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.

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The authors declare that they have no competing financial interests.

위의 프로토콜을 동시에 분리 작업 쥐 심장 글루코스 산화 및 지방산 산화를 통해 기판의 광속을 정량화하는 방법을 자세히. 측정은 (... 등의 부하 변화, 허혈 재관류) 기준선 스트레스 조건 하에서 기판 대사 심장 워크 간의 관계를 결정하기 위해 기록 된 심장 기능 파라미터에 중첩 될 수있다. 이는 대사 / 수축 관계 심부전 및 당뇨병과 같은 기존의 상태에 의해 영향을받는 방법을 평가하?...

임상 관련성 포유 동물의 심장에서 산화 적 대사 경로, ATP 생성과 심장 작품 (1)을 통해 기판의 플럭스 사이에 강한 긍정적 인 관계가있다. 지난 20 년 동안 심장 대사와 기능 사이의 복잡한 링크의 조사는 심장 대사의 변화는 심장 질환 2-4의 다른 유형의 설정에서 수축 기능 장애 가능성이 병리학 적 구조 리모델링에 대한 원인을 인식하게되었다. 따라서, 스트레인 된 심장의 대사 리모델링을 관리하는 메커니즘에 대한 이해 심부전 5-7의 예방 또는 치료를위한 치료 표적의 확인을 초래할 것으로 예상된다. &quot;심장 대사의 평가&quot;에 미국 심장 협회에서 과학적 진술의 최근 간행물은 t에 대한 과학계의 관심이 점점 강조연구 (8) 자신의 분야. 심장 이미징 기술의 진보는 이제 심장 형태와 기능의 신속하고 정확한 평가를 허용 반면, 심장 대사의 생체 내 연구는 제한적이고 부담이 남아있다 : 핵 자기 공명 (NMR) 분광법과 양전자 방출 단층 촬영 (PET) 영상 수 생체 외 날짜 .TO 정상 상태 조건에서 산화 대사에 대한 다양한 기판 기여 9 판별 심장 고 에너지 인산 대사 크렙스 회로 활동을 수행하는데 사용될 수 있지만, 이러한 기술들은 높은 운영 비용으로 자신의 무능력 퍼진 마음의 준비 작업을 동시에 실시간으로, 산화 적 대사 경로 7,9에 수축 기능과 기판의 플럭스에서 공부하는 유일하고 독특한 기술을 사용할 나타냅니다. 다음 프로토콜 래트를 결정하는데 사용될 시약의 제조 및 이용의 지침을 제공하는 것이다격리 된 작업 쥐의 심장에 기판 활용 말이지. 격리 된 작업 쥐 심장 장치 기술은 반세기 이전이지만, 분리 작업 쥐 심장 제제는 심혈관 연구를 선택하는 방법을 남아있다. 랑겐 돌프 심장 제조와 마찬가지로 작용 쥐 심장 신경 호르몬 다른 장기와 다른 순환 계수의 교란 효과는 독립적으로 심장의 다양한 파라미터를 측정 할 수있는 비교적 간단한 신뢰성 있고 저렴한 방법을 제공한다. 그러나 랑겐 돌프 - 관류 마음 대조적으로, 작업 심장에 가까운 생리 심장 연구, 생체 조건에 관련이있는 수준으로 산화 대사 플럭스의 생성을위한 전제 조건을 수행하고 있습니다. 이것은 좌심방 접속 캐뉼라를 통해 좌심실 (LV)로 관류 버퍼를 제공함으로써 달성되고 LV는 채우고 계약으로되어버퍼가 결정된 후 부하 수압에 대한 대동맥 라인을 통해 배출됩니다. 원래 10 이후 Taegtmeyer, HEMS와 크렙스 (11)에 의해 개선 되었으나 닐리와 동료에 의해 기술 된 관류 장치의 디자인은 이후 약간 변경되었습니다. 원래 장치에서 설명한 바와 같이, 수축 기능은 대동맥 측정하기 위해 더 이상 메스 실린더 및 스톱워치를 사용하지 않고, 심 박출량의 측정을 통해 평가 될 수 있고, 관상 동맥은 10, 11을 흐른다. 여러 벤더가 완료 작업 쥐 심장 관류 시스템을 제공합니다. 이들 시판되는 장치 flowprobes, 압력 센서, 압력 볼륨 카테터와 심장 기능의 데이터 수집과 분석에 필요한 모든 장비를 얻을 수있다. 공급 업체는 장비와 새로운 사용자 익숙해 광범위한 문서 및 교육 세션을 제공합니다. 작업 심장기구도에 여러 또한 리뷰 기사 상세 프로토콜[슬라이드 쇼와 카테터의 사용에 설치류 12-15에서 심장 기능을 측정합니다. 이러한 이유로, 우리는 잠시 관류 장치 및 기록 장치의 설정을 언급한다. 본 프로토콜은 다소 동시에 포도당과 장쇄 지방산 산화의 속도는 정상적인 심장의 두 가지 에너지를 제공하는 기판을 측정하도록 구현 될 수있는 방법에 대한 설명과 함께 이미 이용 가능한 정보를 보완하는 것이다. 우리는 데이터 분석 샘플의 회수 및 처리 시약 및 버퍼의 제조에서, 심근 산화 대사 평가 방사성 에너지 기판의 사용에 관련된 현재의 모든 단계를 설명한다. 이 방법의 원리 심근 (glucos를 지방산 (주로 장쇄 지방산)과 탄수화물의 산화 적 인산화의 수축 에너지의 대부분을 생성전자 및 락 테이트). 심장은 매우 정력 보유 제한 및 순환에서 이러한 에너지 제공 기판의 지속적인 공급에 의존하고있다. 당분 경로를 통한 글루코오스의 이화는 미토콘드리아 내막의 피루 베이트 탈수소 효소 복합체에 의해 탈 카복실 화되는 피루브산을 산출한다. 알부민 또는 지단백질 순환하는 트리글리세리드에서 추출한 장쇄 지방산은 제 세포질에서 아실 CoA를 분자로 활성화되고,이어서 베타 산화 경로를 입력 카르니틴 셔틀 통하여 미토콘드리아 기질 내부에 반송된다. 포도당 지방산의 분해 작용에 의해 생성 된 아세틸 -CoA 분자 미토콘드리아 내막을 가로 질러 양성자 원동력을 구축하는 전자 전달계에 사용되는 환원 등가물 (NADH와 FADH 2)를 생성하는 크렙스 회로 연료 및 ATP에 합성 효소의 활동을 통해 ATP를 생성합니다. 물과 이산화탄소의 단부 부산물이다크렙스 사이클 내에서 일어나는 효소 반응. 14 C- 3 H- 방사성 표지 된 기질의 공급은 절연 작업 심장 (예 : C-14 방사성 표지 된 글루코스, 3 H-방사성 올레산) 따라서 14 CO 2, 3 H 2 O의 생산을 유도 할 수있다 정량적 관상 동맥 유출 물로부터 회복 될 수있다. 14 CO 2의 컬렉션은 밀폐 챔버에 격리 관류 심장을 유지하여 그것을 중심을 빠져 나올 때 바로 관상 동맥 유출 물을 회수하여 실시된다. 작은 음이온 교환 컬럼 분리하여 관상 동맥 유출 물로부터 3 H 2 O를 복구하기 위해 사용된다. 처리 된 샘플의 방사능을 액체 섬광 계수기로 측정 한 결과, 사용 된 방사성 표지 된 기질의 특정 활성의 기술로, 개별적으로 포도당과 지방산의 광속을 정량하는 것이 가능하다산화는 16, 17을 경로.

<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

포유류 마음은 ATP의 주요 소비자이며, 수축의 에너지 기판의 지속적인 공급이 필요합니다. 당연히 심근 대사의 변화가 수축 부전 심부전의 개발과 연결되어있다. 따라서, 신진 대사 및 수축 사이의 링크를 푸는 것은 질병 상태에서 심장 적응 또는 부적응을 지배하는 메커니즘의 일부에 빛을 흘렸다한다. 분리 된 래트 심장 작동 준비를 동시에 실시간 심장 수축 기능 및 산화 적 대사 경로에 기판을 제공하는 에너지의 흐름에 따라 사용될 수있다. 본 프로토콜은 포도당 지방산, 심장의 기판을 제공하는 주된 에너지 산화 비율의 정량적 측정을 위해 버퍼의 제조 및 이용에 사용되는 방법에 대한 상세한 설명을 제공하는 것을 목적으로한다. 시료 분석 및 데이터 해석에 사용되는 방법에 대해서도 설명합니다.간단히 말해서,이 기술은 정상 체온 결정질 관류 통해 생체 심장 박동로 (14)의 공급 C- 방사성 포도당과 3 H- 방사성 장쇄 지방산에 기초한다. 14 CO 2, 3 H 2 O, 최종 부산물 이러한 에너지 제공 기판의 사용에 관여하는 효소 반응은 다음 정량적 관상 동맥 유출 물로부터 회수된다. 사용되는 방사성 표지 된 기질의 특정 활성의 기술로, 개별적으로 산화 경로에 포도당과 지방산의 광속을 정량하는 것이 가능하다. 분리 된 심장의 수축 기능은 해당 기록 장치와 병렬로 결정될 직접 대사 플럭스 값과 상관 될 수있다. 이 기술은 사전에로드 및 국소 빈혈, 약물 또는 circula 후 변경 등 다양한 스트레스 조건에 대한 응답으로 대사 / 수축 관계를 연구하는 것이 매우 유용하다유전자 산물의 발현의 변화 다음 팅 계수 나.

두 대표 실험은 아래 그림에 설명되어 있습니다. 두 경우 모두, 16 주령의 수컷 스프 라그 돌리 래트의 심장을 분리하고, 선행하는 프로토콜에 따라 제조 KH 완충액 작업 모드 관류. 각 실험에서, 심장 심장 작업에 영향을 미치는 응력 상태로 하였다. 심장 수축 기능 대동맥 라인의 압력 센서의 삽입을 통해 심장의 전력 측정하여 맥압의 연속 촬영에 의해 평가 하였다. 에너?...

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.

격리 된 작업 쥐의 심장에서 포도당과 지방산 산화의 가격의 결정 방법

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

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14.6K 조회수.  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.

비디오: 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.

랫 중심에 엔지니어링된 조직의 주입

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

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생물학

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.

에 잔 심장을 떠올리 Drosophila</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.

지방산의 정량 Phosphoproteomics은 자극 Saccharomyces cerevisiae</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.

자유롭게 크레이 피쉬 이동에 마음을 Ventilatory 요금 대책

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.

의 전자 현미경을위한 배아의 준비 Drosophila 태아의 심장 관

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.

Ionising 방사선에 대한 응답으로 γH2AX Foci의 부량

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.

이온화 방사선 다음 γH2AX의 공간적 분포 평가

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.

고립 된 혈액 관류 쥐 폐 준비에 단일 미세 혈관 투과성의 양을

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.

마우스 차 간세포에서 지방산 산화 및 지방 생성의 결정

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.

심근 성능 및 산소 소비의 연구에 대한 설치류 작업 심장 모델

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