We appreciate Feifei Zhang for the helpful discussions. We are grateful to Jing Gao and Yixuan Sun for the technical assistance and to Zhengshuai Liu and Fengguang Ma for the animal studies.

모든 동물 실험 프로토콜은 생물 과학에 대한 영양 과학 연구소, 상하이 연구소, 중국 과학 아카데미 (중국 상하이)에서 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. 1. DIO 마우스 모델 <ol><li> HFHS 공급 <ol><li> 40 킬로 칼로리 %의 지방과 40 킬로 칼로리 %의 자당을 포함하는 HFHS으로 8 주 된 수컷 C57BL / 6 마우스 피드. 12 시간 어두운 조명주기 조건을 수용. </li><li> 네 여섯 주 동안 HFHS 다이어트 수유 상태에서 마우스를 유지합니다. 체중 매주을 확인합니다. </li></ol></li><li> 신체 조성 분석 및 의사 컬러 화상 해석 <ol><li> 신체 조성 분석기 분석 생쥐 대상. <ol><li> 의 플라스틱 튜브에 의식하지 마취 마우스를 고정합니다. 이전이 설명 된 바와 같이, 본체 체지방량, 근육량 및 NMR 시스템을 이용하여 유체를 측정한다. </li></ol></li><li> MRI를 시스템을 사용하여 각 마우스의 몸 전체를 스캔합니다. <ol><li> 복강 내 주사 (25 μL / g)을 통해 1 % AVERTIN 가진 쥐를 마취 전문 유리관에서 촬상 분석을 수행한다. 참고 : 이미지의 과정은 각 마우스에 대한 약 0.5 시간 동안 지속됩니다. 측정이 완료되면, 그 케이지에 마우스를 반환한다. </li></ol></li><li> 지느러미, 중간, 복부 층에서 마우스의 전체 몸을 스캔; 수직 방향 간 스캔. 0.55 T 자석과 같은 수단이 매개 변수를 사용하여 스캔 K 공간 = 192 X 256 μm의 섹션 두께 = 3mm, TE = 14.8 MS, TR = 400 밀리, FOVRead = 100mm, FOVPhase = 100mm, 그리고 수를 = 마우스의 모든 그룹에 대해 동일한 자성 조건을 사용하여 제 스캔. </li><li> 신호 강도 9 항에있어서, 의사 컬러 화상에 계조 화상 약도. </li></ol></li><li> 마우스 안락사 <ol><li> dissecti 소독이러한 집게와 가위로 NG 도구. </li><li> 종이 타월에 충분한 이소 플루 란을 붓고 및 이소 플루 란은 유리 항아리에 휘발 할 수 있습니다. 쥐가 이소 플루 란에 의해 안락사되어 있는지 확인합니다. </li><li> 운영 테이블에 마우스를 고정합니다. 75 % 에탄올로 각각 복부 스프레이 가위를 사용하여 칼 모양 프로세스 진피에 절개를하고, 복막을 노출시키기 위해 손으로 중앙선을 통해 길이 진피 찢어. </li><li> 집게와 칼 모양의 과정을 클램프와 내장을 노출 리브의 바닥을 통해 복막 가로를 잘라. </li><li> 심장 천자에 의해 EDTA 코팅 튜브에 혈액을 수집합니다. </li><li> 간을 따라 다이어프램을 잘라 조심스럽게 좋은 가위를 사용하여 enterocoelia에서 간을 제거합니다. 다음과 같은 지질 측정 간 조직의 세 가지를 잘라 : H &amp; E 염색을위한) 6 × 3 × 3mm, b)는 오일 레드 O 염색을위한 6 × 3 × 3mm, 클로로포름에 대한 간 C) 20 ~ 40 밀리그램 / 메탄올 추출. </li></ol></li><li> H &amp; E 염색 <ol><li> 밤새 4 ° C에서 4 % 인산 완충 포르말린 아세테이트 간을 수정 한 후 파라핀 왁스를 포함. </li><li> 이전에 3, 10 설명한 바와 같이, 파라핀 섹션 (5 μm의)를 잘라 H &amp; E 염색 용 유리 슬라이드에 그들을 탑재합니다. </li></ol></li><li> 오일 레드 O 염색법 <ol><li> 리젠트 준비 <ol><li> 10 % 포르말린, 60 % 이소프로판올, 헤 마톡 실린 및 글리세롤 젤리 오일 레드 O 작업 솔루션을 준비합니다. 이소프로판올 100 mL로 오일 레드 O 분말 0.5 g을 용해시키고, 그 0.5 % 스톡 용액을 만들기 위해 60 ° C의 욕에서 가열한다. </li><li> 2 비율 (DDH 2 O 3 원액의 용액 2 ㎖) : 3에서 두 번 증류수 (DDH 2 O)로 희석. 작업 용액을 여과하고, 실온에서 적어도 10 분 동안 정치하자. </li></ol></li><li> AP 통신에 최적의 절삭 온도 OCT () 화합물의 간을 포함lastic 삽입 상자. 30 분 동안 -20 ℃로 동결. , 동결 마이크로톰 8 μm의 섹션으로 잘라 슬라이드에 조직을 탑재하고, 실온에서 30 분 동안 배치. </li><li> 염색 욕조의 바닥에 종이 타월을 놓고, 종이 타월 습윤 10 % 포르말린 소량 붓는다. 염색 욕조에 슬라이드를 넣고 4 ℃에서 5 분 동안 포름 알데히드 증기에 고정 된 부분을 수정합니다. </li><li> 3 초 동안 염색 욕조에 60 % 이소프로판올에 슬라이드를 찍어; 한 번 반복합니다. </li><li> 모든 조직을 커버하고 15 분 동안 배양 슬라이드에 오일 레드 O 작업 용액 몇 방울을 추가합니다. 실온에서 빛으로부터 조직을 보호합니다. </li><li> 3 초 동안 염색 욕조에 새 60 % 이소프로판올에 슬라이드를 씻어; 두 번 반복합니다. </li><li> 조심스럽게 두 번 증류 물을 통해 슬라이드를 씻어 조직 주위에 물을 제거합니다. 조직을 건조하지 않도록해야합니다. </li><li> 1 분 동안 헤 마톡 실린에서 슬라이드를 놓고 5-10에 대한 수돗물로 부드럽게 씻어분. 조심스럽게 두 번 증류수에 슬라이드를 씻어하고 coverslip에 넣어 준비가 될 때까지 증류수에 보관하십시오. </li><li> 전자 레인지에서 글리세롤 젤리를 가열하고, 조직의 상단에 몇 방울을 배치합니다. 조심스럽게 상단에 커버 슬립을 배치하고 거품을 형성하기 위해주의 바랍니다. </li><li> 현미경으로 얼룩을 관찰하고 20X 및 40X 목표를 사용하여 특성 사진을 캡처합니다. </li></ol></li><li> 간 지질의 측정 <ol><li> 새로운 2 mL의 플라스틱 원심 관의 해부 간 조직을 놓습니다. 균질화와 PBS의 1 mL에 균질화하고 새로운 15 mL 유리 튜브 해물 옮긴다. </li><li> 적극적으로 1 분 동안 (1, v / V를 2) 솔루션, 소용돌이 혼합물 및 얼음에 2 시간 동안 서 있도록 클로로포름 / 메탄올 5 mL를 추가합니다. </li><li> 4 ° C에서 10 분 동안 1,650 XG에 원심 분리기; 상부 메탄올 층 중간 단백질 디스크 및 하부 클로로포름 지질 : 혼합물을 3 층으로 분리한다. </li><li> 트란<html> 유리 파스퇴르 피펫을 사용하여 새 유리관에 바닥 sfer 단계; 바닥 분획의 방울을받을 필요가 없다. 얼음에 옆으로 튜브를 설정합니다. </li><li> 적극적으로 이전 튜브와 소용돌이에 남아있는 상층 부분에 클로로포름 4 밀리미터의 MgCl 2, 1.5 mL를 600 μL를 추가합니다. 4 ° C에서 10 분 동안 1,650 XG에 30 분 동안 얼음과 원심 분리기에 품어. </li><li> 파스퇴르 피펫을 이용하여 제 1 바닥 상 관 바닥 상을 결합한다. 상부 및 중간 계층을 폐기하십시오. </li><li> 37 °의 C 조에서 질소 가스 하에서 클로로포름을 증발. 1 % 트리톤 X-100 / 클로로포름 200 μL 충분히 유리관의 측면 워시 (V / V). 질소 기체 하에서 증발시켰다. 최종 에멀젼을 잘 200 DDH 2 O의 μL와 소용돌이와 지질을 용해. </li><li> 제조사의 프로토콜에 따라 중성 지방 또는 콜레스테롤 키트를 사용하여 총 중성 지방 (TG) 및 콜레스테롤 (TC) 수준을 측정외부 참조 &quot;&gt; 3. 주 : 단백질 정량 (3)에 의해 측정 된 지질 값은 초기 파쇄 액의 단백질 농도들로 정규화한다. </li></ol></li></ol> 2. 기본 마우스 간세포 모델 <ol><li> 마우스 차 간세포의 분리 <ol><li> 6 % 클로 랄 하이드레이트 (복강 10 μL / g)와 각 마우스를 마취. </li><li> 이전 10 설명 된대로 기본 간세포를 분리합니다. 오일 레드 O 염색을위한 6 웰 플레이트에 콜라겐 코팅 커버 글라스에 세포를 성장. 지질 추출, 10 cm 세포 배양 접시 (10)에 세포를 시드. </li></ol></li><li> 세포 치료 및 오일 레드 O 염색법 <ol><li> 기아 매체의 2 mL를 매체를 교체하고 24 시간 동안 37 ° C에서 품어. 30 mM의 포도당과 100 nM의 인슐린으로 치료. 24 시간 동안 37 ° C 형 문화 인큐베이터에서 그것을 품어. </li><li> 매체를 기음. 2 mL의 PBS의 추가 매체를 씻어 부드럽게 소용돌이 친다; 한 번 반복합니다. </li><li> 고무 밴드와 슬라이드에 커버 슬립을 수정합니다. 외부 표면에 부착 세포를 노출. (단계 150) 전술 한 바와 같이 오일 레드 O 염색을 수행한다. 참고 : 지질 축적의 기초 수준은 혈청 기아 후 높은 포도당 플러스 인슐린 치료를 자극한다. 지질 방울 오일 레드 O 염색으로 가시화된다. </li></ol></li><li> 지질의 측정 <ol><li> 동일한 조건에서 높은 포도당 플러스 인슐린 (단계 2.2.1)와 간세포 치료. 각 10 cm 세포 배양 접시에 PBS 1 ML을 추가합니다. 세포를 긁어하고 100 μL의 분취 단백질 정량 신선한 1.5mL 용량 튜브에 전송되는 새로운 15 mL 유리 튜브로 수확. </li><li> 세포의 나머지 900 μL에 (1, v / V를 2 개) 4 mL의 클로로포름 / 메탄올을 추가합니다. 대략 20 비프 음에 대한 초음파 처리 한 다음 소용돌이 적극적 출시세포의 지질. 2 시간 동안 얼음 혼합물을 품어. 지질 추출 (단계 1.6)을 수행합니다. </li></ol></li></ol>

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저자는 더 경쟁 재정적 이해 관계가 없음을 선언합니다. 자금 조달 :이 작품은 중국 국가 자연 과학 재단 (제 81270930, 31471129 및 31671224)와 백 재능 중국 과학 아카데미의 프로그램 (2013OHTP04) YL에 대한 보조금에 의해 지원되었다

NAFLD 대사 증후군, 비만, 인슐린 내성이나 2 형 당뇨병 (T2DM) 11와 연관된 프로그레시브 간 질환의 시리즈이다. NAFLD의 특징은 지방간, 간세포에 지방의 축적이다. 여기서, 방법의 스펙트럼은 표현형 및 DIO 마우스 및 마우스 차 간세포를 사용하여 간 지방증의 파라미터를 특성화하기 위해 제공된다. 이 절차는 비 알코올성 지방간 질환 및 기타 관련 대사 질환의 분자 메커니즘을 규?...

비만은 선진국과 개발 도상국의 급성장 건강 문제입니다. NAFLD 환자 (1) 30 % 내지 100 범위의 유병률 자주 알코올성 지방 간 질환 (NAFLD)와 연관된 공존하는 조건 중 하나 인 것으로보고되었다. 인해 지방간, 비만의 강한 상관 관계,식이 유도 비만 (DIO) 마우스 모델은 광범위 NAFLD 2, 3, 4, 5, 6의 개발과 관련된 복잡한 분자 메커니즘을 연구하는데 사용된다. 간 지방증은 NAFLD의 초기 단계이며, 비 알코올성 지방 간염 (NASH), 간경변, 궁극적으로 간암 7 진행할 수있다. 따라서,이 방법의 전반적인 목표는 간 steatotic 조건의 동물 및 세포 모델을 생성하고 홍보하는 것입니다효율적이고 정확한 지질 측정 ovide 상세한 프로토콜. 이 모델과 측정은 또한 인슐린 저항성과 제 2 형 당뇨병 등의 대사 질환의 연구에 유용하다. 비만 NAFLD, 고지방위한 중요한 위험 인자 중 하나 인 것으로 확인 된 바와 같이, 높은 크로스 다이어트 서양식 고지방식이를 모방 (HFHS)는 마우스에서 비만을 유도하기 위해 사용된다. 이어서, 간 지방증의 정도는 다양한 방법을 사용하여 평가 될 수있다. 먼저, 체중 및 핵 자기 공명 (NMR)와 신체 조성 분석 시간 동안 먹이 지질 축적을 나타낸다. 지방 질량과 근육량은 비 침습적 실시간 방식으로 정량화 할 수있다. 또한, 자기 공명 영상 (MRI)는 전신 및 지방 간 분포 모두를 표시하기 위해 사용된다. 자기 공명 영상의 분석의 계조 신호는 명료 한 의사 컬러 화상으로 변환하고, 강도 수그레이 스케일과 컬러는 헤미 - 정량화이다. 이 기술은 살아있는 동물에서 지방 축적의 측정에 독특한 이점을 제공한다. 둘째, 간의 조직 학적 분석 간 지방증을 결정하기 위해 가장 많이 사용되는 방법이다. 오일 레드 O 염색은 간세포에서 지질 소적의 크기와 위치를 나타내고있다 Hematoxylin &amp; Eosin 염색 얼룩, 예컨대 간세포 형태와 같은 식세포 침윤 학적 정보를 제공한다. 셋째, 클로로포름 / 메탄올을 사용하여 추출 지질 함량 분석 간 지질의 정확한 정량적 측정이다. 총 트리글리세리드 및 콜레스테롤 수준은 생화학 적 방법으로 측정 할 수있다. 중요한 지질 추출 분석 및 오일 레드 O 염색은 또한 유전자 조작 또는 약학 간세포 치료에 사용될 수있다. 본 방법의 장점은 간 steatotic 모델을 생성하는 여러 최적화 방법의 사용이다 그종합적 생체 내 및 시험관 내에서 모두 표현형을 특성화. DIO 마우스 모델은 인간 지방간 질환의 병리 및 대사 표현형 요점을 되풀이 할 수있다. 인간의 다른 대사 매개 변수가 아니라 8로이 모델에 복제 할 수 있습니다. 높은 포도당 플러스 인슐린에 응답 steatotic 간세포 모델의 생성에 유용하고 효율적이며, 많은 비용과 시간이 소요 마우스 연구의 한계를 극복한다. 함께 찍은, 이러한 방법은 영양 과부하시 간 지질 장애와 인슐린 저항성의 연구에 대한 충분하고 필수적이다.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">O.C.T compound</td>
			<td style="width:183px;">SAKURA</td>
			<td style="width:163px;">4583</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Oil Red O</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:163px;">O0625-25G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Infinity Triglycerides kit</td>
			<td style="width:183px;">Fisher&#160;Scientific</td>
			<td style="width:163px;">TR22421</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Infinity Cholesterol kit</td>
			<td style="width:183px;">Fisher&#160;Scientific</td>
			<td style="width:163px;">TR13421</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Collagen type I, Rat tail</td>
			<td style="width:183px;">Millipore</td>
			<td style="width:163px;">08-115</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">DMEM (low glucose)</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:163px;">11885-092</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Penicillin / Streptomycin</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:163px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">FBS</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:163px;">10099141</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">PBS</td>
			<td style="width:183px;">cellgro</td>
			<td style="width:163px;">R21-040-CVR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">HBSS</td>
			<td style="width:183px;">cellgro</td>
			<td style="width:163px;">20-021-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Insulin</td>
			<td style="width:183px;">TOCRIS Bioscience</td>
			<td style="width:163px;">3435</td>
			<td>dissolve in PBS, 1mM for stock</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Glucose</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:163px;">G8270-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Microscope</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:163px;">BX53</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Peristaltic pump</td>
			<td style="width:183px;">Longerpump</td>
			<td style="width:163px;">BT100-2J</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">10cm cell culture dish</td>
			<td style="width:183px;">Corning</td>
			<td style="width:163px;">420167</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">6-well-plate</td>
			<td style="width:183px;">Corning</td>
			<td style="width:163px;">3516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">BCA assay</td>
			<td style="width:183px;">Beyotime</td>
			<td style="width:163px;">P0010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Nuclear Magnetic Resonance</td>
			<td style="width:183px;">Niumag technology</td>
			<td style="width:163px;">MiniQMR23-060H-I</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">High fat high surcose diet(HFHS)</td>
			<td style="width:183px;">Research Diets</td>
			<td style="width:163px;">D12327</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimized analysis of in vivo and in vitro hepatic steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

도 1a에 도시 된 바와 같이, 마우스의 체중은 약 차우 다이어트 공급 군보다 1.5 배이다 HFHS 공급, 16 주 후에 45 ± 1.2 g으로 증가시켰다. 핵 자기 공명 신체 조성은 지방 질량과 쥐의 근육량을 보여주는 분석하면 (그림 1B) 표시됩니다. 몸 전체와 간장의 지방 분포는 MRI에 의해 결정하고, 라이브, 의식 생쥐의 대표적인 의사 컬러 이미지는 그림 1C-...

Watch this Scientific Journal Video about Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis at JoVE.com

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

의 최적화 분석<em&gt; 생체</em&gt; 및<em&gt; 체외</em&gt; 간 지방증

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

optimized-analysis-of-in-vivo-and-in-vitro-hepatic-steatosis

Research

JoVE Journal

Medicine

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

15.8K 조회수.  Chinese Academy of Sciences. Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described. 

비디오: Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

급성 간염 모델 신청 : 시각화와 간장 Microcirculation의 혈류와 산소 소비 분석

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

연구

의학

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

세포 추적을위한 생체 19 F MRI에서

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

마우스의 전신과 간장 혈 역동 학적 파라미터의 모니터링

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

암 약물 과민 반응의 효과를 평가<em&gt; 체외</em&gt; 및<em&gt; 생체</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

자궁 원격 측정은 마우스 수축 압력을 측정하는<em&gt; 생체</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

인간 종양 이종 이식에서 마우스 세포의 고갈은 크게 대상 세포의 다운 스트림 분석을 향상

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

생체 외에서 그리고 Vivo에서 인간 세포, C. 선 충, 쥐에서 Mitophagy의 검출

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

구슈강(GSK) 생체내 및 체외 실험을 위한 과립 준비

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

스크래치 마이그레이션 분석 및 등갈 피부 접기 챔버 에서 생체 외 및 상처 치유의 생체 내 분석

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...