定或低于30℃。 </li></ol> 5.远程缺血<ol><li>袖带放气，并保证空气压力阀关闭。伸腿和松散包围在上后肢的袖口。用食指和拇指伸腿和低位保持松软的袖口位置。 </li><li>提高anesthetised动物的袖带压力为160毫米汞柱，而在清醒动物incre酶袖带压力180毫米汞柱。 注:大鼠的anesthetised血压范围从120-140毫米汞柱，并上升到160毫米汞柱时意识。开始的计时器和脚的温度记录一次正确的压力为止。 注:脚的温度应在5分钟的恒压后2℃下降。 </li><li>保持上述动物的"拐点"在整个缺血​​箍的位置。袖带压力将开始几分钟后丢弃，或者如果老鼠的肢体运动。 </li><li>反复泵送充气球在短脉冲以维持期望的袖带压的重复短脉冲抽</li><li>远程缺血可连续进行5至15分钟，被传递。缺血再灌注协议包括2中间有一个5分钟再灌注期5分钟缺血。 </li><li>通过松开空气压力阀袖带放气压力。检查温度变化过缺血亲的过程母育。松开袖带。 </li><li>继续损伤的实验。在麻醉状态下的效果的动物将需要被放置在加热垫上。继续监测动物，直到门诊。动物不能退回住房，直到行走。 </li></ol> 6.光损伤 - 视网膜变性模型<ol><li>暗适应动物过夜（12-15小时）。紧接着远程缺血空调或假远程缺血（动物约束）的地方动物有机玻璃外壳，食物和水。 </li><li>打开上方的有机玻璃外壳在上午9点24小时荧光灯（1000勒克斯）。曝光后，返回动物昏暗灯光循环7天。 </li></ol> 7.后远程缺血程序<ol><li>视觉评估与电图（ERG）: 注:ERG设置和闪光灯协议遵循布兰德利和石26。 <ol><li>一夜之间暗适应动物（12-15小时）。下暗淡红色照明通过腹膜内注射氯胺酮和赛拉嗪（60毫克/公斤和5毫克/公斤，分别）麻醉动物。散瞳（硫酸阿托品1.0％），角膜麻醉剂（丙美卡因0.5％）。 </li><li>应用角膜水化（卡波姆聚合物）的眼睛立即下降到角膜。适用于眼部凝胶，在20分钟的间隔，以维持角膜的​​水化。 </li><li>周围画一个眼球捆绑松散线程，以帮助稳定ERG录音。用直肠探头监测温度，保持动物的体温在37-37.5℃。 </li><li>放置一个Ganzfeld积分球内的头部。 注:Ganzfeld是一个完全可编程的光刺激，可提供统一的白衣闪烁的LED从的眼睛。 </li><li>记录使用定做4毫米铂正极轻轻触摸角膜和一个直径为2毫米的Ag / AgCl颗粒电极插入口的电图。参考两个电极的不锈钢针插入皮下臀部。 </li><li>与0.3-1,000赫兹（-3分贝）带通设置记录的信号，以2 kHz的采集速率（公元仪器）。经过一个稳定的ERG记录建立主题的动物有10分钟的暗适应录音开始前。 </li><li>按照先前由布兰德利和石26中描述的闪光灯协议。 <ol><li>计划闪光的持续时间（我们使用闪烁1-2毫秒的持续时间），并设置其强度为-4.4至2.0日志苏格兰人cd.sm -2。使用明亮的闪烁（2.0日志苏格兰人cd.sm -2，1毫秒）来衡量视网膜功能。在这项研究中是控制，光损伤和光损伤RIP之间的比较。 </li></ol></li></ol></li></ol> 8. TUNEL法<ol><li>通过腹膜内注射苯巴比妥过量（100毫克/千克）的动物实施安乐死。剜除眼睛和4％多聚甲醛固定。 </li><li> cyroprotecting前PBS洗眼睛眼睛过夜蔗糖30％（重量/体积）。嵌入眼睛在OCT化合物中并切成使用低温恒温器20微米的矢状切面。 </li><li>以下Maslim 等人的协议来执行与DAPI染色的视网膜切片的TUNEL法。27 </li><li>使用荧光显微镜的视网膜TUNEL计数。 TUNEL染色的细胞从外核层（ONL）记录;其中包含感光细胞核视网膜的最外层。在这项研究中，TUNEL计数一式三份作了为每只眼睛，用5眼对于每个处理组。 </li><li>使用单因素方差分析统计比较组是指控制，光损伤和光损伤+ RIP只。 </li></ol>

The author has nothing to disclose.

啮齿动物后肢缺血成功诱导带有手动血压计和袖口递送神经保护视网膜的光感受器。一发现与光损伤9,28缺血诱导调节感光保护一致。 从本质上讲，远程缺血引起短暂的缺氧给组织。因此，远程缺血预处理有许多相似之处缺血空调或交替称为缺血耐受，缺氧预处理，并在一定程度上，无氧运动。细胞缺血挑战通过释放大量的各种蛋白质，核苷和转录因子直接提供神经?...

大多数存活，也许所有，在代谢应激的面组织可以通过现有调理用周期亚致死缺血1,2的改进。缺血预处理（IP）在实际应用中是组织的暴露于亚致死缺血，组织经历更严重的压力，如随后的缺血性损伤之前。在动物模型中，IP提供给大脑，视网膜，心脏和肺3-6醒目的保护。与此相对应，在中风患者观察表明先前短暂性缺血发作和更好的临床结果7,8之间的链接。 IP也保护非缺血性损伤9视网膜感光。 知识产权在不同组织和伤害效果表明，它是活化细胞存活存在于所有组织的固有机制。心肌缺血预处理已建议有通过上调保护作用缺氧诱导因子（HIF），已知通过腺苷的释放或通过线粒体ATP钾通道10,11的开口，以调节许多代谢途径。腺苷释放和ATP钾通道有牵连的脑缺血但，调查缺血调理迄今为止的神经保护机制已经被集中在修改反兴奋性毒性，抗凋亡和抗炎通路12,13。总体上，缺血性调理用于保护神经元的分子过程的理解是有限的。 远程缺血预处理试图调节遥远极为重要器官（心脏，脑，肺）通过在较不关键组织产生缺血。使用后肢远程缺血预处理（RIP）已被证明是神经保护性中风14-17的啮齿动物模型。由我们所描述的方法提供了一种简单，可靠和非侵入性PROTocol诱导RIP。 RIP的协议绝大多数涉及后肢，大概是因为股动脉位于上后肢可以很容易地识别和手术夹紧和止血带的应用程序访问。在大脑和皮肤保护的研究侵入性肢体缺血的研究，缺血是由分离腹股沟韧带的股动脉夹闭股动脉致2,15,18。 从任一肢袖套或股动脉夹紧造成的缺血已证实在​​肢体变化，包括损失脉冲，降低氧合和皮肤温度下降。远程缺血可通过脉冲通过使用激光多普勒或超声多普勒17-19损失来确认。皮肤温度可以用作替代多普勒尽管这种关系是非线性的20,21。精确的温度记录是司空见惯的实验室和CAN很容易地纳入远程缺血性研究。 一种替代股骨夹紧手术是缺血使用止血带的诱导。止血带的应用程序产生媲美缺血到实现了与船舶夹紧; Kutchner 等 。相比侵入股动脉夹紧到一个非侵入止血带，发现两种方法停止在皮瓣缺血18整形外科模型血液流向肢体和减少皮肤损伤。掴或腿或臂和提高袖带压力到高于收缩期血压已发现可预防在猪和人类17,19,22局部缺血损伤。 不同止血带接近诱发缺血性远程包括使用血压袖带或松紧带17,22,23的。然而，使用弹性带以诱发局部缺血是一种不安全的方法，有可能引起的压力未调节量在肢，与压力上升到高于500毫米汞柱被记录在人类24。另外，使用弹性带肢体缺血导致肌肉损伤后大鼠除去带23的，如评估通过伊文思蓝染料， 在体内标记的肌纤维渗透率25。与此相反，输送一个受控的压力向止血带可以使用连接到一个血压计17,19,22,26血压袖带来实现。 在这项研究中，光感受器变性的光损伤模型，用于演示的远程缺血预处理的神经保护功效。远程缺血立即被诱导之前光损伤，并阻止随后的光感受器变性证实了视网膜功能测试。随行的视频将展示非侵入性的远程缺血的应用。

<table><tbody><tr><td>Gold series DuraShock hand aneroid sphygmomanometer</td><td>Welch Allyn</td><td>DS56</td><td>Manual Sphygmomanometer</td></tr><tr><td>Neonate [size 2] 1 tube, 10 pack</td><td>Welch Allyn</td><td>5082-102-1</td><td>Limb blood pressure cuff</td></tr><tr><td>Luer lock adaptor</td><td>Welch Allyn</td><td>5082-178</td><td>Adaptor for neonatal cuff</td></tr><tr><td>Thermistor pod</td><td>AD Instruments</td><td>ML 309</td><td>skin tempertature unit</td></tr><tr><td>Skin temperture probe</td><td>AD Instruments</td><td>MLT 422/A</td><td></td></tr><tr><td>Powerlab, 4 channel acquistion hardware</td><td>AD Instruments</td><td>PL 35044</td><td></td></tr><tr><td>Homeothermic blanket system with flexible probe</td><td>Harvard Appartus</td><td>507222F</td><td></td></tr><tr><td>Towel</td><td></td><td></td><td>optional: awake remote ischemia</td></tr><tr><td>Isoflo - 100% Isoflurane (250 ml)</td><td>Abbot Animal Health</td><td>05260-05</td><td>optional: inhaltion anaesthetic remote ischemia</td></tr><tr><td>Ketamil - ketamine 100 mg/ml (50 ml)</td><td>Troy Laboratories Pty Ltd</td><td></td><td>optional: injectable anaesthetic remote ischemia</td></tr><tr><td>Xylium - Xylazine 100 mg/ml (50 ml)</td><td>Troy Laboratories Pty Ltd</td><td></td><td>optional: injectable anaesthetic remote ischemia</td></tr></tbody></table>

remote limb ischemic preconditioning:  a neuroprotective technique in rodents

亚致死缺血保护免受随后，更严重的缺血组织通过内源性机制在受影响的组织的上调。亚致死缺血也已表明上调保护机制在远程组织。缺血（5-10分钟）在哺乳动物后肢的简短期间诱导的脑，肺，心脏和视网膜自我保护反应。该效应被称为远程缺血预处理（RIP）。它是保护重要器官的治疗有希望的途径，并且已经根据对心脏和脑损伤的临床试验。该出版物表明使肢体的控制，微创方法 - 大鼠的具体后肢 - 缺血。在人类新生儿开发用于一种血压箍被连接到一个手动血压计和用于施加围绕后肢的上部160毫米汞柱的压力。设计成检测皮肤温度的探针被用来验证ischemi一，通过记录下降引起的腿动脉压引起的闭塞皮肤温度和温度升高随后的压脉袋的释放。 RIP的这种方法提供保护，以对抗明亮光损伤和退行性变大鼠视网膜。

升高到高于160毫米汞柱的血压停止血流到后肢如在图1B中清楚地看到。组织氧合的缺乏导致了减少动物的脚温度为缺血再灌注协议（ 图2）。脚的温度（33℃）比核心温度低，可靠地减少在袖带压力升高（31℃）时，袖带放气（32℃）上升。单1000勒克斯的光损伤交付黯淡引发白鼠带或不带远程缺血性预处理。视网膜功能被记录并使用电图（ERG）进行评估。 <p class="jove_conte...

Watch this Scientific Journal Video about Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents at JoVE.com

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

远程缺血预处理（RIP）是调节组织反对破坏性压力的方法。我们已经建立了远程缺血的方法在后肢，由充气血压计袖带5-10分钟。 RIP的神经保护能力已经被证明在视网膜变性的啮齿类动物模型。

远程肢体缺血预适应:在啮齿动物的保护技术

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

remote-limb-ischemic-preconditioning-neuroprotective-technique

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

11.0K 次观看.  University of Sydney. 远程缺血预处理（RIP）是调节组织反对破坏性压力的方法。我们已经建立了远程缺血的方法在后肢，由充气血压计袖带5-10分钟。 RIP的神经保护能力已经被证明在视网膜变性的啮齿类动物模型。 

视频: Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Strategies for Study of Neuroprotection from Cold-preconditioning

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-&alpha; to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.

We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.

冷预处理神经保护作用的研究策略

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

科研

神经科学

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

双侧颈总动脉闭塞足够的预处理刺激诱导局灶性脑缺血早期缺血耐受

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

医学

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

肢体缺血小鼠模型

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

生物学

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

可重放截瘫胸主动脉阻断脊髓缺血再灌注的小鼠模型

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

使用电生理评估功能，在啮齿动物的视网膜和远程肢体缺血预适应的保护作用

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

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

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

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 ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

诱导和缺血再灌注损伤评估中的Langendorff灌注鼠心

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Methods for Acute and Subacute Murine Hindlimb Ischemia

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators&#39; ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

对急性和亚急性鼠下肢缺血的方法

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for neuroprotection, postconditioning treatments have proven to be promising therapies against cerebral ischemia. However, complicated procedures and potential safety issues limit their clinical application. To overcome these disadvantages, we have developed acidic postconditioning (APC) as a therapy for experimental focal cerebral ischemia. APC refers to the mild acidosis treatment by inhaling CO2 during reperfusion following ischemia. Here we present two models to execute APC in vitro and in vivo, respectively. The oxygen-glucose deprivation (OGD) treatment of mice and the corticostriatal occlusion and middle cerebral artery occlusion (MCAO) of mice were employed to mimic cerebral ischemia. APC can be simply achieved by transferring brain slices to acidic buffer bubbled with 20% CO2, or by mice inhaling 20% CO2. APC showed significant protective effects against cerebral ischemia, as reflected by tissue viability and brain infarct volume.

Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO2 after middle cerebral artery occlusion in vivo.