우리는이 연구의 그들의 지원을 위해 Boettcher 재단과 루 경화 증 협회 (#17-II-344)에 감사.

쥐와 관련 된 모든 절차는 콜로라도의 대학-Anschutz 의료 캠퍼스 기관 동물 관리 및 사용 위원회에 의해 승인 되었다 (91813(05)1D).
 1. 주 1: 기본 (1 °)와 소 혈 청 알 부 민 (BSA) 차단 Immunostaining 마우스 hindlimb 근육 (예를 들어, 앞쪽에 tibialis, soleus)의 <ol><li>Air-dry 냉동된 섹션에 대 한 청구 슬라이드에 장착 ~ 30 분 11. 소수 성 장벽을 PAP 펜을 사용 하 여 섹션 주위에 테두리를 그립니다.</li>
	<li>장소 ~ 5 %BSA / 인산 염 버퍼 식 염 수 (BSA/PBS) 일반적인 항 체 바인딩 차단 하 각 슬라이드의 250 µ L. 1 헤에 대 한 실 온에서 슬라이드를 품 어</li>
	<li>차단, 하는 동안 준비 1시 50분의 5%에서 표면에 뜨는 1 ° 항 체 희석 BSA/PBS (모든 마우스 단일 클론 항 체는; 표 1 6 , 12 참조); 250 µ L의 준비 슬라이드 당 솔루션입니다. 얼음에 1 ° 항 체 주식 및 희석 유지</li>
</ol> <img alt="Table 1" src="/files/ftp_upload/56024/56024table1.jpg" /> 표 1: MyHCs를 구별 하는 데 사용 하는 기본 항 체.
<ol start="4"> <li>5 %BSA / PBS 차단 솔루션의 열망에 따라 적절 한 슬라이드에 1 ° 항 체 희석의 250 µ L을 추가. 추가 250 µ L 5 %BSA / PBS 보조 (2 °) 항 체 전용 부정적인 제어 슬라이드.</li>
	<li>품 습 한 환경에서 24-48 h 4 ° C에서. 
	참고: 페 트리 접시 약화 필터 종이와 알루미늄 호 일에 커버 수 있습니다 역할을 적합 한 보육 실.</li>
</ol> 2. 주 2: 2 ° 형광 항 체와 Immunostaining <ol><li>1 ° 항 체 솔루션을 발음 하거나 5 %BSA / PBS 솔루션 제어. 워시 모든 슬라이드 3 번, 5 %BSA 10 분.</li>
	<li>세척, 동안 준비 1: 200의 5%에서 정화, fluorophore 활용 2 ° 항 체 희석 BSA/PBS (표 2 참조 하십시오 모두 염소 반대로 마우스). 슬라이드 당 솔루션의 250 µ L를 준비 합니다. 얼음에 어둠 속에서 2 ° fluorophore 활용 된 항 체를 유지</li>
</ol> <img alt="Table 2" src="/files/ftp_upload/56024/56024table2.jpg" /> 표 2: Fluorophore 활용 된 이차 항 체 MyHCs 하는의 시각화 기본 항 체 인식 하는 데 사용.
<ol start="3"> <li>5 %BSA / PBS 차단 솔루션의 세 번째 응용 프로그램의 포부, 다음 모든 슬라이드에 2 ° 항 체 희석의 250 µ L을 추가. 어두운, 습 한 환경에서 실 온에서 90 분 동안 품 어.</li>
	<li>Aspirate 2 ° 항 체 솔루션입니다. 세척 모든 슬라이드 3 번, 10 분 5 %BSA / PBS.</li>
	<li>린스 PBS입니다. 10 분 동안 건조</li>
	<li>비 영구, 저 점도 수성 장착 매체 coverglass 탑재.</li>
	<li>건조 하면, 매니큐어와 슬라이드 가장자리 물개. 건조 하 고 어두운 slidebox에 저장.</li>
</ol> 3. Epifluorescence 현미경 검사 법으로 슬라이드 이미징 <ol><li>는 작은, 70% 에탄올 배어 연구소-삭제 기능으로 슬라이드를 청소.</li>
	<li>사진 장치 (재료의 표 참조)를 갖춘 epifluorescence 현미경을 사용 하 여 immunostained 근육 섹션의 디지털 이미지를 얻기.
	<ol><li>약 1 m m 2 (10 X 목표, 1.4 나)의 영역을 선택 합니다.</li>
		<li>보기는 505 통해 2 ° 알 렉 사 488 활용 된 항 체 형광 nm 긴 통과 필터. 595 nm 긴-패스 필터를 통해 2 ° 알 렉 사 594 활용 된 항 체의 흥분에 의해 생성 된 형광 볼. 호환 하는 이미징 소프트웨어를 갖춘 PC 컴퓨터와 이미지를 디지털화 합니다. 나중에 분석 소프트웨어 이미징에서 눈금 막대 포함.</li>
	</ol></li></ol> 4. 피지와 이미지의 분석 참고: 분석을 계속 하기 전에 https://imagej.net/Fiji/Downloads를 통해 피지 (는 건강의 국가 학회, 베 데스 다, 메릴랜드에서 자유롭게 사용 가능)를 설치 해야 합니다. 또한,이 과정에서 사용 하는 매크로 추가 파일에 제공 됩니다. 매크로 파일을 쉽게 액세스할 수 있는 디렉터리에 배치.
피지에서 선택한 섹션의 <ol><li>부하는 명시 이미지. 플러그인으로 이동 &#8594; 세분화 &#8594; Trainable Weka 세분화.</li>
	<li>섹션의 세포질 영역을 규정 하기 위해 라인에는 섬유, 그리고 클릭 &#34; 클래스 1에 추가 &#34;. 그런 다음, 섬유, 사이 공간에 선을 그릴을 클릭 &#34; 클래스 2에 추가 &#34; 세포 외 도메인을 표시. 각 클래스에 있는 5-10 레이블이 될 때까지 반복 합니다.
	<ol><li>클릭 기차 분류자입니다. 결과 빨간색과 초록색 오버레이에 추가 더 많은 레이블을 수동으로 (단계 4.2 참조) 이미지, 필요한 경우의 잘못 된 조각. 섬유 (레드) 섬유 (녹색) 사이의 공간에서 적절 하 게 분리 될 때까지 반복.</li>
	</ol></li> <li>클릭 &#34; 얻을 확률 &#34; 새로 레이블이 지정 된 폴더에 이미지를 저장 하 고.</li>
	<li>같은 필드에서 가져온 형광 이미지 (4.2-4.3 단계) 위의 절차를 반복 합니다. 으로 immunostained 섬유 라벨 &#34; 클래스 1 &#34;로 모든 비 형광 지역 &#34; 클래스 2 &#34;. 결과 확률 지도 처리 명시 이미지 저장 된 동일한 폴더에 저장.</li>
	<li>피지에서 이전에 저장 된 확률 지도 중 하나를 엽니다. 이미징 소프트웨어에 의해 제공 눈금 막대에 직선을 그립니다. 이동 분석 &#8594; 규모 설정. 눈금 막대에 의해 주어진 그들의 적절 한 값을 매개 변수 (즉, 알려진된 거리, 길이 단위)를 변경 후 확인은 &#34; 글로벌 &#34; 표준화 각 이미지에 대 한 규모를 상자.</li>
	<li>탐색 플러그인을 &#8594; 매크로 &#8594; 실행 &#8594; Tyagi 외. 섬유 부 량 macro.ijm (보조 파일 참조). 즉시, 탐색 창에 표시 됩니다. 이미지를 열고 &#39; s 호스트 폴더; 대화 상자가 표시 됩니다. 각 대화 상자에 대 한 변경 된 &#34; 배경 &#34; 드롭다운 값을 &#34; 빛. &#34; 참고: 폴더 이제 채워집니다 섬유 윤곽선의 이미지와 스프레드시트 소프트웨어 파일 (체코와 Feret 직경은 가장 관련 된 결과의 측정 섬유 형태; 측정 된 매개 변수를 조정할 수 있습니다 설정 측정 분석). 스프레드시트 소프트웨어 파일에 대 한 두 형광 및 밝은 분야 이미지 피지의 기 공 분석 기능에 의해 생성 하는 섬유 형태 데이터를 자동으로 옮겨질 것 이다.</li>
	<li>계산 하는 필드에 지정 된 MyHC 필드에 해당 필드 밝은 이미지에 총 섬유의 수에 의해 형광 섬유의 수를 분할 하 여 표현 하는 섬유의 분수.</li>
</ol>

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저자는 공개를 전혀 상반 되는 관심사를 있다.

여기, 우리 골격 근육 섬유 종류의 식별에 유용한 방향 제공. 이렇게, 우리는 데이터의 분석을 위한 새로운 알고리즘을 설명 합니다.
때문에 우리의 결과 크게 이전 보고서5,,810 의 확인 하 고 우리 자신의 수동 측정을 반영, 알고리즘은 정확 하 게 나타납니다. 아직도, 우리는 유물 단면에서 결과 불분명 섬?...

그것은 골격 근육 많은 종류1의 단일 섬유의 구성 된 시간에 대 한 알려져 있다. 처음, 섬유의 2 개 그룹 그들의 수축 성의 속성에 따라 특징 이었다 하 고, 적절 하 게, 느린-트 위치 (유형 I) 및 빠른 트 위치 (유형 II). 이러한 범주 추가 섬유 물질 대사에 바탕으로 구별 했다. 이후 유형 I 섬유는 미토 콘 드리 아에서 풍부 하 고 산화 물질 대사에 의존, 그들은 견고 하 게 긍정적인 니코틴아미드 아데닌 디뉴클레오티드-tetrazolium 환 원 효소 (NADH-TR) diaphorase2 또는 호박 효소 (SDH)3 에 의해 해명 되었다 얼룩. 대조적으로, 유형 II 섬유 전시 덜 NADH TR diaphorase 또는 SDH의 가변도 얼룩이 지 고 두 빠른 트 위치 하위 그룹으로 분할 되었다 (유형 IIA 및 IIB 입력) 그들의 상대적인 산화 용량에 따라 다소 조잡. 섬유 사이의 이러한 구분 myosin ATPase 얼룩에 의해 보다 효과적으로 시각화 되어 있다 어디 유형 I 섬유는 pH 4.0에서 사전 부 화 후 어두운 얼룩 및 유형 IIB 섬유 흡수 유형 IIA 섬유와 pH 10.0에서 촉진 다음 사전 부 화 4얼룩 판매인.
더 최근에, myosin 무거운 체인 (MyHC) isoforms의 immunohistochemical 얼룩이 섬유 유형5의 미세한 판별자로 떠오르고 있다. 유형 I, IIA를 입력 하 고 유형 IIB 섬유 확인 될 수 있다 모두 그들의 MyHC 프로 파일에 따라 정밀. 또한, 또 다른 빠른 트 위치-중급 metabolically 섬유 종류, IIX, 입력 되었습니다 확인된6. 하나 이상의 MyHC을 표현 하는 하이브리드 섬유도 확인된5,,78있다. 고양이 개 코 원숭이 등 일부 종 표현 유형 IIB MyHCs6하지 알려져 있습니다. MyHC immunostaining는 현재 근육 구성의-의 상태--예술 평가,이 기술을 통해 얻은 데이터의 분석은 복잡 하 고 자동화 된 도움 없이 소모. 이 위해 이러한 데이터를 분석 하는 반자동된 방법의 개발된5,,910되었습니다. 여기, 우리 소설 반자동된 함께 근육 섬유 타입5,,78,10, 의 immunohistochemical 식별에 대 한 상대적으로 표준 프로토콜 제시 섬유 유형 및 섬유 형태학의 분석 정확도와 가속 알고리즘입니다.

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	<tbody>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Bovine Serum Albumin</td>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Microscope Slides</td>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Coverglass</td>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Immumount</td>
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			<td class="xl68" style="width:172pt" width="229">9990402</td>
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			<td class="xl65" height="41" style="width:226pt;height:30.75pt" width="301">Nikon Eclipse TE-200 Inverted Fluorescence and Brightfield Microscope</td>
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			<td class="xl65" style="width:172pt" width="229">Discontinued</td>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Dell Optiplex</td>
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			<td class="xl70">monoclonal mouse IgG2b; 1:50</td>
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			<td class="xl65" height="61" style="width:226pt;height:45.75pt" width="301">SC-71 Primary Antibody</td>
			<td class="xl65" style="width:140pt" width="187">Developmental Studies Hybridoma Bank at the University of Iowa</td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<td class="xl70">monclonal mouse IgG1; 1:50</td>
		</tr>
		<tr height="61" style="height:45.75pt">
			<td class="xl65" height="61" style="width:226pt;height:45.75pt" width="301">BF-F3 Primary Antibody</td>
			<td class="xl65" style="width:140pt" width="187">Developmental Studies Hybridoma Bank at the University of Iowa</td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<td class="xl70">monoclonal mouse IgGM; 1:50</td>
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			<td class="xl65" height="61" style="width:226pt;height:45.75pt" width="301">6H1 Primary Antibody</td>
			<td class="xl65" style="width:140pt" width="187">Developmental Studies Hybridoma Bank at the University of Iowa</td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<td class="xl70">monoclonal mouse IgGM; 1:50</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Alexa Fluor 594 anti-IgG2b</td>
			<td class="xl65" style="width:140pt" width="187">Invitrogen</td>
			<td class="xl65" style="width:172pt" width="229">A21145</td>
			<td class="xl66">goat anti-mouse; 1:200</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Alexa Fluor 488 anti-IgG1</td>
			<td class="xl65" style="width:140pt" width="187">Invitrogen</td>
			<td class="xl65" style="width:172pt" width="229">A21121</td>
			<td class="xl66">goat anti-mouse;1:200</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Alexa Fluor 594 anti-IgGM</td>
			<td class="xl65" style="width:140pt" width="187">Invitrogen</td>
			<td class="xl65" style="width:172pt" width="229">A21044</td>
			<td class="xl66">goat anti-mouse;1:200</td>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">OCT</td>
			<td class="xl65" style="width:140pt" width="187">Sakura Finetek</td>
			<td class="xl68" style="width:172pt" width="229">4583</td>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">isopentane</td>
			<td class="xl65" style="width:140pt" width="187">Fisher Scientific</td>
			<td class="xl65" style="width:172pt" width="229">O3551-4</td>
			<td class="xl66">cool with liguid nitrogen</td>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">PBS</td>
			<td class="xl65" style="width:140pt" width="187">Fisher Bioreagents</td>
			<td class="xl65" style="width:172pt" width="229">BP665-1</td>
			<td class="xl66">10X, dilute to 1X</td>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Kim wipes</td>
			<td class="xl66">Kimberly-Clark</td>
			<td class="xl65" style="width:172pt" width="229">06-666A</td>
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semi-automated analysis of mouse skeletal muscle morphology and fiber-type composition

수년간, 골격 근육 섬유 유형 사이 구분 했다 myosin ATPase 얼룩에 의해 최고의 시각 됩니다. 더 최근에, myosin 무거운 체인 (MyHC) isoforms의 immunohistochemical 얼룩이 섬유 타입의 미세한 판별자로 떠오르고 있다. 유형 I, IIA, IIX 입력 입력 하 고 유형 IIB 섬유는 정밀 프로필 기반으로 MyHC; 지금 확인 될 수 있다 그러나, 이러한 데이터의 수동 분석 느리고 다운 오른쪽 수 지루한. 이와 관련, 섬유 형 구성과 형태학의 신속, 정확한 평가 매우 바람직한 도구입니다. 여기, 우리는 최신의 immunohistochemical 마우스 hindlimb 근육 섬유 유형 및 섬유 형태 분석 가속 소설 반자동된 알고리즘으로 콘서트에서에서 얻은 냉동된 섹션에 MyHCs의 얼룩에 대 한 프로토콜을 제시. 표시 soleus 근육 유형 I에 얼룩이 지 고 입력 IIA 섬유, 예상 대로, 하지만 형식 IIX 또는 유형 IIB 섬유. 다른 한편으로, 앞쪽에 tibialis 근육 유형 IIX 및 유형 IIB 섬유, 유형 IIA 섬유의 작은 일부분의 주로 구성 되었고 거의 또는 전혀 유형 I 섬유. 여러 이미지 변환 섬유 형태 (즉, 횡단면 지역 (CSA), 최대 및 최소 Feret 직경)의 다양 한 측면을 측정 하기 위해 확률 지도 생성 하기 위해 사용 되었다. 이러한 매개 변수 값이 수동으로 얻은 다음 비교 했다에 대 한 얻은 값입니다. 체코, 최대 또는 최소 Feret 직경에 관하여 분석의 두 모드 사이의 중요 한 차이가 관찰 되었다 (모든 p &#62; 0.05), 우리의 방법의 정확도 나타내는. 따라서, 우리의 immunostaining 분석 프로토콜 노화와 myopathy의 많은 모델에서 근육 구성에 대 한 효과의 조사에 적용할 수 있습니다.

알 수 없는 나이의 남성 C57BL/6 마우스 해 부 Hindlimb 근육 (즉, 앞쪽에 tibialis, soleus) 액체 질소 냉각 isopentane 화합물 10 월에는 근육을 포함 하는 플라스틱 금형을 침 몰 하 여 냉동 플래시 했다. 그런 다음는 cryotome를 사용 하 여, 8-10 µ m 직렬 섹션-20 ° C에서 잘라 되었고 다른 긍정적으로 위탁 유리 슬라이드12전송.
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Watch this Scientific Journal Video about Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition at JoVE.com

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

골격 근육 섬유 타입의 최신의 판별자로 떠오르고 있다 Immunohistochemical myosin 무거운 체인 isoforms의 얼룩 (즉, 유형 I, 유형 IIA, IIX 입력, IIB를 입력). 여기, 우리는 섬유 유형 및 섬유 형태학의 급속 한 평가 용이 하 게 하는 소설 반 자동화 알고리즘 함께 얼룩 프로토콜을 제시.

마우스 골격 근육 형태와 섬유 형 구성의 반 자동화 된 분석

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

semi-automated-analysis-mouse-skeletal-muscle-morphology-fiber-type

Research

JoVE Journal

Medicine

10.3K 조회수.  University of Colorado School of Medicine. 골격 근육 섬유 타입의 최신의 판별자로 떠오르고 있다 Immunohistochemical myosin 무거운 체인 isoforms의 얼룩 (즉, 유형 I, 유형 IIA, IIX 입력, IIB를 입력). 여기, 우리는 섬유 유형 및 섬유 형태학의 급속 한 평가 용이 하 게 하는 소설 반 자동화 알고리즘 함께 얼룩 프로토콜을 제시.

비디오: Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

절연 골격근의 묘 - 기계 분석

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

연구

의학

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Proteomics에서 골격근 성별 동종 이형

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

경 흉부 심 초음파에 의한 폐동맥 수축의 마우스 모델에서 마우스 오른쪽 심실 구조 및 기능의 평가

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

수정 된 버그 스트롬 기술을 이용하여 인간의 골격 근육 생검 절차

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

골격근 질환의 정량적 자기 공명 영상

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

사람의<em&gt; 광근의 Lateralis</em&gt; 웨일 - Blakesley Conchotome를 사용하여 골격 근육 생검

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

유도와 인간의 운동시 골격근 손상의 평가

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

마우스의 심장 형태학의 echocardiographic 및 조직학 검사

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

신체 구성 및 높은 지방 연 준 쥐에서 대사 배우면 분석

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

부드러운 근육 세포 계보 추적 마우스의 고급 동맥 경 화성 병 변에서 세포 성분의 정량 분석

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...