This work was supported by National Institutes of Health Grants R00 HL112952 (to R. H.), R01 HL108618 (to J.P.G.), P01 HL051971, and P20 GM104357. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

<ol>
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The authors declare that they have no competing financial interests.

前述协议的细节的方法，通过在隔离工作大鼠心脏葡萄糖氧化和脂肪酸氧化同时量化底物的通量。然后测量可以叠加到所记录的心功能参数，以确定基线和压力条件下，底物的代谢和心脏工作的关系（在工作量的变化，缺血再灌注等 ）。它也可以评估如何新陈代谢/收缩关系由先前存在的条件，如心脏衰竭和糖尿病的影响。此外，转基因啮齿动物的心脏可用于询问特定蛋白质对心脏代谢和?...

临床相关性在哺乳动物心脏，有基材的通量之间通过氧化代谢途径，ATP的生成和心脏工作1较强的正相关关系。在过去的二十年中，心脏代谢和功能之间的错综复杂的链路的调查已经导致认识到，在心脏代谢的改变是用于在不同类型的心脏疾病2-4的设置收缩功能障碍和可能的病理结构重建的原因。因此，可以预期，我们的管辖强调心脏的代谢重构机制的理解会导致心脏衰竭5-7的预防或治疗的治疗靶的鉴定。从美国心脏协会科学语句的"评估心脏代谢"的最近的出版物强调了科学界的T的兴趣与日俱增他的研究领域8。不过，虽然在心脏成像的技术进步现在允许心脏形态和功能的一个快速，准确的评估，心脏代谢的体内研究仍然有限和繁重:核磁共振（NMR）谱和正电子发射断层扫描（PET）成像可以用来跟随心脏高能磷酸代谢和克雷布斯循环的活性，但这些技术都受到高的操作费用和他们无法困扰，以确定各种基材在稳态条件9的贡献氧化代谢。对此日期体外工作心脏制备代表鞋底和独特的技术可用来研究，同时，实时，收缩功能和底物的助熔剂装入氧化代谢途径7,9。以下方案的目的在于提供在用于确定大鼠试剂的制备和使用的指南在大鼠离体心脏基板利用率上课。 隔离工作鼠类心脏仪器虽然技术是近半个世纪之久，隔离工作大鼠心脏的准备仍然首选心血管研究的方法。由于与心脏的Langendorff编制，工作啮齿动物心脏提供了一个相对简单，可靠和廉价的方式从其他器官，神经内分泌等循环因子的混杂效应独立测量范围广泛的心脏参数。但与此相反的的Langendorff灌注心脏，工作心脏继续执行近生理心脏工作，为氧化代谢通量的产生是相关的体内条件级的先决条件。这是通过经由连接到左心房的插管递送灌注缓冲液至左心室（LV）的实现，并作为低压填充和合同，该缓冲器是通过对一个确定的负荷静水压力主动脉线喷射。最初由尼利和他的同事描述的10后来被Taegtmeyer，折边和克雷布斯11好转，但灌注设备的设计至今变化不大。如在原始设备描述的，收缩功能可以通过测定心输出量的评估，使用不超过量筒和秒表来测量主动脉和冠状动脉流10,11。一些厂商现在提供完整的工作啮齿动物心脏灌注系统。这些可商购的装置可与flowprobes，压力传感器，一个压力 - 体积导管和必需的所有心功能数据采集和分析的设备来获得。该供应商提供大量的文档和培训课程，熟悉他们的设备新用户。一些评论文章还详细协议的工作心脏仪器和设备行进，而对使用导管来测量在啮齿类动物12-15心脏功能。因为这个原因，我们将只简要地提及灌注装置和记录设备的设置。本协议，而旨在用的，可以被实现同时测量葡萄糖和长链脂肪酸的氧化的速率，在正常心脏两大能量提供衬底的方法的描述，以补充已经可用的信息。我们在这里描述的所有参与利用放射性标记的能量底物的心肌氧化代谢的评估的步骤，从试剂和缓冲液，以样品的回收和处理的制备中，对数据的分析。 该方法的原理心肌细胞产生它们的能量的对收缩从脂肪酸（主要的长链脂肪酸）和碳水化合物的氧化磷酸化的体（glucose和乳酸）。心脏有非常有限的精力充沛的储量依赖于从循环这些能源提供基板的稳定供应。葡萄糖通过糖酵解途径分解代谢产生丙酮酸，然后通过线粒体内膜的丙酮酸脱氢酶复合脱羧。长链脂肪酸，从循环白蛋白或脂蛋白甘油三酯萃取，首先活化成酰基-CoA分子在细胞质中，并随后输送的线粒体基质内通过肉碱穿梭进入β-氧化途径。由葡萄糖和脂肪酸分解代谢产生的乙酰-CoA分子燃料三羧酸循环来产生其用于通过电子传递链来构建跨线粒体内膜的质子动力的还原当量（NADH和FADH 2）和通过ATP合酶的活性产生的ATP。水和二氧化碳是的端部的副产物酶反应发生的克雷布斯循环回路内。的14 C和3 H-放射性标记的底物的供给（如14 C-放射性标记的葡萄糖和3 H-放射性标记油酸）的分离工作心脏将因此导致生产14 CO 2和3 的 H 2的O其可以定量地从冠脉流出液回收。 14 CO 2的集合由保持离体灌注心脏成密封室以及由冠状动脉流出物在离开心脏立即恢复进行。一个小的阴离子交换柱用于分离和从冠状动脉流出物中回收3 H 2 O操作。从经处理的样品的放射活性用液体闪烁计数器测定，并与所使用的放射性标记的底物的比活性的知识，那么有可能单独定量葡萄糖和脂肪酸中的磁通氧化通路16,17。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>BP366</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic (KH2PO4)</td>
			<td>Fisher Scientific</td>
			<td>P284</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate (MgSO4*7H2O)</td>
			<td>Fisher Scientific</td>
			<td>M63</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Fisher Scientific</td>
			<td>S233</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td></td>
		</tr>
		<tr>
			<td>AG 1-X8 resin, chloride form, 100-200 dry mesh size, 500 g</td>
			<td>Bio-Rad</td>
			<td>1401441</td>
			<td>This item can be replaced by purchasing directly the hydoxide form&#160; (see reference below), but this will cost almost 8 times more</td>
		</tr>
		<tr>
			<td>AG 1-X8 resin, hydroxide form, 100-200 dry mesh size, 100 g</td>
			<td>Bio-Rad</td>
			<td>1432445</td>
			<td>Purchasing this item allows to bypass the conversion of the anion exchange resin from the chloride form to the hydroxide form (See section 1.2 of protocol)</td>
		</tr>
		<tr>
			<td>Glass Microanalysis Vacuum Filter Holder</td>
			<td>Fisher Scientific</td>
			<td>09-753-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>S318</td>
			<td>Corrosive. Consult the product MSDS for appropriate handling and storage.</td>
		</tr>
		<tr>
			<td>Gas Dispersion Tube with Fritted Cylinder</td>
			<td>Fisher Scientific</td>
			<td>11-138B</td>
			<td></td>
		</tr>
		<tr>
			<td>Probumin Bovine Serum Albumin Fatty Acid Free, Powder</td>
			<td>EMD Millipore</td>
			<td>820027</td>
			<td>We recommend the use of a charcoal-defatted BSA, as other purification process such as cold ethanol fractionation may leave residues toxic for the heart.</td>
		</tr>
		<tr>
			<td>Sodium Oleate</td>
			<td>Sigma-Aldrich</td>
			<td>O7501</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic Acid, [9,10-3H(N)]-</td>
			<td>PerkinElmer</td>
			<td>NET289005MC</td>
			<td>Radioactive material. Follow your Institution&#39;s radiation safety office guidelines for ordering and handling.</td>
		</tr>
		<tr>
			<td>Dialysis Membrane Tubing, 29 mm diameter</td>
			<td>Fisher Scientific</td>
			<td>08-667E</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose, D-[14C(U)]-</td>
			<td>PerkinElmer</td>
			<td>NEC042B005MC</td>
			<td>Radioactive material. Follow your Institution&#39;s radiation safety office guidelines for ordering and handling.</td>
		</tr>
		<tr>
			<td>Humulin R U-100</td>
			<td>Eli Lilly and Company</td>
			<td>NDC 0002-8215-01 (HI-210)</td>
			<td></td>
		</tr>
		<tr>
			<td>Inactin Hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>T133</td>
			<td>Controlled substance on USDEA Schedule III</td>
		</tr>
		<tr>
			<td>3-0 Silk Black Braid</td>
			<td>Roboz Surgical</td>
			<td>SUT-15-3</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Hyamine Hydroxide</td>
			<td>PerkinElmer</td>
			<td>6003005</td>
			<td>Highly toxic and causes severe burns. Consult the product MSDS for appropriate handling and storage</td>
		</tr>
		<tr>
			<td>20 mL Glass Scintillation Vials</td>
			<td>Fisher Scientific</td>
			<td>03-341-25E</td>
			<td>Use glass vials for quantitative recovery of 14CO2</td>
		</tr>
		<tr>
			<td>20 mL HDPE Scintillation Vials</td>
			<td>Fisher Scientific</td>
			<td>03-337-23B</td>
			<td>Use HDPE vials for quantitative recovery of 3H2O</td>
		</tr>
		<tr>
			<td>Red Rubber Sleeve Stoppers</td>
			<td>Fisher Scientific</td>
			<td>14-126DD</td>
			<td>Fit 20 mL scintillation vials; Reusable</td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle 23G x 40 mm</td>
			<td>BD</td>
			<td>305194</td>
			<td>Use to inject perchloric acid through the rubber sleeve stopper of the CO2 trap</td>
		</tr>
		<tr>
			<td>Perchloric Acid, 60%</td>
			<td>Fisher Scientific</td>
			<td>A228</td>
			<td>Highly corrosive and may act as an oxidizer and/or cause an explosion hazard. Consult the product MSDS for appropriate handling and storage</td>
		</tr>
		<tr>
			<td>Ultima Gold, Scintillation Cocktail</td>
			<td>PerkinElmer</td>
			<td>6013327</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Wool</td>
			<td>Fisher Scientific</td>
			<td>AC38606</td>
			<td></td>
		</tr>
		<tr>
			<td>Decon Dri-Clean Detergent Powder</td>
			<td>Fisher Scientific</td>
			<td>04-355</td>
			<td>For cleaning of glassware, plastic parts, and tubing</td>
		</tr>
		<tr>
			<td>Alconox Tergazyme Enzyme-Active Powered Detergent</td>
			<td>Fisher Scientific</td>
			<td>16-000-115</td>
			<td>For cleaning of &#34;hard to reach&#34; surfaces (tubing, glassware) contaminated by fatty acid-BSA residue</td>
		</tr>
	</tbody>
</table>

methods for the determination of rates of glucose and fatty acid oxidation in the isolated working rat heart

哺乳动物的心脏是ATP的主要消费和需要能量底物进行收缩的稳定供应。不足为奇的是，心肌代谢的改变已被链接到的收缩功能障碍和心脏衰竭的发展。因此，解开新陈代谢和收缩之间的联系应该在某些领导在疾病状态下心脏适应或适应不良的机制线索。分离的工作大鼠心脏制剂可用于跟踪，同时和实时，心脏收缩功能和能量提供基板到氧化代谢途径的通量。本协议的目的是提供在缓冲器的氧化的葡萄糖和脂肪酸，主要能量提供所述心脏的基板用率的定量测量的制备和利用所采用的方法的详细描述。还讨论了用于样品分析和数据解释的方法。简言之，该技术是基于通过常温晶体灌注的14供给的C放射性标记的葡萄糖和3 H-放射性标记长链脂肪酸对离体心脏跳动。14 CO 2和3 H 2 0，结束副产物参与这些提供能量的基板的利用酶反应，然后定量地从冠状动脉流出物中回收。与所使用的放射性标记的底物的比活性的知识，那么有可能单独定量葡萄糖和脂肪酸的通量在氧化途径。离体心脏的收缩功能可以并行确定与适当的记录设备和直接相关代谢通量的值。该技术是研究响应于各种胁迫条件的代谢/收缩关系极为有用，如在前和负荷和缺血，药物或circula之后改变廷因子，或者在基因产物的表达的改变如下。

两个代表性实验在下面的图中描述。在这两种情况下，一个16周龄的雄性Sprague Dawley大鼠的心脏中分离，并与根据前述方案制备KH液的工作模式灌注。在每个实验中，将心脏经受应力条件影响心脏的工作。心脏收缩功能是由脉压的连续记录通过主动脉线压力传感器的插入和通过测定心肌力量评估。在提供能量的底物的利用每个应力条件下的后果是同时由葡萄糖和油的氧化速?...

Watch this Scientific Journal Video about Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart at JoVE.com

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

为葡萄糖和脂肪酸氧化率在大鼠离体的测定方法

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

methods-for-determination-rates-glucose-fatty-acid-oxidation-isolated

Research

JoVE Journal

Biology

14.6K 次观看.  University of Mississippi Medical Center. The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

视频: Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

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

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 ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an impending predator attack.  Although it is not known whether invertebrates posses an autonomic nervous system identical to that of vertebrates, evidence shows invertebrates have a  sympathetic-like  response to regulate the internal environment and ready the organism to act behaviorally to a given stimuli.  Furthermore, this physiological response can be feasibly measured and it acts as a biological index for the animal's internal state.  Measurements of the physiological response can be directly related to internal and external stressors through changes in the central nervous system controlled coordination of the cardio-vascular and respiratory systems.   More specifically, monitoring heart and ventilation rates provide quantifiable measures of the stress response not always behaviorally observed. Crayfish are good model organisms for heart and ventilatory rate measurements due to the feasibility of recording, as well as the rich history known of the morphology of the crayfish, dating back to Huxley in 1888, and the well-studied typical behaviors.

Invertebrates show an autonomic sympathetic-like response similar to that described for vertebrates. The coordination of the cardio-vascular and ventilatory systems allows for measurement of a biological index in which to assess an organism internal state.

在自由移动的小龙虾的心脏和换气率的措施

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an ...

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 ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

在离体血液灌注大鼠肺备量化单微血管通透性

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

在小鼠原代肝细胞测定脂肪酸氧化和脂肪生成的

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

灭鼠工作心脏模型心肌的性能和氧耗的研究

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...