Name: using laser doppler imaging and monitoring to analyze spinal cord microcirculation in rat
Uploaded: 2018-05-30T13:00:00.000+00:00
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涉及实验动物的动物议定书遵循了国家卫生研究院 (NIH) 制定的指导方针, 并得到了首都医科大学动物保育和使用委员会的批准。
使用以下描述的激光多普勒设备介绍 SCI 和测量脊髓 BF 的程序在发布的研究1中使用。
1. 手术准备
<ol><li>在生理盐水中准备戊巴比妥钠溶液 3% (w/v), 在35毫克/千克的剂量下进行管理。 注意: 戊巴比妥钠是一种受控物质。应保存详细记录, 并将解决方案存储在安全、锁定的位置。</li>
	<li>消毒设备, 准备手术区。<ol>
<li>用以下步骤清洁手术设备: 75% 乙醇清洗, 然后在121摄氏度的蒸压釜30分钟, 然后在60摄氏度烤箱过夜。用75% 酒精消毒手术区。</li>
	</ol>
</li>
</ol>2. 手术大鼠的制备
<ol><li>麻醉大鼠腹腔注射戊巴比妥钠 (35 毫克/千克)。整个过程应采取 30-40 分钟, 包括手术, 高炉测量, 缝合。</li>
	<li>从下背部到颈部剃去老鼠的背部区域。头发应尽可能短。把老鼠放在40摄氏度的加热垫上, 保持恒定的体温。</li>
</ol>3. 椎板切除术与脊髓震荡
注意: 要只为假组执行椎板切除术, 请按照步骤3.1 至3.6。
<ol><li>将动物背侧向上定位。用碘消毒剃光部分, 然后用无菌棉球75% 乙醇。做一个皮肤切口 (4 厘米) 与手术刀在椎板切除部位覆盖胸椎 T7 到 T11。</li>
	<li>从 T8 到 T10, 将两侧连接的肌肉切开, 以暴露棘突过程、椎板和关节。</li>
	<li>使用手术刀, 使切口断开 T10 和 T11 之间的连接。通过仔细解剖肌肉层以暴露骨骼, 进一步暴露连接。</li>
	<li>使用剪刀进一步清除肌肉远离叶片和周围的椎弓根小剪。这将在 T10 和 T11 的椎骨之间打开一个小空间 (图 1A)。慢慢地, 微妙地插入一个止血钳进入这个缺口, 并打破椎弓根 (图 1B)。确保钳的曲率始终处于横向, 远离脐带。在另一边重复。</li>
	<li>暴露脊髓 (图 1C) 并小心地提起并折断叶片。确保不要留下任何游离或锯齿状的骨骼碎片。</li>
	<li>重复该过程以进一步去除 T9 和 T8 板。</li>
	<li>将动物移动到撞击器设备表中, 并使用连接在表上的一对 Adson 钳, 通过夹紧 T7 和 T11 的棘突过程来稳定动物的脊椎, 然后调整镊子以伸直脊柱 (图 1D)。</li>
	<li>把动物放在撞击器下, 瞄准被暴露的脊髓中心的触击杆, 并将杆降低到脊髓表面3-5 毫米以内。</li>
	<li>设置撞击力 (160 KD) 和停留时间 (1 秒) 的影响参数<ol>
<li>通过单击软件界面上的 "开始实验" 按钮, 然后在下面的接口上单击 "是" 以自动启动影响。在影响之后, 软件将显示设置参数旁边的影响的实际数据, 检查数据以确保它靠近设置点 (图 1E)。 注: 实验成功的一个典型标志是短周期的非自愿尾部摆动和肢体运动后的影响。刺激尾巴检查肢体反射也可以做。然而, 运动评估, 如低音, 贝蒂, 和 Bresnahan (BBB) 运动规模22,23是必要的, 以确定诱发伤害的有效性。</li>
	</ol>
</li>
</ol>4. 激光多普勒扫描
<ol><li>有关本研究中使用的激光多普勒扫描仪的详细信息, 请参阅材料表。要扫描裸露的脊髓, 将大鼠背侧放在黑色的、不反光的背景上。</li>
	<li>
设置扫描参数:打开扫描软件, 单击 "测量" 以输入测量图形用户界面, 然后单击 "扫描仪设置" 按钮打开扫描仪安装界面。要扫描诸如本实验中的小区域, 请在 "扫描大小和显示选项" 下选择 "高分辨率", 以提高分辨率 (256 x 256 磅, 覆盖 4 x 10 厘米2) (图 2A)。单击 "图像扫描" 选项以检查扫描周长 (图 2B)。</li>
	<li>单击 "视频和距离" 选项以检查实时视频图像。将扫描仪放置在手术窗口上方10-13 厘米处, 并将背景与动物一起移动到扫描窗口 (图 2C) 上的裸露脊髓中心。</li>
	<li>使用 "自动远程" 功能微调扫描高度, 注意在实验图 2C中, 扫描高度应保持一致。</li>
	<li>使用不会反射盖与窗口, 只暴露手术区域, 以进一步减少背景和标记动物的方向。</li>
	<li>点击 "重复扫描", 设置扫描次数 (我们使用8重复扫描在这种情况下), 然后单击 "确定" 打开重复扫描界面。单击 "开始" 按钮开始扫描, 整个过程将需要大约 3-4 分钟 (图 2D)。</li>
</ol>5. 激光多普勒监测
<ol><li>我们使用了一个扫描仪显示器与 VP3 钝针端交付探针监视 BF, 因此2随着时间的推移。将激光多普勒探头与立体定向仪器连接在一起, 建立监测设备。</li>
	<li>把老鼠放在立体定向的器械上, 在必要时给动物垫上一小块泡沫塑料, 以使裸露的脊髓水平。</li>
	<li>将探头放在脊髓上以监测 BF。 注: 步骤5.3 对于测量的重现性至关重要, 因为数据读数对探头施加的压力敏感, 因此需要格外小心, 以不超过或不定位探头。<ol>
<li>检查切口和清除任何过量的液体或血液使用无菌棉垫。</li>
		<li>使用该仪器的 X 和 Y 轴定位探针到2毫米延髓到中心点的暴露脊髓或病灶点, 并避免中心静脉。</li>
		<li>使用 Z 轴慢慢地将探针降低到刚刚触及脊髓表面的水平。探头应该只是触摸脊髓的表面, 但不那么松散, 以允许任何明亮的光线从接触点一侧逃脱。</li>
	</ol>
</li>
	<li>
数据记录
	<ol>
<li>打开数据采集软件, 点击 "新实验" 按钮打开安装界面。在 "常规" 选项中检查系统配置并单击 "下一步" (图 3A), 在显示安装程序中选择 BF 的通道, 因此2 , 然后单击 "下一步" (图 3B)。</li>
		<li>输入文件信息并单击 "下一步" (图 3C) 输入数据录制界面, 单击绿色三角形按钮开始从探头记录数据 (图 3D)。</li>
		<li>信号稳定后, 连续8分钟记录数据。然后抬起探头, 将动物从立体定向装置中取出, 缝合切口, 将动物置于术后护理中。</li>
	</ol>
</li>
</ol>6. 缝合和术后护理
<ol><li>缝合切口: 在切口两侧的肌肉上插入缝合针。拉线通过, 拉组织在一起, 从而覆盖裸露的脊髓在被删除的叶片的地方。使用针架, 拉整个线程通过, 形成三平方节和修剪线程尽可能接近的节点。</li>
	<li>用与缝合切口相同的方式缝合皮肤 3-4 平方节, 然后从结节中修剪大约1厘米的螺纹。</li>
	<li>把老鼠放在笼子的一侧, 避免手术部位和笼子底部的接触。笼子应该放在暖气垫上。</li>
	<li>监测动物, 直到它从麻醉中醒来, 以确保没有手术后出血和缝合仍然关闭。</li>
	<li>皮下注射苄青霉素钠在大鼠手术后3天, 每天120毫克/千克。腹腔在手术后立即注射丁丙诺啡 (0.05 毫克/千克), 每6小时后手术1天。</li>
	<li>为了确保动物能够获得足够的食物和水, 装上长嘴的水瓶, 把食物靠近笼子里的动物。 注: 我们进行了脑屏障评分表, 以评估动物24小时后的后肢运动功能, 以排除在0以上的动物的脑屏障评分, 从而确保该动物瘫痪的诱发伤害。</li>
	<li>手术后, 如果需要, 每天两次轻轻按压腹部, 提供人工膀胱排空。</li>
</ol>

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Z., 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=Dynamics+of+Microvascular+Blood+Flow+and+Oxygenation+Measured+Simultaneously+in+Human+Skin.">Dynamics of Microvascular Blood Flow and Oxygenation Measured Simultaneously in Human Skin.</a> Microcirculation. 21 (6), 562-573 (2014).</li><li>Li, Z. Y., 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=Post+pressure+response+of+skin+blood+flowmotions+in+anesthetized+rats+with+spinal+cord+injury.">Post pressure response of skin blood flowmotions in anesthetized rats with spinal cord injury.</a> Microvascular Research. 78 (1), 20-24 (2009).</li><li>Muck-Weymann, M. E., 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=Respiratory-dependent+laser-Doppler+flux+motion+in+different+skin+areas+and+its+meaning+to+autonomic+nervous+control+of+the+vessels+of+the+skin.">Respiratory-dependent laser-Doppler flux motion in different skin areas and its meaning to autonomic nervous control of the vessels of the skin.</a> Microvasc Res. 52 (1), 69-78 (1996).</li><li>Stefanovska, A., Bracic, M., Kvernmo, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wavelet+analysis+of+oscillations+in+the+peripheral+blood+circulation+measured+by+laser+Doppler+technique.">Wavelet analysis of oscillations in the peripheral blood circulation measured by laser Doppler technique.</a> Ieee Transactions on Biomedical Engineering. 46 (10), 1230-1239 (1999).</li></ol>

作者没有什么可透露的。

执行此协议时应注意一些细节。首先, 应尽可能快速、优雅地进行麻醉和手术, 尽量减少对动物的压力。为了减少对结果的干扰, 保持动物处于相对平静和稳定的状态。其次, 在使用激光多普勒设备测量时, 应更加注意出血, 因为血液可能会干扰阅读。最后, 在数据记录过程中, 应将动物保存在温度控制的环境中, 避免因温度变化而产生不一致的结果。
研究人员在使用激光多普勒?...

脊髓损伤 (SCI) 的组织是高血管化的, 对缺氧非常敏感。我们以前的研究表明, 脑震荡后脊髓血流明显减少1,2, 这可能与运动功能的缺陷有关。最近的研究表明, SCI 后血管的完整性与感官运动功能的改善有很好的相关性3。据报道, 改良的血管可能会拯救白质, 间接导致改善功能4。因此, 维持损伤后脊髓灌注似乎是至关重要的维持生存能力和功能。
许多研究者在 sci5、6、7的实验模型中使用多种技术, 对 sci 后的各种治疗效果进行了研究。激光多普勒作为一种公认的技术, 无疑是定量研究几种动物和人体实验的有效方法8,9,10,11。该技术是基于测量的多普勒移位12诱导的红色血细胞到照明光。自二十世纪八十年代代初技术商业化以来, 激光技术、光纤和信号处理在激光多普勒仪器测量中的应用已经取得了很大的进展, 这使得该方法成为一种可靠的技术.
本研究采用激光多普勒测量方法对震荡大鼠脊髓血流 (BF) 进行评价。由于该技术的无创性和简单的设置, 我们的协议提供了一个敏感, 快速, 可靠的方法, 对高炉测量脊髓。更重要的是, 这种方法允许对高炉后震荡 SCI 的纵向研究没有动物牺牲在每个时间点。
由于有能力评估高炉的组织和快速灌注变化的刺激, 有可能应用此协议评估脑 BF14,15以及测量其他组织, 如肝16,17、皮肤18、19和肠道20。在大脑中动脉短暂闭塞的大鼠模型中, 采用激光多普勒读数, 以确保 BF 率适当地降低到缺血性半影14中预期的水平。在发生严重肢体缺血 (cli) 诱导的大鼠中, 应用激光多普勒扫描观察了术前和术后的后肢 BF, 并在治疗后的不同时期进行了21。此外, 一些药物的生物利用度和代谢清除依赖于肝 BF, 这是由16检测到的。因此, 可广泛应用于实验模型、药效学和药动学评价。

<table><tbody><tr><td>Laser Doppler Line Scanner</td><td>Moor Instruments</td><td>moorLDLS2</td><td></td></tr><tr><td>Laser Doppler Monitor</td><td>Moor Instruments</td><td>moorVMS-LDF</td><td></td></tr><tr><td>Probe for Monitor</td><td>Moor Instruments</td><td>VP3</td><td>Blunt needle end delivery probe</td></tr><tr><td>Impactor</td><td>Precision Systems and Instrumentation</td><td>IH-0400</td><td></td></tr><tr><td>Phenobarbital sodium</td><td>Sigma-Aldrich</td><td>P3761</td><td></td></tr><tr><td>Buprenorphine</td><td>Sigma-Aldrich</td><td>B-908</td><td></td></tr><tr><td>Syringe</td><td>Becton Dickinson Medica (s) Pte.Ltd</td><td>300841</td><td></td></tr><tr><td>Surgical suture needles with thread</td><td>Shanghai Pudong Jinhuan Medical Products Co., Ltd</td><td></td><td>18T0329 (batch number) /4-0</td></tr><tr><td>Scalpel</td><td>Operation instrument factory of Shanghai Medical Instrument Co., Ltd.</td><td>J11030 4#</td><td></td></tr><tr><td>Scalpel blade</td><td>Operation instrument factory of Shanghai Medical Instrument Co., Ltd.</td><td>J12130 20#</td><td></td></tr><tr><td>Ophthalmic forceps</td><td>Operation instrument factory of Shanghai Medical Instrument Co., Ltd.</td><td>JD1040</td><td></td></tr><tr><td>Hemostatic forceps</td><td>Operation instrument factory of Shanghai Medical Instrument Co., Ltd.</td><td>J31050</td><td></td></tr><tr><td>Benzyl penicillin sodium</td><td>North China Pharmaceutical Co., Ltd</td><td></td><td>F6072116 (batch number)</td></tr><tr><td>75% alcohol</td><td>Dezhou Anjie Gaoke disinfection products Co., Ltd</td><td></td><td>150421R (batch number)</td></tr><tr><td>Iodine</td><td>Shandong Lierkang Medical Technology Co., Ltd</td><td></td><td>20170102 (batch number)</td></tr><tr><td>Rat</td><td>Laboratory Animal Center, The Academy of Millitery Medical Sciences</td><td></td><td>Sprague-Dawly (rat strain)</td></tr></tbody></table>

using laser doppler imaging and monitoring to analyze spinal cord microcirculation in rat

激光多普勒血流仪是一种无创的血流 (BF) 测量方法, 它更适合于测量脊髓微循环的变化。在本文中, 我们的目标是使用激光多普勒成像和监测来分析在脊髓损伤后 BF 的变化。激光多普勒图像扫描器和探头/显示器都被用来获取每个读数。LDPI 的数据提供了高炉的局部分布, 对损伤部位的灌注进行了概述, 使高炉在不同地点进行了比较分析。通过在一段时间内对探测区域进行强烈测量, 采用联合探针同时测量脊髓的 BF 和氧饱和度, 显示整体脊髓灌注和供氧量。它本身有一些局限性, 如相对通量, 运动敏感性和生物零信号。然而, 由于高炉的简易设置和快速测量, 该技术已在临床和实验研究中得到应用。

LDPI 用于测量脊髓中的 BF, 这是通过提取线性轮廓 (图 4) 在脊髓的延髓-尾鳍轴上进行量化的。图 5A和图 5B分别代表了假组和 SCI 组脊髓的通量成像。图 5C和图 5D分别代表了假组和 SCI 组脊髓的延髓-尾轴的改变。图 5A和图 5B</s...

Watch this Scientific Journal Video about Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat at JoVE.com

Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat

在这里, 我们提出了激光多普勒灌注成像 (LDPI) 和激光多普勒灌注监测 (LDPM) 来测量脊髓局部血流量和氧饱和度 (因此,2), 以及一个标准化的程序, 脊髓的介绍老鼠受伤了

激光多普勒成像与监测在大鼠脊髓微循环分析中的应用

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory ...

using-laser-doppler-imaging-monitoring-to-analyze-spinal-cord

Research

JoVE Journal

Behavior

10.0K 次观看.  China Rehabilitation Research Center. 在这里, 我们提出了激光多普勒灌注成像 (LDPI) 和激光多普勒灌注监测 (LDPM) 来测量脊髓局部血流量和氧饱和度 (因此,2), 以及一个标准化的程序, 脊髓的介绍老鼠受伤了

视频: Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

优化系统的脑灌注监测在中风模型大鼠的大脑中动脉闭塞腔内

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

科研

医学

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

神经科学

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

超声造影成像脊髓血流量的评估实验脊髓损伤

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

在体内挫伤脊髓损伤后影像血管动力学的双色法

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained using laser Doppler flowmetry (LDF). This paper demonstrates a closed skull preparation that allows cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window. To evaluate autoregulatory mechanisms, a model of controlled blood pressure reduction via graded hemorrhage can be utilized while simultaneously employing LDF. This enables the real time tracking of the relative changes in the blood flow in response to reductions in arterial blood pressure produced by the withdrawal of circulating blood volume. This paradigm is a valuable approach to study cerebral blood flow autoregulation during reductions in arterial blood pressure and, with minor modifications in the protocol, is also valuable as an experimental model of hemorrhagic shock. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow.

This article demonstrates the use of laser Doppler flowmetry to evaluate the ability of the cerebral circulation to autoregulate its blood flow during reductions in arterial blood pressure.

使用激光多普勒流测量测量大鼠脑血流自动调节的评估

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

激光多普勒灌注成像在鼠标欣德利姆

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

猪缺血/再灌注模型中脊髓微灌注的实时评估

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...

<meta charset="utf8"></head><body>眶周激光多普勒血流测定法治疗啮齿动物 MCAO 诱导的缺血性卒中

Periorbital Placement of a Laser Doppler Probe for Cerebral Blood Flow Monitoring Prior to Middle Cerebral Artery Occlusion in Rodent Models

Middle cerebral artery occlusion (MCAO) is the gold-standard method for preclinical modeling of ischemic stroke in rodents. However, successful occlusion is not guaranteed by even the most skilled surgical hands. Errors primarily occur when the filament is not placed at the correct depth and include instances of either no infarction or vessel perforation, which can cause death. Laser Doppler flowmetry (LDF) is a reliable technique that provides real-time feedback on regional cerebral blood flow (CBF) during the MCAO procedure. Here we demonstrate a rapid technique for periorbital placement of a laser Doppler probe for measurement of CBF in both mice and rats. Our rationale was to simplify LDF implementation, encouraging widespread usage for improved surgical reliability. The technique eliminates the need for skull thinning and specialized equipment, with placement at the periorbital region rather than dorsal placement, promoting efficiency and ease of adoption. The protocol described here encompasses presurgical preparations, periorbital Doppler probe placement, and post-operative care. Representative results include visual depictions of procedural elements along with representative LDF tracings illustrating successful MCAO surgeries, with instances of unsuccessful filament placement leading to complications. The protocol illustrates LDF in confirming proper filament placement and offers a simplified procedure compared to alternative methods.

<meta charset="utf8"></head><body>作者聚焦:通过啮齿动物模型中区域脑血流的眶周多普勒监测推进中风研究

A minimally invasive surgical procedure is shown here, which involves placing the laser Doppler probe onto the skull over the distal region of the middle cerebral artery (MCA), a periorbital location suitable for rats and mice, to assess blood flow during transient MCA occlusion.

啮齿动物模型中激光多普勒探头在大脑中动脉闭塞前眶周放置用于脑血流监测

Middle cerebral artery occlusion (MCAO) is the gold-standard method for preclinical modeling of ischemic stroke in rodents. However, successful occlusion ...

激光多普勒成像与监测在大鼠脊髓微循环分析中的应用

摘要

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此视频中的章节

优化系统的脑灌注监测在中风模型大鼠的大脑中动脉闭塞腔内

一个

超声造影成像脊髓血流量的评估实验脊髓损伤

在体内挫伤脊髓损伤后影像血管动力学的双色法

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

使用激光多普勒流测量测量大鼠脑血流自动调节的评估

激光多普勒灌注成像在鼠标欣德利姆

猪缺血/再灌注模型中脊髓微灌注的实时评估

啮齿动物模型中激光多普勒探头在大脑中动脉闭塞前眶周放置用于脑血流监测

啮齿动物模型中激光多普勒探头在大脑中动脉闭塞前眶周放置用于脑血流监测