우리는 베보 2100 초음파 시스템의 제공을 위해 홍콩 폴리 테크닉 대학교의 생명 과학 (ULS)의 대학 연구 시설을 인정하고 싶습니다. 교육 및 촉진을위한 생명 공학, 홍콩 폴리 테크닉 대학의 학제 부문에서 박사 Chunyi 원 총리 특별 감사. 이 작품은 홍콩 정부 RGC 부여 GRF 17127215, 764812 빌리 KC 차우에 HKU6이 / CRF는 / 11G에 의해 지원되었다.

이 프로토콜은 홍콩 대학의 교육 및 연구 (CULATR)에서 라이브 동물의 사용에 대한위원회의 가이드 라인을 따른다. 1. 작업 현장 준비 <ol><li> 75 % 이소 프로필 알코올로 사업장 소독. 마음의 변화와 피사체 동물의 호흡 속도를 피하기 위해 37 ° C까지 가열 패드 온도를 조정합니다. </li><li> 30 분 동안 또는 다른 적절한 방법으로 121 ° C에서 중력 변위 멸균기에서 멸균 수술 도구를 사용합니다. </li><li> 도 1에 도시 된 바와 같이 결찰 조직 분리 및 권취 장치로 사용하기위한 도구를 만들기 위해 4 개의 30 게이지 바늘과 바늘 홀더 곡선을 2 - 가라. 수술에 사용되는면 도포에 지금은 곡선 바늘을 탑재합니다. </li><li> 이는 니들 홀더 90º 27 게이지 바늘과 곡선 블런트. 결찰 단계에서 0.4-mm 스페이서를 만들기 위해 하드 표면을 문질러 끝을 부드럽게. </li></ol><ol><li> 음의 꼬리 핀치 반사와 마취 확인 100 ㎎ / ㎏ + 10 ㎎ / ㎏ IP : 체중과 관련하여 적절한 케타민과 자일 라진의 혼합물 2 개월, 남성, 22~25g C57BL / 6N 마우스를 마취 . </li><li> 제모 크림의 2 ~ 3 분의 응용 프로그램과 함께 전방 목과 가슴에 머리를 제거합니다. 따뜻한 물과면 주걱으로 크림을 제거합니다. </li><li> 각막에서 건조를 방지하기 위해 동물의 눈 안과 용 연고 또는 겔을 적용한다. </li><li> 앙와위에서 가열 패드에있는 동물을 놓고 매 15 분 간격으로 해당 문서를 유지해야되고, 수술 중 모니터링을 실시하고 있습니다. </li><li> 수술시에 올바른 위치에 동물을 유지하기 위해 상기 가열 패드 사지를 고정 수술 종이 테이프를 사용한다. </li><li> 알코올, 포비돈 - 요오드 용액을 3 회 교대로 피부를 소독. </li><li> 멸균 DR을 사용하여노출 작업 필드 원숭이 오염을 방지 할 수있다. 각 마우스 및 필요에 따라 새로운 멸균 장갑을 사용합니다. </li></ol> 3. 내고 절차 <ol><li> 메스와 목과 가슴의 중간 선 위치에서 마우스의 피부를 엽니 다. </li><li> 부드럽게 무딘 가위로 결합 조직을 분리하여 머리를 향해 갑상선를 당깁니다. </li><li> 만곡 된 바늘 양쪽 방향 중심선에서 기관의 근육 층을 분리한다. </li><li> 무딘 가위를 사용하여, 상기 제 리브 (약 5 ㎜)에 흉골 잘라. 트랙터 또는 곡선 집게로 잘라을 엽니 다. </li><li> 서로 곡선 바늘과 결합 조직을 분리하여 하단 가슴 벽에서 흉선 로브를 분리; 가로 대동맥 궁과 두 개의 경동맥 동맥이 시점에서 명확하게 볼 수 있습니다. </li><li> 아치 아래 곡선 바늘을 삽입하고 고름하여 다른 쪽의 용기의 벽과 결합 조직 사이에 관통곡선 머리를 힝. </li><li> 곡선 바늘을 사용하여 대동맥 궁 아래에 6-0 모노 필라멘트 봉합사를 잡아 당깁니다. </li><li> 루프에 스페이서를 놓고 이중 매듭 장소에 봉합을 수정합니다. 부드럽게 스페이서를 제거합니다. </li><li> 매듭의 위치에 성공적으로 수축을 확인하고 봉합의 끝을 잘라. </li><li> 간단한 중단 봉합 패턴으로 6/0 실크 봉합사를 사용하여 흉벽을 닫습니다. 연속 봉합 패턴의 6/0 모노 필라멘트 봉합사로 피부를 닫는다. </li></ol> 4. 수술 후 케어 <ol><li> 봉합 사이트에 포비돈 - 요오드 솔루션을 적용하고 회복과 수술 후 모니터링을위한 사전 예열 새장에 동물을 배치합니다. </li><li> 동물이 통증을 완화하고 적절한 부드러운 음식 마우스를 제공하기 위해 의식을 회복 한 후 - (0.1 ㎎ / ㎏ 사우스 캐롤라이나 모든 12 시간 0.05), 부 프레 노르 핀을 주입한다. </li><li> 전체 복구 후 12 시간 빛 / 어둠 사이클 방에 동물을 돌려줍니다. </li></ol> 5.심 초음파 : 성공적인 결찰 및 측정의 확인 <ol><li> 1 L / min의 산소 유속, 유지 보수를 위해, 1.5 % - 유도, 5 % 이소 플루 란, 및 1 - 일주 후 3 밴드 이전 마우스를 마취. </li><li> 제모 크림을 사용하여 가슴 털을 제거합니다. 철저 따뜻한 물과면 주걱으로 크림을 제거합니다. </li><li> 37 ° C에서 유지되는 동물 처리 플랫폼으로 누운 자세에서 마우스를 고정합니다. 발톱에 전극 젤을 적용하고는 전극 패드에 테이프. </li><li> 마우스의 가슴에 초음파 젤을 적용합니다. 40 MHz의 MS550D 프로브와 고주파 초음파 시스템은, 대동맥 궁에뿐만 아니라 좌측 및 우측 경동맥 컬러 도플러 및 펄스 파 도플러 검색을 수행한다. </li><li> B 모드를 사용하여 좌심실을 찾을. M 모드를 이용하여 수축기 및 이완기의 벽 두께를 측정 할 수있다. 필요에 따라 사용 시네 저장 및 프레임 스토어 데이터와 이미지를 저장합니다. </li><li> 유, B 모드를 노래 분기점 전에 왼쪽 경동맥을 찾습니다. 흐름을 평가하기 위해 펄스 파 및 색 도플러를 사용합니다. 비슷한 지점에서 오른쪽 경동맥과 반복합니다. 나중에 분석 할 수 시네 저장 및 프레임 스토어 모드에서 이미지를 저장합니다. </li><li> 마우스 왼쪽 욕창 위치에 있도록 맨 왼쪽에 동물 처리 플랫폼을 기울입니다. 대동맥 궁을 찾기 위해 X와 Y를 마우스의 흉골 위치에 머리에 위치 평행로부터의 오프셋 (offset) 30 °에 프로브를 배치하고 조작 할 수 있습니다. </li><li> 수축을 찾습니다. 두 개의 경동맥 동맥은 대동맥 궁으로부터 분기 어디 혈액이 점 사이의 좁은 채널을 통해 유입되는 바와 같이, 더 볼 수축의 사이트로 만들려면 컬러 도플러 모드를 사용합니다. </li><li> 최대 유속을 측정하기 위해, 펄스 파형 및 컬러 도플러를 사용한다. 필요에 따라 사용 시네 저장 및 프레임 스토어 데이터와 이미지를 저장합니다. </li><li> 동물이 발생하기 쉬운 위치에 가열 패드에 복구 할 수 있습니다.완전히 마취에서 회복 후 12 시간 빛 / 어둠 사이클 방에 원래 케이지에 동물을 돌려줍니다. </li></ol>

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	<li>Rockman, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+atrial-specific+and+inducible+expression+of+an+atrial+natriuretic+factor+transgene+in+an+in+vivo+murine+model+of+cardiac+hypertrophy.">Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy.</a> Proc Natl Acad Sci U S A. 88 (18), 8277-8281 (1991).</li><li>de Almeida, A. C., van Oort, R. J., Wehrens, X. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transverse+aortic+constriction+in+mice.">Transverse aortic constriction in mice.</a> J Vis Exp. (38), (2010).</li><li>Hu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+invasive+aortic+banding+in+mice:+effects+of+altered+cardiomyocyte+insulin+signaling+during+pressure+overload.">Minimally invasive aortic banding in mice: effects of altered cardiomyocyte insulin signaling during pressure overload.</a> Am J Physiol Heart Circ Physiol. 285 (3), H1261-H1269 (2003).</li><li>Shimizu, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+cardiac+insulin+signaling+exacerbates+systolic+dysfunction+induced+by+pressure+overload+in+rodents.">Excessive cardiac insulin signaling exacerbates systolic dysfunction induced by pressure overload in rodents.</a> J Clin Invest. 120 (5), 1506-1514 (2010).</li><li>Tarnavski, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+surgical+models+in+cardiovascular+research.">Mouse surgical models in cardiovascular research.</a> Methods Mol Biol. 573, 115-137 (2009).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+murine+model+for+the+study+of+reverse+cardiac+remodelling.">A modified murine model for the study of reverse cardiac remodelling.</a> Exp Clin Cardiol. 18 (2), e115-e117 (2013).</li><li>Hartley, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doppler+estimation+of+reduced+coronary+flow+reserve+in+mice+with+pressure+overload+cardiac+hypertrophy.">Doppler estimation of reduced coronary flow reserve in mice with pressure overload cardiac hypertrophy.</a> Ultrasound Med Biol. 34 (6), 892-901 (2008).</li><li>Scherrer-Crosbie, M., Thibault, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiography+in+translational+research:+of+mice+and+men.">Echocardiography in translational research: of mice and men.</a> J Am Soc Echocardiogr. 21 (10), 1083-1092 (2008).</li><li>Faerber, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+heart+failure+by+minimally+invasive+aortic+constriction+in+mice:+reduced+peroxisome+proliferator-activated+receptor+gamma+coactivator+levels+and+mitochondrial+dysfunction.">Induction of heart failure by minimally invasive aortic constriction in mice: reduced peroxisome proliferator-activated receptor gamma coactivator levels and mitochondrial dysfunction.</a> J Thorac Cardiovasc Surg. 141 (2), 492-500 (2011).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Cardiac+Morphological+and+Functional+Changes+in+Mouse+Model+of+Transverse+Aortic+Constriction+by+Echocardiographic+Imaging.">Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging.</a> J Vis Exp. (112), (2016).</li></ol>

The authors declare that they have no competing financial interests.

록맨 연구팀은 심장에 압력 과부하를 연구하는 방법으로 대동맥 밴딩을위한 첫 번째 방법을 고안했다. 이 활성화 연구는 고혈압과 심장 장애 1의 세포 및 분자 결과를 발견하기 시작합니다. 개선 된 방법, 침습적 횡 대동맥 협착 (MTAC)은 종래의 횡 대동맥 협착 (9)의 단점없이 좌심실 압력 과부하 및 심부전의 유도에 대한 의무, 시간에 따른 방법을 제공한다. TAC?...

<html> 이 문서의 전체 목적은 쥐 대동맥 협착 (TAC)가 간단하고 편리한 수술을 생성하도록 변형 될 수있는 방법을 횡 보여주는 것이다. TAC는 제 1991 (1)에 도입되고 조작 게놈 2, 3, 4, 5, 6, 특히 소 동물에 좌심실과 심부전 비대증으로 이어지는 세포 및 분자 경로를 연구하는 분자 생물학을 활성화시켰다. 원하는 압력 과부하를 제조에서 효능에도 불구하고,이 기술은 궁극적 절차를 수행 및 낮은 생존율로 어려움에 의해 제한된다. TAC 전도성 외과 기관 내 삽관, 개흉술을 수행하고 폐 붕괴 일을 방지하기 위해 인공 호흡기를 사용할 수 있어야한다. 콘텐츠 &quot;&gt; 종종 간단하고 덜 침습적 방법이 요구된다. 결과적으로, 최소 침습 횡 대동맥 협착 (MTAC)는 3을 고안 하였다. 그 결과 (즉, 좌심실 비대)는 동일하지만, 절차는 상당히 더 적은 수의 장치를 포함 상기 동물에게 덜 손상. MTAC 90 % 이상, 더 생존율을 갖는 바람직한 방법으로 증명하고, 또한 기인 할 수 성공적인 수술 후 사망의 15 분 내지 25. 많지 않은 경우의 동작 시간을 갖는 엄청난 좌심실 과부하 및 신장 등 중요 장기에 혈액 공급의 심각한 감소의 결과 대동맥 궁의 조인 다구 수축. 절차가 완료된 후, 초음파 검사는 밴딩 심장 형태와 기능의 성공 여부를 평가할 수있다. 고주파, 초음파 시스템은 직경을 측정하고, 좌우의 변화를 흐르게하는 데에 이용 될 수있다최대 유속 측정 대동맥 궁 수축을 시각화하고, 좌심실 벽 두께 및 기능적 파라미터 (7, 8)를 결정하기 위해, 경동맥 동맥. 안락사 후 장기도 헤 마톡 실린과 에오신 염색 Picrosirius 콜라겐 염색 조직 학적 연구를 위해 수집 될 수 후자 섬유증의 관찰에 특히 유용한.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 inch 30 Gauge Needles</td>
			<td>BD, Franklin Lakes, NJ 07417, USA.&#160;</td>
			<td>305128</td>
			<td>Curved as in the procedure and Figure 1</td>
		</tr>
		<tr>
			<td>27 Gauge Needle</td>
			<td>BD, Franklin Lakes, NJ 07417, USA.&#160;</td>
			<td>301629</td>
			<td>Make blunt and smooth for spacer</td>
		</tr>
		<tr>
			<td>6/0 Dafilon</td>
			<td>B. Braun, 34212 Melsungen, Hessen, Germany.</td>
			<td>C0933066</td>
			<td></td>
		</tr>
		<tr>
			<td>6/0 Silkam</td>
			<td>B. Braun, 34212 Melsungen, Hessen, Germany.</td>
			<td>C0762067</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gloves</td>
			<td>A.R. Medicom, Inc (Asia), Hong Kong.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Applicator</td>
			<td>Mannings, Quarry Bay, Hong Kong.</td>
			<td></td>
			<td>Local Shopping Center</td>
		</tr>
		<tr>
			<td>Delapitory Cream</td>
			<td>Veet (Hong Kong), Kwun Tong, Kowloon, Hong Kong</td>
			<td></td>
			<td>Local Shopping Center</td>
		</tr>
		<tr>
			<td>Lexer-Baby Scissor</td>
			<td>FST, British Columbia V7H 0A6, Canada.</td>
			<td>14078-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Iris Forcep</td>
			<td>FST, British Columbia V7H 0A6, Canada.</td>
			<td>11065-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Olsen-Hegar Needle Holder</td>
			<td>WPI, Sarasota, FL 34240, USA.&#160;</td>
			<td>501989</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Micrioscope</td>
			<td>WPI, Sarasota, FL 34240, USA.&#160;</td>
			<td>PZMIII-BS</td>
			<td></td>
		</tr>
		<tr>
			<td>GenTeal Gel</td>
			<td>Novartis, East Hanover, NJ 07936, USA.&#160;</td>
			<td></td>
			<td>Local Pharmacy</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>Kent Scientific Corporation, Torrington, CT 06790, USA.&#160;</td>
			<td>DCT-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td>Laboratory Animal Unit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine and Xylazine</td>
			<td>Laboratory Animal Unit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Laboratory Animal Unit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Laboratory Animal Unit</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

minimally invasive transverse aortic constriction in mice

최소 침습 가로 대동맥 수축 (MTAC)는 표준 오픈 가슴을 가로 대동맥 수축 (TAC)보다 마우스의 가로 대동맥의 수축을위한 더 바람직한 방법입니다. 횡 대동맥 협착이 좌심실 높은 압력을 유도하는 고기능 방법이지만 인한 기관 내 삽관과 인공 호흡기의 사용에 더 어렵고 긴 절차이다. TAC는 새로운 방법, MTAC로도 자주 덜 존속 없다, 둘 환기 설치와 갈비와 늑간 근육이나 기관 삽관의 절단을 필요로한다. 흉강에 액세스 할 수 개흉술에 반대 MTAC에서, 대동맥 궁은 ​​앞쪽 목의 중간 선 절개를 통해 도달한다. 갑상선은 흉골 노치를 나타 내기 위해 다시 당겨진다. 흉골이어서 제 리브 수준까지 절단하고, 대동맥 궁는 결합 조직 및 흉선을 분리하는 것만으로 도달된다. 에서거기에, 봉합 아치를 감싸 및 스페이서와 연결 한 후 흉골 절단 및 피부 닫을 수 있습니다 할 수 있습니다. MTAC 좌심실 고혈압을 유도하는 매우 빠르고 덜 침습적 방법은 높은 처리량 연구 가능성을 가능하게한다. 수축의 성공은 대동맥의 흐름 속도를 결정하기 위해 고주파 트랜스 흉부 심장 초음파, 특히 컬러 도플러 및 펄스 파 도플러를 이용한 검증 좌우 될 수 있고 우측 경동맥 동맥 혈관의 치수 및 좌심실 기능 및 형태. 성공적인 수축은 또한 간질과 혈관 주위 섬유화와 심장 근육 세포의 비대와 같은 중요한 조직 병리학 적 변화를 트리거합니다. 여기서, MTAC의 과정 경동맥에서 얻어진 유량 변화가 심장 초음파, 총 형태학 및 심장 조직 학적 변화를 관찰 할 수있는 방법을 보여주는 설명한다.

MTAC는 적시에 좌심실에서 중요한 고혈압의 유도 할 수 있습니다. 폴리 우레탄 튜브 대신에 구부러져 열리는 4 30 게이지 바늘 -도 1에 도시 된 바와 같이, 절차는 기본 도구의 제조는이 요구를 간단하고 최소한이다. 수술 자체는 TAC에 비하여, 용이하게 수행 될 수 있기 때문에, 또한 덜 복잡하다. 갑상선이 수축되면,...

Watch this Scientific Journal Video about Minimally Invasive Transverse Aortic Constriction in Mice at JoVE.com

Minimally Invasive Transverse Aortic Constriction in Mice

기관 삽관과 인공 호흡기의 사용을 제거하면서 최소 침습 가로 대동맥 수축 (MTAC)는 일반 가로 대동맥 수축 (TAC)의 본질을 절약합니다. 그것은 특히 병진 연구에서 좌심실 과부하에 높은 처리량 연구를위한 매우 바람직한 방법으로 증명한다.

마우스의 최소 침습 가로 대동맥 수축

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard ...

minimally-invasive-transverse-aortic-constriction-in-mice

Research

JoVE Journal

Medicine

20.3K 조회수.  The University of Hong Kong.  기관 삽관과 인공 호흡기의 사용을 제거하면서 최소 침습 가로 대동맥 수축 (MTAC)는 일반 가로 대동맥 수축 (TAC)의 본질을 절약합니다. 그것은 특히 병진 연구에서 좌심실 과부하에 높은 처리량 연구를위한 매우 바람직한 방법으로 증명한다. 

비디오: Minimally Invasive Transverse Aortic Constriction in Mice

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

마우스를 사용 Tracheal 수축의 체외 측정에서

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

연구

의학

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50&#8211;60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.

We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.

압력 과부하 심장 비대 모델의 창조를위한 쥐의 대동맥 수축 오름차순

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

쥐과 동 소성 방광 이종 이식의 최소 침습 설립

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

지방 조직의 종점에 이식 캡슐화 열 발생 Preadipocytes

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

심 초음파 이미징에 의해 가로 대동맥 수축의 마우스 모델에서 심장 형태 학적 평가 및 기능 변경

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

왼쪽된 심 실 비 대의 유도 대 한 생쥐에서 최소 침 습 가로 대동맥 수축의 기술

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Endochondral 골절 표준화 된 Biomechanical 조건 하에서 쥐에서 치유를 분석 하는 최소 침 습 모델

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

마우스에 가로 대동맥 수축 유도를 폐쇄 가슴 모델

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

재건 미세 수술에서 허혈 재관류 손상의 연구를위한 전 임상 쥐 모델

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Evaluation of Capnography Sampling Line Compatibility and Accuracy when Used with a Portable Capnography Monitor

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment of end-tidal CO2 (ETCO2), its accuracy is commonly validated using commercial kits composed of a capnography monitor and its matching disposable nasal cannula sampling lines. The purpose of this study was to assess the compatibility and accuracy of cross-paired capnography sampling lines with a single portable bedside capnography monitor. A series of 4 bench tests were performed to evaluate the tensile strength, rise time, ETCO2 accuracy as a function of respiratory rate, and ETCO2 accuracy in the presence of supplemental O2. Each bench test was performed using specialized, validated equipment to allow for a full evaluation of sampling line performance. The 4 bench tests successfully differentiated between sampling lines from different commercial sources and suggested that due to increased rise time and decreased ETCO2 accuracy, not all nasal cannula sampling lines provide reliable clinical data when cross-paired with a commercial capnography monitor. Care should be taken to ensure that any cross-pairing of capnography monitors and disposable sampling lines is fully validated for use across respiratory rates and supplemental O2 flow rates commonly encountered in clinical settings.

The goal of this study was to evaluate the accuracy of capnography sampling lines used in conjunction with a portable bedside capnography monitor. Sampling lines from 7 manufacturers were evaluated for tensile strength, rise time, and ETCO2 accuracy as a function of respiratory rate or supplemental oxygen flow rate.

휴대용 Capnography 모니터와 함께 사용할 때 Capnography 샘플링 라인 호환성 및 정확도 평가

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment ...