이 작품은 미국 심장 협회 (American Heart Association) 14FTF20370058 (DMA)와 NIH T32 HL007101-35 (DMA)에 의해 지원됩니다.

1. 컨덕턴스 카테 테르 준비 및 압력 교정 <ol><li> 혈역학 적 카테터 모듈에 전도 카테터를 연결합니다. 전자적으로 카테터 모듈에 설정 프리셋 압력과 부피를 기록하여 압력과 부피 측정을 교정. 0mm 수은 25 mmHg로 (그림 1A)의 추적을 기록하고 모두 압력 트레이싱 (그림 1B 및 1C)에 전압을 할당합니다. 마찬가지로, 5 RVU 25 RVU (그림 1D)의 볼륨 추적을 기록하고 두 볼륨 트레이싱 (1E 및 1 층)에 전압을 할당합니다. </li><li> 수은주 혈압계를 사용하여 수동 압력 교정과 전자 압력 보정을 확인합니다. 3 방향 스톱 콕 혈압계 커프 포트를 장착한다. 측면 포트를 사용하여 RT 물 종종 관상 동맥 확장술에 사용될 3 포트 지혈 밸브 시스템을 채우기. <ol><li> 유체 가득 지혈 밸브에 전도성 카테터의 팁을 놓고 부드럽게 기지 확보심은 카테터를 비틀면. 혈압계로 지혈 밸브를 연결하고 200 mmHg로에 팽창과 3 방향 스톱 콕을 잠급니다. 측정 된 압력이 혈압계에 팽창 압력에 해당 여부를 검사합니다. </li></ol></li><li> 식염수 장소 카테터 운영 필드 및 측정 압력 레벨 인 37 ° C로 가열 하였다. 기록 된 압력이 제로가 될 때까지 압력 제어를 조정합니다. </li></ol> 2. 마취 / 삽관 <ol><li> 케타민 / 자일 라진 복강 내 주입 (-1 80 ~ 100 / 10 mg을 kg)을 관리합니다. 참고 : 대체 마취제를 사용할 수 있습니다. 마취제의 광범위한 목록은이 기술을 11, 12의 이전 검토에 제공됩니다. 적절한 마취는 부드러운 꼬리 핀치에 의해 확인 될 수있다. </li><li> 일단 마취, 온수 패드에 머리를 가위와 장소 목과 가슴을 면도. 36.5-37.5 ºC에서 마우스 직장 온도를 유지한다. 낮은 체온우울 심장 박동수가 발생합니다. 건조 함을 방지하기 위해 눈 연고를 적용 </li><li> 목의 정중선 절개를 확인하고 기관을 노출 멀리 기관 근육을 해부하다. 삽관을 보장하기 위해 기관을 시각화하는 동안, 입을 통해 기관 내 튜브를 배치하고 인공 호흡기에 연결합니다. </li><li> 절차를 수행하는 동안 인공 호흡기에 마우스를 유지한다. 이전에 기술 된 바와 같이 11으로 설정 호흡기 설정 동물 중량을 기준. 호흡량 (㎖) 6.2 × (kg 동물의 무게) 1.01와 호흡 속도 = 53.5 × (kg 동물의 무게) -0.26을 =. </li></ol> LV 챔버 컨덕턴스 카테터 3. 배치 <ol><li> 경동맥 접근 <ol><li> 불임을 보장하기 위해, 무균 수술 도구의 두 세트의 초기 피부 절개 및 개흉술에서 작동하는 하나 used- 하나입니다. 악기는 개별 수술 세션 동안 동물의 건조 살균기를 사용하여 오염을 제거해야한다ND 각 수술 당일의 끝에서 멸균. </li><li> 피부 클로르헥시딘 + 알콜 피부 스크럽 (0.5 % 클로르헥시딘 / 70 % 알코올 용액)의 3주기와 정화 된 후, 흉골 하악에서 오른쪽 경동맥을 통해 절개를. 오른쪽 경동맥을 노출 주변 조직 해부 및 경동맥에 인접 실행 미주 신경을 잘랐다. </li><li> 넥타이, 보안, (떨어져 가슴에서) 경동맥의 말단부 주위 멸균 6-0 실크 봉합사를 놓습니다. 느슨하게 중간 봉합을 묶는 첫 번째 봉합에 (가슴에 가까운) 경동맥 근위부 아래에 두 개의 추가 봉합을 놓습니다. 부드럽게 근위 봉합사를 당겨 피부에 클램프로 고정합니다. 경동맥이 진행하기 전에 모두 근위 및 원위 고정되어 있는지 확인하십시오. </li><li> 첫 번째 봉합에 작은 R.의 경동맥에 절개, 근위을 확인하고 가슴을 향해 길이 방향으로 연장된다. </li><li> 전도 카테터 팁을 삽입,미리 절개를 통해 용기에 30 분 동안 따뜻한 식염수에 침지하고 중간 봉합사를 이용하여 카테터를 고정. </li><li> 정확한 위치를 보장하기 위해 추적 압력 볼륨 루프를 보면서 부드럽게 경동맥을 통하여 좌심실로 카테터를 전진. 참고 : 구형 나타나는 압력 볼륨 루프를 수득한다 카테터의 최적 배치 (도 2 참조). 카테터의 흐름에 대한 저항이 발생하면, 조심스럽게 당겨 부드러운 압력으로 다시 진행합니다. 카테터의 부드러운 회전은 좌심실에 배치에 도움이 될 수 있습니다. 전도 카테터를 강제하는 것은 심각한 심혈 관계 합병증 또는 카테터가 손상 될 수 있습니다. </li><li> 녹화 기준 압력 볼륨은 10 ~ 카테터 배치 분 후 및 정상 상태를 달성 (도 2) 루프. </li></ol></li><li> 혀끝 접근 <ol><li> 마취 및 통풍이 마우스에서, 절개 FR을OM 칼 모양의 프로세스와 조리개가 가시 측방까지 가슴 벽을 통해 절단. </li><li> 다이어프램 비록 잘라 마음의 정점을 시각화. </li><li> 근위 전극 막 심실 내에까지 (25-30 G 바늘 사용) 바늘 자상 통해 좌심실의 정점에 컨덕턴스 카테터를 삽입한다. </li><li> 녹화 기준 압력 볼륨은 10 ~ 카테터 배치 분 후 및 정상 상태를 달성 루프. </li></ol></li></ol> 일시적인 대동맥 폐색을 사용하여 4. 변하는 후 부하 <ol><li> 과도 대동맥 폐색을 수행하기 위해, 상부 가슴에 작은 절개를 수평 횡 대동맥을 노출 주변 조직 해부. </li><li> 가로 대동맥 아래에 6-0 실크 봉합사를 놓습니다. 압력 볼륨 루프가 기본으로 돌아 한 후, 부드럽게 천천히, 바늘 클램프 봉합사의 양쪽 끝을 걸쇠 1-2 초 이상 봉합사를 제기, 천천히 긴장을 풀어. </리&gt; <li> 세 개의 최적의 녹음이 같은 동물로 만든 때까지이 절차를 반복합니다. 주 : 최적의 기록은 적어도 5 압력 볼륨 루프 사이클과 봉합사 (도 3a 및도 3c)에 장력을인가에 단부 수축기 압력에 꾸준히 증가가 있어야한다. </li></ol> 5. 변하는 예압 과도 하대 정맥 폐쇄를 사용하여 (IVC) <ol><li> 과도 하대 정맥 폐쇄를 수행하려면, IVC를 노출 다이어프램 아래, 칼 모양의 과정을 아래에 수평 절개를합니다. </li><li> IVC 아래에 6-0 실크 봉합사를 놓습니다. 압력 - 부피 루프가 기준선으로 복귀 한 후에, 바늘 클램프 봉합사의 양단 걸쇠; 부드럽게 천천히 1-2 초 이상 봉합사를 제기, 천천히 긴장을 풀어. IVC 흡장도 면봉 스쳐면을 이용하여 압력을 완만 애플리케이션에 의해 수행 될 수있다. </li><li> 세 개의 최적의 녹음 M 때까지이 과정을 반복같은 동물에서 ADE. 주 : 최적의 기록은 적어도 5 압력 볼륨 루프 사이클과 봉합 장력의인가에 좌심실 이완 기말 압 꾸준히 감소 가져야 (도 4a 및도 4b). </li></ol> 6. 식염수 교정 <ol><li> 연구의 결론에서, 병렬 컨덕턴스 (V 용 P) 값은 경정맥을 통해 동물에 고장 성 식염수 (15 %)의 10 μL 러스 주입함으로써 얻을 수 (도 5a 및도 5b) 할 수있다. 참고 :이 덩어리는 압력의 변화없이 볼륨에 명백한 증가의 원인이됩니다. 이 겉보기 부피 변화에 의한 부피 증가를 실제 혈액 풀 컨덕턴스보다는의 변화의 결과이다. 고장 성 식염수 부정적인 효과 13 수축성을 갖는 한 DP / DT 최대의 일시적인 감소는 관찰 될 수있다. 계산 V p를 따라 압력 볼륨 루프 분석 소프트웨어에 입력 할 수있다큐벳 교정 매개 변수 및 마이크로 리터에 RVUs 변환하는 방법. </li></ol> 7. 큐벳 교정 <ol><li> 큐벳 보정을 수행하기 위해, 37 ºC 가온 가열 패드 또는 수조에 제조업체에서 제공하는 공지 된 직경의 웰과 큐벳 장소. 혈역학 적 평가를받은 쥐에서 신선한 따뜻한 헤파린 혈액을 가진 첫번째 4-5 구멍을 채 웁니다. 참고 : 큐벳 교정은 마우스의 혈액을 이용하여 좌심실의 혈액 풀의 정확한 평가를 할 수 있으며 마이크로 리터에 RVUs에서 볼륨 데이터의 변환이 가능합니다. </li><li> 모든 전극이 침수 될 때까지, 첫 번째 우물에 전도 카테터를 삽입합니다. 부드럽게 변화 RVUs를 생성하는 우물에 카​​테터를 이동합니다. </li><li> RVUs에 볼륨 채널의 전도도 변화를 기록한다. 교정에 대한 가장 높은 R​​VU를 선택합니다. 주 : 웰의 체적은 하나 있습니다 volum 대한 식을 이용하여) (1)에 의해 계산 될 수있다반경이 큐벳 잘 길이의 인 실린더의 전자는 내부 두 감지 전극 또는 2) 제조업체의 지침에 확인 사이의 길이를 기반으로합니다. 컨덕턴스 출력은 11 마이크로 리터로 RVUs에서 데이터를 변환 보정 방정식을 개발하기 위해 알려진 부피와 상관 될 수있다. </li></ol> 8. 안락사 <ol><li> 프로토콜의 결론에서, 마우스는 마취 동안 경추 탈구하여 안락사시켰다. </li><li> 보장하기 위해 죽음,받은 자궁 전위를 갖는 마우스는 안락사의 보조 방법을 거쳐야합니다. 우리는 마취하에 실험을위한 심장 조직의 수확, 또는 양자 개흉술과, 마취 하에서 방혈을 사용합니다. </li></ol> 9. 데이터 분석 사용 압력 볼륨 루프 분석 소프트웨어 <ol><li> 식염수 교정에서 V p를 계산 <ol><li> 선택 압력과 volu날은 고장 성 식염수 토출 동안 획득 루프 (도 5a 및도 5b) </li><li> 보 압력 볼륨 분석 소프트웨어로 루프. 식염수 교정에 대한 옵션을 선택합니다 (그림 5C) </li><li> 계산 된 V의 p 값 (그림 5D)를 기록한다. 참고 : V의 p 값이 식염수 교정에서 최종 수축기 볼륨 대 1) 최종 이완기 볼륨의 교회법을 확인하고 2) 최종 이완기 볼륨 = 최종 수축기 볼륨 라인으로 계산된다. 이러한 라인들의 교차 압력 볼륨 루프 분석 소프트웨어에 의해 계산 된 P V를 제공한다. </li></ol></li><li> 볼륨에 전도성을 입력 (RVU) 볼륨 채널 옵션에 관계 (그림 1 층) </li><li> 기준 압력 볼륨 루프 관계를 측정 <ol><li> 정상 상태가 달성되면 압력과 부피 채널에서 8-10 심장주기를 선택합니다 (그림 2A) 및 소프트 분석에 수출도자기. 5-6 기말 루프 (그림 2B)를 식별 </li><li> 병렬 전도성을 보정하기 위해 V의 p 값을 사용합니다. &quot;정상 상태&quot;옵션을 선택하고 혈역학 요약 테이블을 생성한다 (그림 2C) </li></ol></li><li> 대동맥 수축하는 동안 측정 압력 볼륨 루프 관계 <ol><li> 이완 기말 압력 (그림 3A) 및 분석 소프트웨어 수출의 증가에 앞서 대동맥 수축에 해당하는 압력과 부피 채널에서 8-10 심장주기를 선택합니다. 5-6 기말 루프 (그림 3C)를 식별 </li><li> 최종 수축기 압력 볼륨 관계를 계산합니다 선택 &quot;수축성&quot;분석 (그림 3B), (ESPVR) </li></ol></li><li> IVC 수축하는 동안 측정 압력 볼륨 루프 관계 <ol><li> (Figur을 IVC 수축에 해당하는 압력과 부피 채널에서 8-10 심장주기를 선택분석 소프트웨어에 전자 4A) 및 수출. 5-6 기말 루프 (그림 4B)을 식별 </li><li> &quot;수축성&quot;분석 예압 Recruitable 스트로크 작업을 계산합니다 (그림 3B), (PRSW) (그림 4C)을 선택, EDV (그림 4D)뿐만 아니라 ESPVR 및 최종 이완기 압력 볼륨 관계 대 DT 최대 DP / (그림 4E) </li></ol></li></ol>

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저자가 공개하는 게 없다.

우리는 심장 수축과 이완 모두의 광범위한 분석을 유도하기 위하여, 마우스에서 컨덕턴스 카테터를 사용하여 압력 볼륨 루프 perfoming 분석을위한 방법을 설명한다. 스가, 사가와와 동료 압력 볼륨 심장 수축력의 조치, 특히 ESPVR의 기울기, 또는 최종 수축기 elastance (전자들) 및 E 최대를 정의하는 루프를 이용했다. 볼륨 (P / V를)에 압력의 비율에 의해 정의 Elastance은 수축기의 기간에...

심장 압력 볼륨 루프 분석은 심장 기능의 상세한 정보를 제공하고 기능 평가 1 금 표준이다. 이러한 심 초음파 또는 심장 MRI 등의 영상 기술이 작동하는 수단을 제공하지만, 이러한 조치는 하중 조건에 크게 의존한다. 심장의 수축력과 이완의 부하에 관계없이 조치 프리로드와 후 부하의 범위에서 심실의 압력과 부피의 관계의 동적 측정을 필요로한다. 압력 볼륨 관계의이 이해는 사가와 익스프레스와 동료 2,3의 혁신적인 작업에서 발생한다. 그들은 압력 볼륨 루프 유도 수축 대책 하중 조건 (4) 독립적이라고 생체 관류 개 마음에 보여 주었다. 이러한 분석의 생체 내 응용 프로그램에서 1980 년대에 전도 카테터의 개발을 가능하게되었다. 카 허용이 기술 사전SS와 동료들은 인간 5,6 압력 볼륨 루프 분석을 수행 할 수 있습니다. 1990 년대 후반의 7 전도성 카테터 및 수술 기법의 향상의 소형화 수행 할 유전 적, 약리학 적 연구를 허용, 설치류 심장 기능의 분석이 가능했다. 이 사전은 압력 볼륨 루프 분석의 광범위한 사용으로 이어질 때문에 가지고 있으며, 포유류의 심장 생리학에 대한 통찰력의 큰 거래를 생성했습니다. 컨덕턴스 카테터의 사용과 이로부터 획득 된 데이터의 해석에 중요한 개념은 볼륨 및 컨덕턴스의 관계이다. 컨덕턴스 반비례 근위 전극 배치, 보통 LV 에이펙스 8에서, 대동맥판 아래에 배치하고, 원심으로 카테터를 사용하여 측정되는 전압과 관련되어있다. 전압 또는 전도도의 변화는 근위부에서 원위부 전극에 흐르는 전류의 변화에​​ 의해 측정된다. 혈액 풀 기여하지만크게 전도에이야, 심실 벽의 기여, 병렬 전도도 (V P는)라고, 측정 전도도가 절대 LV 볼륨 측정을 얻기 위해 감산해야합니다. 이 보정 방법을 수행하기 위해, 아래에서 설명하는 프로토콜, 식염수 보정을했다. 반 및 동료에 의해 기술 전도도 및 볼륨 사이의 수학적 관계는 해당 볼륨 = 1 / α이다 (ρ의 L 2) (피 GG), 여기서 α = 균일 필드 보정 계수, ρ = 혈액 저항률, L = 전극 간 거리 G = 컨덕턴스 G의 p = 비 혈액 컨덕턴스 9. 참고로, 마우스에서 균일 필드 보정 계수 인해 스몰 챔버 볼륨 (10)에 접근 1.0. 압력 트랜스 듀서와 결합 컨덕턴스 카테터 실시간 동시 압력 볼륨 데이터를 제공한다. 심장 pressu그들은 하중 조건과 심장 박동의 독립적 인 심실 기능의 측정을 허용로 다시 볼륨 분석은 심장 기능의 다른 방법을 통해 특정 장점을 제공한다. 최종 수축기 혈압 볼륨 관계 (ESPVR), D의 P / D T의 최대 -end-이완기 볼륨과 관련하여, 최대 elastance (E 최대) 및 recruitable 스트로크 일 (PRSW를) 미리로드 : 수축력의 특정 부하에 관계없이 심장 지수 등이 있습니다. 확장기 함수의 부하에 관계없이 측정은 확장 기말 압력 볼륨 관계 (EDPVR) (11)이다. 프로토콜은 다음과 경동맥 혀끝 접근법 모두 사용하여, 심장 압력 볼륨 루프 분석의 수행을 설명한다. 이러한 연구를 수행하는 방법이 상세히 이전 8,11에서 설명 하였지만, 우리는 두 식염수 큐벳 교정 정정 포함한 정밀한 압력 볼륨 측정을 획득하기 위해 중요한 단계를 검토하고 살전의 시각 증명을 제공한다전자 절차. 이 연구 수행 동물 연구 승인 프로토콜과 듀크 대학 의료 센터의 기관 동물 관리 및 사용위원회의 동물 복지 규정에 따라 처리한다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
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			<td>AnaSed (xylazine)&#160;</td>
			<td>Lloyd Laboratories</td>
			<td>NADA no. 139-236</td>
			<td>Anesthetic</td>
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			<td>Matrx Medical Inc.</td>
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			<td>Anesthesia machine</td>
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			<td>Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>Model 683</td>
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			<td>Tubing kit</td>
			<td>Harvard Apparatus</td>
			<td>72-1049</td>
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			<td>Homeothermic Blanket&#160;</td>
			<td>Kaz Inc.</td>
			<td>5628</td>
			<td>Surgical Equipment</td>
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			<td>Stereo microscope</td>
			<td>Carl Zeiss Optical Inc.</td>
			<td>Stemi 2000</td>
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			<td>Illuminator</td>
			<td>Cole&#8211;Parmer</td>
			<td>41720</td>
			<td>Surgical Equipment</td>
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			<td>Dumont no. 55 Dumostar Forceps&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>11295-51</td>
			<td>Surgical Instruments</td>
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			<td>Graefe forceps, curved&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>11052-10</td>
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			<td>Moria MC31 forceps&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>11370-31</td>
			<td>Surgical Instruments</td>
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			<td>Mayo scissors&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>14512-15</td>
			<td>Surgical Instruments</td>
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			<td>Iris scissors&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>14041-10</td>
			<td>Surgical Instruments</td>
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			<td>Fine Science Tools Inc</td>
			<td>12501-13</td>
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			<td>Olsen&#8211;Hegar needle holder&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>12002-12</td>
			<td>Surgical Instruments</td>
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			<td>spring scissors</td>
			<td>Fine Science Tools Inc</td>
			<td>15610-08</td>
			<td>Surgical Instruments</td>
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			<td>disposable underpads</td>
			<td>Kendall/Tyco Healthcare</td>
			<td>1038</td>
			<td>Surgical Supplies</td>
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			<td>Sterile gauze sponges, sterile&#160;</td>
			<td>Dukal</td>
			<td>62208</td>
			<td>Surgical Supplies</td>
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			<td>Cotton-tipped applicators, sterile&#160;</td>
			<td>Solon</td>
			<td>368</td>
			<td>Surgical Supplies</td>
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			<td>Surgical suture,&#160; silk, 6-0&#160;</td>
			<td>DemeTECH</td>
			<td>FT-639-1</td>
			<td>Surgical Supplies</td>
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			<td>1 cc Insulin syringes&#160;</td>
			<td>Becton Dickenson</td>
			<td>329412</td>
			<td>Surgical Supplies</td>
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			<td>Access-9&#8482; Hemostasis Valve</td>
			<td>Merit Medical&#160;</td>
			<td>MAP111</td>
			<td>Hemodynamic equipment</td>
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			<td>320</td>
			<td>Hemodynamic equipment</td>
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			<td>ADInstruments Inc</td>
			<td></td>
			<td>Hemodynamic equipment</td>
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			<td>ADInstruments Inc</td>
			<td>SPR-839</td>
			<td>Hemodynamic equipment</td>
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			<td>Wahl</td>
			<td>8787-450A</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Intradermic tubing PE-10 (Becton Dickinson, cat. no. )</td>
			<td>Becton Dickenson</td>
			<td>427401</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Intradermic tubing PE-50 (Becton Dickinson, cat. no.)</td>
			<td>Becton Dickenson</td>
			<td>427411</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Needle assortment (18, 25 and 30 gauge; Thomas Scientific)</td>
			<td></td>
			<td></td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>0.9% (wt/vol) sodium chloride injection, USP , cat. no. )</td>
			<td>Hospira</td>
			<td>NDC no. 0409-4888-50</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td></td>
			<td></td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Alconox (Alconox Inc.) for catheter cleaning</td>
			<td>ADInstruments Inc.</td>
			<td></td>
			<td>Miscellaneous</td>
		</tr>
	</tbody>
</table>

cardiac pressure-volume loop analysis using conductance catheters in mice

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.

압력 - 부피 루프 분석은 연구 약물 (16)을 거치고, 유전자 변형 마우스 (14, 15) 또는 마우스에서 심장 기능을 측정 할 수있다. 주제 압력 볼륨 루프가 ß-아레스의 효과를 조사 이전에 출판 일 (16)로부터 제공되는 TRV120023, AT1R 리간드를 바이어스. TRV120023은 생체 내에서 심장 기능에 영향을 미치는 여부를 테스트하기 위해, 압력 볼륨 루프 분석은 기존 및 신규 안?...

Watch this Scientific Journal Video about Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice at JoVE.com

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters.

마우스에서 컨덕턴스 카테터를 사용하여 심장 압력 볼륨 루프 분석

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular ...

cardiac-pressure-volume-loop-analysis-using-conductance-catheters

Research

JoVE Journal

Biology

19.3K 조회수.  Duke University Medical Center. Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters. 

비디오: Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

이온 채널 유학을위한 패치 클램프 및 재관류 기법으로 표현 Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

연구

생물학

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Immunodeficient 마우스의 생체 공학 인간 Microvascular 네트워크

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

의 낭성 섬유증의 Transmembrane의 전도성 조절 단백질의 표현과 정제 Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

증발 플럭스 방법 (EFM)를 사용 조사량과 탈수에 잎 유압 전도성과 Stomatal 전도성과 응답 측정

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

주변 줄기 세포 이주의 분석을위한 마우스 Cremaster 근육에있는 미세 혈관의의 intravital 현미경

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

로 표현 낭포 성 섬유증 막 횡단 전도력 규칙 단백질의 정제<em&gt; 사카로 마이 세스 세레 비시 애</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

그레이 스케일 분석을 사용하여 마우스의 탈모의 정량 분석​​을위한 새로운 기술

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

잎 기능 형질에 기공 컨덕턴스 관련된

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

마우스의 심장 수축력의 조사에 대 한 격리 유두 근육의 수축력 측정

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

마우스에 압력 볼륨 루프 측정

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...