本次工作得到了四川省重大研发专项（2022YFS043）和成都中医药大学青年基金会发展人才专项（QJJJ2022029）的支持。

该动物实验方案经成都中医药大学实验动物福利伦理委员会审核通过（备案号：2023-04）。本研究使用雄性 Sprague Dawley （SD） 大鼠（280 ± 20 g，8-10 周龄）。老鼠被关在动物室里，可以自由饮水和进食。
1.溶液制备
<ol><li>准备 0.9% NaCl 溶液，用于保持工作区域充分湿润。</li>
	<li>要制备 20% 高渗 NaCl 溶液，将 2 g NaCl 溶解在 10 mL 双蒸水 （ddH2O） 中。为了确定心肌的平行电导率，有必要改变脑室内液体的电导率。</li>
	<li>制备1%的酶活性粉末洗涤剂溶液。在实验完成时使用它，将压力容积导管浸入溶液中1-2小时。</li>
	<li>通过将 10 mg 阿魏酸溶解在 20 mL ddH2O 中来制备 FA 溶液，通过 0.22 μm 膜过滤溶液。向大鼠注射1mL / kg阿魏酸溶液。</li>
</ol>2.传感器的准备
<ol><li>在实验开始前，将压力体积传感器浸入37°C的0.9%NaCl溶液中约30-60分钟，这有助于实验数据的稳定性。</li>
	<li>连接实验设备。测量压力-容积回路的系统由一根压力-容积导管、两个控制单元、一个记录单元和计算机运行软件组成。软件中的压力-体积回路模块将提供参考实验程序。</li>
	<li>按 下“开始 ”按钮，软件将自动记录来自压力体积传感器的监测数据。</li>
	<li>使用 Miro-Tip 压力体积 （MPVS） 软件校准压力和电导率。</li>
</ol>3.实验前大鼠制备
<ol><li>通过肌肉注射给大鼠氯胺酮（50mg / kg）和芬太尼（0.25mg / kg）5。</li>
	<li>捏住大鼠的脚趾，通过没有反射来验证麻醉深度。情绪会影响大鼠的生理状态，疼痛会导致心脏功能的改变16.使用小动物剃须刀和脱毛膏去除手术部位的毛发。使用碘磷和 75% 酒精擦拭皮肤以保持无菌。</li>
	<li>将完全麻醉的大鼠固定在等温加热板上，背部与加热板接触。</li>
	<li>将涂有凡士林的温度探头插入大鼠的直肠中。通过调节加热板将大鼠体温保持在37°C±0.5。 注意：在实验过程中必须保持气道畅通。</li>
</ol>4. 将导管插入右颈动脉
<ol><li>纵向切开大鼠颈部正中线右侧的皮肤。做一个 4 厘米的切口，用镊子分离肌肉和结缔组织。位于气管右侧的颈动脉是可见的。大鼠的右颈动脉呈暗红色，脉动强烈，并有一条与之平行的白色迷走神经。</li>
	<li>使用镊子将颈动脉与其他组织和神经分开。在干净的颈动脉下方放置三条 5-0 手术线。将无菌的0.9%氯化钠溶液滴入手术区域，以保持颈动脉的湿润。</li>
	<li>切开左锁骨上方的皮肤，剥离颈静脉周围的组织。然后，在左颈静脉下方放置一个 5-0 手术线。</li>
	<li>使用动脉夹暂停近端的血流，使用微型剪刀剪断血管中血流停止的部分。伤口横截面出现少量血液是正常的。如果血液快速间歇性地流出血管，请抬起近端手术管并再次使用动脉钳。</li>
	<li>将导管从横截面沿颈动脉深入左心室。确保进入左心室后的最低收缩压值接近 0 mmHg。</li>
	<li>为了获得合理的压力-容积关系，稍微调整心室中的压力-容积导管。为防止大量失血和导管因心跳而改变位置，请结扎手术管路的近端。 注意：在此过程中，大鼠的体温、麻醉水平、压力信号和电导信号应保持稳定。大鼠的呼吸道应保持畅通。</li>
</ol>5.药物注射和电导率校准
<ol><li>保持压力容积导管在心室腔室中的位置，数据稳定后，结扎颈静脉远端的手术管线结扎并缓慢注射至1mL / kg阿魏酸溶液。观察5-10分钟。</li>
	<li>从左颈静脉注射 50 μL 20% NaCl 溶液以去除心肌产生的平行电导。平行电导的体积范围约为 130-280 μL5。以 2 分钟的间隔重复此操作 3 次。</li>
	<li>在大鼠进行心室压力和容量测试后，使用采血针从大鼠腹主动脉抽血。将收集的血液放入肝素钠收集管中，上下倒置 2 次以防止血液凝固。通过左颈静脉注射120mg / kg戊巴比妥钠对实验大鼠实施安乐死。</li>
	<li>使用大鼠容量校准管将测得的电导转换为实际血容量。将血液与肝素钠混合的血液依次放入校准管的孔中，导管检测不同孔中血液的电导值并将其记录在压力-容积监测模块中。</li>
</ol>6. 数据分析
<ol><li>通过将已知体积血液的测量电导率值添加到指定位置，软件会自动绘制曲线并推断血液的电导率。使用至少三组血液电导率值来推断被测大鼠的血液电导率。血液电导率是个体化的。对于测试下的每只大鼠，分别执行此程序。</li>
	<li>高渗校准：通过将三次高渗盐水注射获得的数据添加到指定位置，软件计算平行电导平均值并自动校准实验数据。</li>
	<li>利用血压和电导值稳定的区域分析大鼠的左心室功能。</li>
	<li>点击 “分析 ”，软件会根据所选区域自动计算各种参数，包括EF（左心室射血分数）、SW（卒中功）和CO（心输出量）等。</li>
</ol>

使用压力-体积电导技术监测大鼠的心脏功能

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作者声明，该研究是在没有任何可被解释为潜在利益冲突的商业或财务关系的情况下进行的。

对于不同的心脏功能状态，必须采用合理的给药策略。压力-容积电导导管技术是研究左心室功能最直观的方法5.这种方法可以从整体角度研究药物对心脏功能的影响。我们详细描述了实验的各个阶段。这将为心脏功能的研究提供一些便利。
压力容积电导率导管技术是最全面、最严格的方法。在一次实验中可以获得多达30个指标的信息，包括绝对值（压力和体...

心血管疾病是世界上死亡率最高的国家 1.其病因包括冠状动脉狭窄（心肌缺血）、冠状动脉阻塞（心肌梗死）和缺血再灌注损伤2。由于心脏处于恒定的收缩期和舒张期，它是身体最需要能量的部位之一。因此，当冠状动脉难以维持足够的能量和氧气时，心脏功能不可避免地下降，从而对其他器官产生负面影响3,4。心脏是循环系统中的动力源，需要理性评估心脏功能。
通过心室压力和容量关系评估心脏功能被认为是一种综合方法5。在整个心动周期中，心室压力和容积的实时变化构成了压力-容积环。心室压力-容积环可以根据心室的不同阶段和能量对心脏功能和储备能力进行定量分析。正常心室收缩末期容积小，搏动功好，效率高5,6,7。
压力容积传导导管技术是一种检测左心室状态的侵入性方法。它可用于获得连续的实时压力-体积回路8。 压力容积电导率导管是强大的工具，良好的处理程序对于可重复和可靠的结果至关重要，包括在生理盐水校准期间对心肌平行电导率进行 体内 分析，以及在比色皿校准中对血液电导率进行 体外 测量3。
阿魏酸（FA）是一种酚酸，广泛分布于植物界，如燕麦和女贞子9,10。阿魏酸具有降低血压和心律失常的药理作用。FA是一种具有多种功能的生物活性天然产物。FA能抵抗氧化损伤，减少炎症反应，抑制血小板聚集，预防冠心病和动脉粥样硬化11。然而，大多数关于阿魏酸的研究都集中在心脏的一个方面，很少评估阿魏酸在循环系统中的作用12,13,14,15。在这里，我们描述了一种异氟醚麻醉联合氯胺酮 （50 mg/kg） 的闭胸方法，重点是颈静脉注射过程中对阿魏酸溶液的心脏反应。
我们将描述在闭胸条件下使用该工具的完整程序，包括溶液制备、换能器制备、实验前大鼠制备、导管插入右颈动脉和数据分析。实验的持续时间通常小于4小时，由不同的实验方案决定。在单个实验中，我们可以获得详细的心脏信息，例如左心室压力、容量和心率。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>Sartorius AG, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal temperature maintainer</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>69020</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual Bio Amp</td>
			<td>Millar, Inc., USA</td>
			<td>DA-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzyme-Active Powdered Detergent</td>
			<td>Alconox Inc., USA</td>
			<td>1104</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferulic acid</td>
			<td>&#160;Macklin Biochemical Co., Ltd,Shanghai, China</td>
			<td>F900027</td>
			<td></td>
		</tr>
		<tr>
			<td>Mikro-Tip Catheter Transducers, SPR-838NR</td>
			<td>Millar, Inc., USA</td>
			<td>SPR-838NR</td>
			<td></td>
		</tr>
		<tr>
			<td>Millar Miro-Tip Pressure Volume (MPVS) Ultra</td>
			<td>Millar, Inc., USA</td>
			<td>SPR-869</td>
			<td></td>
		</tr>
		<tr>
			<td>Pet electric clippers</td>
			<td>Jinyun County New Concept Home Supplies Co., Ltd.</td>
			<td>&#160;-</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Lab 8 / 35</td>
			<td>Millar, Inc., USA</td>
			<td>PL3508</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride, NaCl</td>
			<td>&#160;Kelong Chemical Reagent, Chengdu, China</td>
			<td>KX829463</td>
			<td></td>
		</tr>
		<tr>
			<td>Veet hair removal cream</td>
			<td>Shanghai Songqi E-commerce Co., Ltd.</td>
			<td>3226470</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time detection of ferulic acid effects on rat left ventricle using pressure-volume conductivity catheter

心脏功能下降会对其他器官产生负面影响。左心室压力-容积关系被认为是评估心脏功能的有效方法。心脏功能的实时监测对于药物评估非常重要。在闭胸条件下，微型换能器是压力容积导管的重要组成部分，通过右颈动脉进入大鼠的左心室。该设备以压力-容积环的形式可视化实验过程中心脏功能的变化。通过将50μL20%氯化钠溶液注射到大鼠的左颈静脉中来改变血液的电导率来计算心室的实际体积。通过使用压力 - 体积电导导管测量已知体积中血液的电导率来计算大鼠心室腔的实际体积。该协议允许持续观察药物对心脏的影响，并将促进在心血管疾病中使用特殊种族药物的基本原理。

每个测试 （n = 3） 都基于压力容积电导率导管进入左心室。当导管从颈动脉进入左心室时，信号会发生显著变化，例如压力范围明显增加（图 1）。通过绘制 Y 轴上的体积 （μL） 和 X 轴上的压力 （mmHg） 来完成压力-体积关系的图形分析。大鼠左心室压在10-105 mmHg以内，体积电导值在65-115 μL以内。
完整的心动周期由逆时针压力-容积环形成（<strong class="x...

Watch this Scientific Journal Video about Author Spotlight: Advantages of Pressure-Volume Loop Measurement Method in Cardiovascular Research at JoVE.com

作者聚焦：压力-体积环测量方法在心血管研究中的优势

该协议描述了一种使用压力 - 体积电导技术测量左心室压力和体积的方法。这种方法可以连续实时监测药物对心脏的影响。

使用压力-容积电导导管实时检测阿魏酸对大鼠左心室的影响

Decreased cardiac function can have a negative impact on other organs. The left ventricular pressure-volume relationship is considered to be a valid ...

麻醉是动物实验中不可避免的问题。在本实验中，不同程度的麻醉对实验结果有显著影响。与开胸法相比，这种压力-容积环测量方法更易于学习，包括更少的实验工具和更少的实验人员学习时间。我们的实验室将研究中医对心血管疾病的影响，探索其对心脏健康的广泛但尚未完全了解的影响。

real-time-detection-ferulic-acid-effects-on-rat-left-ventricle-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

Real-Time Detection of Ferulic Acid Effects on Rat Left Ventricle Using Pressure-Volume Conductivity Catheter

398 次观看.  Chengdu University of Traditional Chinese Medicine. 麻醉是动物实验中不可避免的问题。在本实验中，不同程度的麻醉对实验结果有显著影响。与开胸法相比，这种压力-容积环测量方法更易于学习，包括更少的实验工具和更少的实验人员学习时间。我们的实验室将研究中医对心血管疾病的影响，探索其对心脏健康的广泛但尚未完全了解的影响。

视频: 作者聚焦：压力-体积环测量方法在心血管研究中的优势

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

多光谱实时荧光成像术在妇科肿瘤前哨淋巴结的检测

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

科研

医学

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac substrates such as left atrial flutter. This protocol describes all the clinical and invasive interventional steps performed during a human electrophysiological study and ablation to assess the accuracy, safety and real-time navigation of the Catheter Guidance, Control and Imaging (CGCI) system. Patients who underwent ablation of a right or left atrium flutter substrate were included. Specifically, data from three left atrial flutter and two counterclockwise right atrial flutter procedures are shown in this report. One representative left atrial flutter procedure is shown in the movie. This system is based on eight coil-core electromagnets, which generate a dynamic magnetic field focused on the heart. Remote navigation by rapid changes (msec) in the magnetic field magnitude and a very flexible magnetized catheter allow real-time closed-loop integration and accurate, stable positioning and ablation of the arrhythmogenic substrate.

This report provides a detailed description of a new remote navigation system based on magnetic driven forces, which has been recently introduced as a new robotic tool for human cardiac electrophysiology procedures.

遥控磁导航导管定位准确，实时和心脏电生理消融程序

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac ...

Catheter Ablation in Combination With Left Atrial Appendage Closure for Atrial Fibrillation

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and disordered electrical activity in the atria gives rise to palpitations, fatigue, dyspnea, chest pain and dizziness with or without syncope 4, 5. Patients with AF have a five-fold higher risk of stroke 6.
Oral anticoagulation (OAC) with warfarin is commonly used for stroke prevention in patients with AF and has been shown to reduce the risk of stroke by 64% 7. Warfarin therapy has several major disadvantages, however, including bleeding, non-tolerance, interactions with other medications and foods, non-compliance and a narrow therapeutic range 8-11. These issues, together with poor appreciation of the risk-benefit ratio, unawareness of guidelines, or absence of an OAC monitoring outpatient clinic may explain why only 30-60% of patients with AF are prescribed this drug 8.
The problems associated with warfarin, combined with the limited efficacy and/or serious side effects associated with other medications used for AF 12,13, highlight the need for effective non-pharmacological approaches to treatment. One such approach is catheter ablation (CA), a procedure in which a radiofrequency electrical current is applied to regions of the heart to create small ablation lesions that electrically isolate potential AF triggers 4. CA is a well-established treatment for AF symptoms 14, 15, that may also decrease the risk of stroke. Recent data showed a significant decrease in the relative risk of stroke and transient ischemic attack events among patients who underwent ablation compared with those undergoing antiarrhythmic drug therapy 16.
Since the left atrial appendage (LAA) is the source of thrombi in more than 90% of patients with non-valvular atrial fibrillation 17, another approach to stroke prevention is to physically block clots from exiting the LAA. One method for occluding the LAA is via percutaneous placement of the WATCHMAN LAA closure device. The WATCHMAN device resembles a small parachute. It consists of a nitinol frame covered by fabric polyethyl terephthalate that prevents emboli, but not blood, from exiting during the healing process. Fixation anchors around the perimeter secure the device in the LAA (Figure 1). To date, the WATCHMAN is the only implanted percutaneous device for which a randomized clinical trial has been reported. In this study, implantation of the WATCHMAN was found to be at least as effective as warfarin in preventing stroke (all-causes) and death (all-causes) 18. This device received the Conformit&eacute; Europ&eacute;enne (CE) mark for use in the European Union for warfarin eligible patients and in those who have a contraindication to anticoagulation therapy 19.
Given the proven effectiveness of CA to alleviate AF symptoms and the promising data with regard to reduction of thromboembolic events with both CA and WATCHMAN implantation, combining the two procedures is hoped to further reduce the incidence of stroke in high-risk patients while simultaneously relieving symptoms. The combined procedure may eventually enable patients to undergo implantation of the WATCHMAN device without subsequent warfarin treatment, since the CA procedure itself reduces thromboembolic events. This would present an avenue of treatment previously unavailable to patients ineligible for warfarin treatment because of recurrent bleeding 20 or other warfarin-associated problems.
The combined procedure is performed under general anesthesia with biplane fluoroscopy and TEE guidance. Catheter ablation is followed by implantation of the WATCHMAN LAA closure device. Data from a non-randomized trial with 10 patients demonstrates that this procedure can be safely performed in patients with a CHADS2 score of greater than 1 21. Further studies to examine the effectiveness of the combined procedure in reducing symptoms from AF and associated stroke are therefore warranted.

Catheter ablation is combined with placement of the WATCHMAN Left Atrial Appendage Closure Device to prevent ischemic stroke in a patient with non-valvular atrial fibrillation.

结合消融房颤左心耳封堵

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

实时数字成像大鼠提睾肌缺血再灌注损伤（IRI）的白细胞 - 内皮细胞相互作用

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

的运动再培训采用实时反馈的性能

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

脑脊液的microRNA分析采用实时定量PCR技术

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

在小鼠肺压力容积曲线的测量

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.