这项工作得到了福田医疗技术基金会（ET和GN）和JSPS KAKENHI科学研究资助21K08047（ET）的研究资助。

该动物方案已获得机构动物护理和使用委员会的批准，并遵循东京大学动物实验和相关活动的规定。本研究以8-12周龄雄性C57/Bl6J小鼠为研究对象。这些动物是从商业来源获得的（见 材料表）。如前所述，通过与NG-硝基-L-精氨酸甲酯一起给予高脂肪饮食15周来建立HFpEF模型10。
1. 导管准备和压力/体积校准
<ol><li>将电导导管放入生理盐水中，并将其连接到由 PowerLab 8/35 和压力容积单元（MPVS 模块，参见 材料表）组成的模块上。</li>
	<li>通过在 MPVS 模块 3,11 上记录预定压力（0 mmHg 和 100 mmHg）和体积参数（这些因 MPVS 模块而异）以电子方式校准压力和体积（另请参阅制造商的说明）。</li>
</ol>2. 准备动物进行导尿
<ol><li>麻醉和通气<ol><li>插管前 5-10 分钟腹膜内注射 5 mg/kg 依托咪酯和 500 mg/kg 氨基甲酸乙酯（见 材料表）。 注:聚氨酯虽然在动物研究中作为麻醉剂有效，但被怀疑对人类致癌。因此，当氨基甲酸乙酯是实现实验目标所必需的，并且没有替代剂足以满足时，必须谨慎处理。必须采取适当的保护措施，例如戴手套和口罩以及在准备过程中使用通风柜。作为一种可能的替代方法，可以使用氯胺酮（80 mg/kg，ip）。</li>
		<li>将小鼠置于先前用2%异氟烷饱和的麻醉室中，并在麻醉诱导时将动物转移到保持在38°C至40°C之间的预热加热垫中。</li>
		<li>剃除手术区域。然后，用三轮交替的甜菜碱和酒精对手术部位进行消毒。</li>
		<li>在颈部做一个水平切口（1-2厘米），切除气管肌，露出气管。将手术 2-0 丝线缝合在气管下方，将其抬高，并做一个小切口（1-2 毫米）将其打开。</li>
		<li>将气管插管插入气管，并将其连接到呼吸机，呼吸机提供 100% 氧气和 2% 异氟烷的混合物（后来减少到 0.5% 至 1%）。</li>
	</ol></li>
	<li>中心静脉 （CV） 导管插入和液体注入<ol><li>找到胸锁乳突肌下方的颈内静脉3.</li>
		<li>将中心静脉导管插入颈静脉，该导管由连接到30G针头的PE-10硅橡胶管（参见 材料表）组成。</li>
		<li>在 3 分钟内推注 5-6 μL/g 体重的 10% 白蛋白/NaCl，然后以 5-10 μL/min 的恒定输注速率。 注意:此步骤对于预防麻醉引起的外周血管舒张引起的低血压至关重要。颈内静脉位于胸锁乳突肌和颈动脉之间，颜色比动脉深。</li>
	</ol></li>
</ol>3. 左心室导管插入术（开胸入路）的外科手术
<ol><li>剃除麻醉小鼠的手术区域。然后，用三轮交替的甜菜碱和酒精对手术部位进行消毒。</li>
	<li>通过捏脚趾来确认麻醉深度。然后，在剑突下方做一个水平切口（2-3厘米），并用钝剪刀将皮肤与胸壁分开。</li>
	<li>使用电烧术在两侧横向切开胸壁（参见 材料表）。</li>
	<li>通过切开横膈膜暴露心脏，然后用镊子轻轻地从心脏中取出心包。</li>
	<li>将一根 27 G 针插入左心室 （LV） 的顶点，然后 通过 穿刺孔逆行将电导导管插入左心室。</li>
	<li>调整导管位置，以获得方形压力-容积环。</li>
	<li>通过检查下腔静脉 （IVC） 闭塞期间 PV 环的形状，验证当负荷条件发生变化时导管不会接触肌。 注意:充分的心脏暴露有助于手术并有助于获得清晰的视野。</li>
</ol>4. 记录PV回路数据，确定收缩末期压力-容积关系（ESPVR）和舒张末期压力-容积关系（EDPVR）
注意:通过 IVC 闭塞减少预紧力可以确定 ESPVR 和 EDPVR。
<ol><li>在信号稳定后（插管后5-10分钟），使用LabChart软件（参见 材料表），PowerLab和MPVS模块记录和分析基线压力 - 体积（PV）回路。</li>
	<li>通过用镊子按压IVC进行IVC闭塞，并在IVC闭塞期间记录至少20个心动周期的PV环。通过使用 LabChart 软件将曲线线拟合到 PV 环的收缩末点和 EDPVR 中，通过拟合一条曲线穿过 PV 环的舒张末期点来确定 ESPVR。 注意: 在 IVC 闭塞期间停止呼吸机以防止肺运动伪影。当肺运动过度时，泮库溴铵 （0.5-1 mg/kg） 等麻痹剂可能有帮助，只有在确认麻醉平面稳定后才应使用。</li>
</ol>5.经食管起搏
<ol><li>将 2-Fr 四极电极导管插入食管，将导管连接到脉冲刺激器（参见 材料表），并确定心房捕获阈值（通常，刺激幅度为 3 mA，脉冲宽度为 1 ms）。</li>
	<li>使用腹膜内给药的20mg / kg伊伐布雷定（参见 材料表）将HR减慢至400次/ min以下。</li>
	<li>稳定后，以 400 次/分钟至 700 次/分钟的不同起搏率获取 20 个连续心动周期的 PV 循环，增量为 100 次/分钟;以每个起搏率获得超过 5 分钟的周期。</li>
</ol>6. 生理盐水校准和主动脉血流校准
<ol><li>使呼吸机失活，并通过 CV 导管静脉注射 5-10 μL 高渗盐水溶液。</li>
	<li>记录盐水注射过程中压力和体积的波动，并使用 PowerLab 3,11 计算 Vp 值。</li>
	<li>重复盐水校准以提高准确性和可重复性。</li>
	<li>将鼠标转到左侧，以免干扰音量信号。</li>
	<li>在Th3至Th5之间朝向脊柱进行侧向开胸术，并用镊子轻轻解剖降主动脉的一小部分。</li>
	<li>在主动脉上放置一个血管流量探头（参见 材料表）以测量心输出量。 注意:绝对容积的准确计算需要使用两种类型的校准:盐水校准和主动脉血流校准。重要的是要认识到与动物受试者高渗盐水输注相关的潜在风险，因为过量的盐负荷会导致收缩力下降。</li>
</ol>7. 安乐死
<ol><li>研究结束后， 通过 颈椎脱位在麻醉剂过量下对小鼠实施安乐死。 注意:为确保生命功能完全停止，采用安乐死的辅助方法，例如在麻醉下放血，随后摘取心脏组织。</li>
</ol>

使用心房起搏方案探索 HR 依赖性心脏功能

<ol>
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没有相互竞争的经济利益。

我们提出了一种方法来评估压力-容积与经食管起搏应用的关系。运动不耐受是HFpEF的关键特征之一，但尚无技术可用于评估运动期间小鼠的心脏功能。我们的起搏方案为检测舒张功能障碍提供了有价值的工具，这在静息条件下可能并不明显。
为了获得准确和一致的质量的PV回路，必须仔细执行以下步骤3,4,5,7,8,11,12,13,14:（1）必须小心地<sup clas...

心力衰竭是全球住院和死亡的主要原因，射血分数保留型心力衰竭 （HFpEF） 约占所有心力衰竭诊断的 50%。HFpEF 的特征是舒张功能障碍和运动耐量受损，相关的血流动力学异常，如舒张功能障碍，可以通过运动应激血流动力学测试或 MRI 扫描 1,2 清楚地检测到。
然而，在实验模型中，用于评估与 HFpEF 相关的生理异常的可用模式是有限的 3,4。跑步机运动试验 （TMT） 用于确定跑步时间和距离，这可能反映运动应激心脏血流动力学;然而，这种方法容易受到体重、骨骼肌力量和精神状态等外来变量的干扰。
为了规避这些限制，我们设计了一种心房起搏方案，该方案根据心率 （HR） 检测舒张性能的细微但关键的变化，并验证了其在 HFpEF5 小鼠模型中的有用性。有几个生理因素会影响与运动相关的心脏功能，包括交感神经和儿茶酚胺反应、外周血管舒张、内皮反应和心率6.然而，其中，心率-压力关系（也称为鲍迪奇效应）被称为心脏生理特征的关键决定因素 7,8,9。
该方案涉及在基线时进行常规压力-容积分析，以评估收缩压和舒张功能，包括压力发展速率 （dp/dt）、收缩末期压力-容积关系 （ESPVR） 和舒张末期压力-容积关系 （EDPVR） 等参数。然而，应该注意的是，这些参数受心率的影响，由于动物的内在心率不同，心率可能因动物而异。此外，还应考虑麻醉对心率的影响。为了解决这个问题，通过与伊伐布雷定同时进行心房起搏来标准化心率，并以增量心率进行心脏参数测量。值得注意的是，HR依赖性心脏反应将HFpEF小鼠与对照组小鼠区分开来，而在基线PV环测量（使用固有心率）中未观察到显着差异5。
虽然这种起搏协议可能看起来相对复杂，但当它被充分理解时，它的成功率超过 90%。该协议将为研究HFpEF小鼠模型中舒张功能障碍的潜在机制提供有用的方法，并有助于开发针对这种情况的新疗法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-0 silk suture, sterlie</td>
			<td>Alfresa Pharma Corporation, Osaka, Japan</td>
			<td>62-9965-57</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>2-Fr tetrapolar electrode catheter</td>
			<td>Fukuda Denshi, Japan and UNIQUE MEDICAL, Japan</td>
			<td>custom-made</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>Albumin Bovine Serum</td>
			<td>Nacalai Tesque, Inc., Kyoto, Japan</td>
			<td>01859-47</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>C57/BI6J mouse</td>
			<td>Jackson Laboratory</td>
			<td></td>
			<td>animals</td>
		</tr>
		<tr>
			<td>Conductance catheter</td>
			<td>Millar Instruments, Houston, TX</td>
			<td>PVR 1035</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical cautery, Electrocautery Knife Kit</td>
			<td>ellman-Japan,Osaka, Japan</td>
			<td>1-1861-21</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>Etomidate</td>
			<td>Tokyo Chemical Industory Co., Ltd., Tokyo Japan</td>
			<td>E0897</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Grass Instrument S44G Square Pulse Stimulator</td>
			<td>Astro-Med, West Warwick, RI</td>
			<td></td>
			<td>Pacing equipment</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Viatris Inc., Tokyo, Japan</td>
			<td>8803998</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Ivabradine</td>
			<td>Tokyo Chemical Industory Co., Ltd., Tokyo Japan</td>
			<td>I0847</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>LabChart software</td>
			<td>ADInstruments, Sydney, Australia</td>
			<td>LabChart 7</td>
			<td>Hemodynamic equipment</td>
		</tr>
		<tr>
			<td>MPVS Ultra</td>
			<td>Millar Instruments, Houston, TX</td>
			<td>PL3516B49</td>
			<td>Hemodynamic equipment</td>
		</tr>
		<tr>
			<td>Pancronium bromide</td>
			<td>Sigma Aldrich Co., St. Louis, MO</td>
			<td>15500-66-0</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>PE10 polyethylene tube</td>
			<td>Bio Research Center&#160; Co. Ltd., Tokyo, Japan</td>
			<td>62101010</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>PowerLab 8/35</td>
			<td>ADInstruments, Sydney, Australia</td>
			<td>PL3508/P</td>
			<td>Hemodynamic equipment</td>
		</tr>
		<tr>
			<td>PVR 1035</td>
			<td>Millar Instruments, Houston, TX</td>
			<td>842-0002</td>
			<td>Hemodynamic equipment</td>
		</tr>
		<tr>
			<td>Urethane (Ethyl Carbamate)</td>
			<td>Wako Pure Chemical Industries, Ltd., Osaka, Japan</td>
			<td>050-05821</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Vascular Flow Probe</td>
			<td>Transonic, Ithaca, NY</td>
			<td>MA1PRB</td>
			<td>Surgical Supplies</td>
		</tr>
	</tbody>
</table>

a pacing-controlled procedure for the assessment of heart rate-dependent diastolic functions in murine heart failure models

射血分数保留型心力衰竭 （HFpEF） 是一种以舒张功能障碍和运动不耐受为特征的疾病。虽然运动应激血流动力学测试或 MRI 可用于检测舒张功能障碍和诊断人类的 HFpEF，但这种方式在使用小鼠模型的基础研究中受到限制。跑步机运动试验通常用于小鼠的此目的，但其结果会受到体重、骨骼肌力量和精神状态的影响。在这里，我们描述了一种心房起搏方案，用于检测舒张性能的心率（HR）依赖性变化，并验证其在HFpEF小鼠模型中的有用性。该方法包括麻醉、插管和进行压力-容积 （PV） 环分析，同时进行心房起搏。在这项工作中， 通过 左心室心尖入路插入电导导管，并在食管中放置心房起搏导管。在用伊伐布雷定减慢心率之前收集基线 PV 环。 通过 心房起搏以 400 bpm 至 700 bpm 的 HR 增量收集和分析 PV 环路。使用该协议，我们在代谢诱导的HFpEF模型中清楚地证明了HR依赖性舒张功能障碍。与对照组小鼠相比，弛豫时间常数（Tau）和舒张末期压力-容积关系（EDPVR）均随着HR的增加而恶化。总之，这种心房起搏控制方案可用于检测 HR 依赖性心功能障碍。它为研究HFpEF小鼠模型中舒张功能障碍的潜在机制提供了一种新方法，并可能有助于开发针对这种情况的新疗法。

基线PV环路数据如图1和表1所示。在基线（没有起搏的情况下），对照组和HFpEF小鼠之间的舒张期参数（如弛豫时间常数（Tau）、最小压力变化率（dP / dt min）和EDPVR没有显着差异。然而，HFpEF小鼠表现出更高的血压和动脉弹性（Ea），如图1所示，并在心室收缩期间表现出典型的山形PV环。这应与心室肌肉直接接触压力传感器引起的尖峰区分开...

Watch this Scientific Journal Video about Investigating HR-Dependent Cardiac Function in Mouse Models Through a Novel Atrial-Pacing Approach at JoVE.com

作者聚焦:通过新型心房起搏方法研究小鼠模型中的 HR 依赖性心脏功能 

本方案描述了通过经食管起搏获得压力-容积关系，这是评估心力衰竭小鼠模型舒张功能的宝贵工具。

用于评估小鼠心力衰竭模型心率依赖性舒张功能的起搏控制程序

Heart failure with preserved ejection fraction (HFpEF) is a condition characterized by diastolic dysfunction and exercise intolerance. While ...

心率相关关系对于确定心脏特征很重要，尤其是在 HFpEF 中。该协议旨在评估心率依赖性心脏功能。任何实验模型，用于评估心率相关心脏功能的可用模式，仅限于跑步机运动测试。然而，跑步机测试会受到体重等变量的影响。为了克服这些局限性，我们围绕心房起搏方案进行了开发，以检测与心率相关的后期表现的细微但显着的变化。我们的方案提供了一种用于识别心功能障碍的可变工具，表现为心率变化。它还提出了一种研究舒张功能障碍的多种方法，用于镜像心力衰竭模型，特别是在 HFpEF 中。

a-pacing-controlled-procedure-for-assessment-heart-rate-dependent

Research

JoVE Journal

Biology

A Pacing-Controlled Procedure for the Assessment of Heart Rate-Dependent Diastolic Functions in Murine Heart Failure Models

1.2K 次观看.  The University of Tokyo Hospital. 心率相关关系对于确定心脏特征很重要，尤其是在 HFpEF 中。该协议旨在评估心率依赖性心脏功能。任何实验模型，用于评估心率相关心脏功能的可用模式，仅限于跑步机运动测试。然而，跑步机测试会受到体重等变量的影响。为了克服这些局限性，我们围绕心房起搏方案进行了开发，以检测与心率相关的后期表现的细微但显着的变化。我们的方案提供了一种用于识别心...

视频: 作者聚焦:通过新型心房起搏方法研究小鼠模型中的 HR 依赖性心脏功能 

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

大鼠心脏中植入的工程组织

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

科研

生物学

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

小鼠肾移植程序

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

荧光标记的果蝇心结构

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

可视化心脏跳动果蝇</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

评价以下电离辐射的空间分布的γH2AX

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...