박사 칸 NIH/NICHD K12HD052892, 알파-1 재단, 힐만 재단, 그리고 피츠버그 간 연구 센터에서 보조금 지원을 인정 한다. 박사 Michalopoulos는 NIH/NIDDK P01DK096990에서 보조금 지원을 인정합니다. 앤 오 어의 우수 하 고 관대 한 기술 지원 부탁 드립니다. 우리는 또한 이식/설치류 미세 실험실에 대 한 액세스를 제공 하기 위한 박사 데이비드 A. 겔러에 감사입니다.

모든 절차는 기관 동물 관리 및 사용 위원회 피츠버그 대학교의 윤리 지침에 따라 실시 했다.
1. 기본 기술 및 공통 절차
<ol><li>압력솥 사용 하기 전에 모든 microsurgical 장비.</li>
	<li>마우스 마 취에 의해 살해를 방지 하려면 신중 하 게 그것의 호흡 패턴을 확인 합니다.</li>
	<li>터치 기관 및 내장 기관 손상 및 출혈을 일으키는 방지 하기 위해 가능한 한 작은. 젖은 면봉 이나 거 즈 스폰지 출혈 포인트를 누릅니다.</li>
	<li>젖은 면봉을 사용 하 여 장기의 부드러운 조작. 3.5 × 3.5 cm 및 6.0 × 6.0 cm. 작은 하나를 사용 하 여 전체 간 안전 거리를, 젖은, 비-짠 거 즈 스폰지 2 개 크기에서 사용 된다. 더 큰 하나를 사용 하 여 크고 작은 창 자 하.</li>
</ol>2. presurgical 준비
<ol><li>남성 c57/b6 쥐, 부분적 담 관 결 찰에 대 한 (때 몸 무게는 약 20-35 g) 나이의 8-12 주를 사용 합니다.</li>
	<li>마 취의 유도까지 쥐 물과 음식에 대 한 무료 액세스를 허용 합니다.</li>
</ol>3. 부분 담 관 결 찰 수술
<ol><li>1.5-2.0 %isoflurane 및 1 L/min 산소의 혼합으로 유도 챔버에서 동물을 넣어 마우스를 anesthetize. 호흡 속도 관찰 하 여 동물을 신중 하 게 모니터링 합니다. 동물의 호흡 속도 초당 한 호흡, 마우스 잘 마 취입니다. 앞 다리 철수 반사는 더 빠르게 사라집니다, 이후 철수 반사의 완전 한 부재를 확인 뿐만 아니라 뒷 다리 발 핀치. 눈 각 막 궤 양을 방지 하기 위해 안과 윤 활 에이전트와 기름을 바른 다 한다. 참고 진통제의 그 첫 번째 복용량 통증 수용 체의 자극을 방지 하기 위해 수술 전 주어질 수 있다.</li>
	<li>헤어 클리퍼 스로 복 벽을 면도 하 고 수술 현미경 수술 테이블에 마우스를 놓습니다. 테이프와 함께 테이블에 모든 사지를 수정 합니다. Isoflurane 흡입을 사용 하 여 전신 마 취 아래 마우스를 유지.<ol>
<li>Isoflurane 기화 기를 조정 하 고 유도 2.0-3.0 %isoflurane 사용 합니다.</li>
	</ol>
</li>
	<li>복 벽을 소독 멸 균 면봉을 사용 하 여, 먼저 0.3-0.5 mL povidone-요오드 응용 몸에 붙이기, 70% 에탄올에 의해 따라. 동물 살 균 드 레이프와 함께 처리 되어야 합니다.</li>
	<li>긴 중간 복 부 절 개를 고 칼에서 Adson 집게와가 위를 사용 하 여 액을 확인 합니다. 복 부 절 개 후, 줄일 isoflurane 0.8-1.0% 유지 보수에 대 한.</li>
	<li>마이크로-견인을 사용 하 여 복 부 구멍을 노출 합니다. 모기 겸 자는 칼 잡고 하 고 마우스의 머리 쪽으로 철회. 모기 겸 자 부드러운 흙을 사용 하 여 장소를 수정 했다.</li>
	<li>따뜻하게 (37 ° C)의 1-2 mL를 주입 멸 균된 인산 염 버퍼 식 염 수 (PBS) 복 부 장기 부상 방지 하려면 복 부 구멍으로. 젖은 면봉으로 소장 누른 부드럽게 마우스의 머리 쪽으로 그들을 취소 합니다. 작은 창 자, 작은 창 자 (그림 1A) 부상 피하려고 retroperitoneum 사이 인 대를 잘라.</li>
	<li>랩, 비-짠 젖은 거 즈 스폰지와 크고 작은 창 자. 복 부 구멍의 꼬리 쪽에 넣어. 멸 균 거 즈에 싸서 내장 오염을 피하기 위하여 메 마른 draped 필드에 배치할 수 있습니다.</li>
	<li>부드럽게 간 hilum와 성질과 십이지 장 인 대 (그림 1B)에 작은 젖은 비 짠 거 즈 스폰지를 사용 하 여 전체 간으로는 칼을 철회.</li>
	<li>간장 hilum의 것인지 확인 합니다. 일반적인 담 관은 포털 정 맥의 복 부 측에 있습니다. 오른쪽 및 왼쪽된 간 담 관 간 hilum에서 주요 담 즙 합류에 드레인의 위치를 확인 합니다.</li>
	<li>왼쪽된 간 담 즙 덕트는 왼쪽된 엽 빼낸 식별 하 고 10-0 나일론 봉합 봉합 바늘 (그림 2A)의 무딘 사이드를 사용 하 여 함께 그것을 둘러싸 자. 문맥, 간 동맥, 및 간 표면 부상 하지 마십시오.</li>
	<li>10-0 나일론 왼쪽된 담 관 선 (그림 2B)입니다.</li>
	<li>젖은 면봉을 사용 하 여 크고 작은 창 자 작업 이전에 같은 위치에 배치 합니다.</li>
	<li>장 비틀림 및 내부 복 부 출혈, 확인 후 근육과 4-0 vicryl와 2 개의 층에 피부를 닫습니다. 꼰된 봉합 (Vicryl) 피부 폐쇄를 위해 사용 되었다 그러나 monofilament 감염의 가능성을 줄이기 위해 선호 된다. 참고: 액, 하 칼에서 지속적인된 봉합 하 여 근 막 닫고 액에서 칼에 계속된 봉합 하 여 피부를 닫습니다.</li>
	<li>절 개를 닫은 후 마 취를 중지 합니다.</li>
</ol>4. 수술 후 치료 및 후속
<ol><li>그냥 수술 후, 마우스의 등 쪽 피부를 꼬 집 고 주사 Buprenex (0.1 mg/kg) 및 Cefazolin (100mg/kg), 피하 주입에 의해 Buprenex 모든 12 h 3 일, 그리고 Cefazolin에 대 한 모든 24 h 3 일 뒤에 각각11,12 ,13. 참고 진통제의 그 첫 번째 복용량 통증 수용 체의 자극을 방지 하기 위해 수술 전 주어질 수 있다. Cefazolin 및 다른 요정 요원 항생제만 이식 연구에 사용 된 같은 면역 억제 동물에 대 한 필요 하다. 참고: 경우에 수술을 성공적으로 수행 하 고, 마우스 일반적으로 복구 30 분 이내 쥐의 활동 거의 수술 전 상태와 동일 있을 것입니다.</li>
	<li>고통의 어떤 표시 든 지를 위한 매일 쥐를 확인 하십시오.<ol>
<li>PBDL 후 간 및 정의 된 시간 점 (즉, 7, 14, 21 일)에서 다른 조직 샘플을 조달 전신 마 취하에 자 궁 경부 전위에 의해 쥐를 희생.</li>
	</ol>
</li>
</ol>

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저자 아무 공개 있다.

CBDL cholestasis 폐쇄성에 대 한 가장 일반적인 실험 기술 이지만, 그것은 설치류에 여러 과제를 제시할 수 있습니다. 끝점을 달성 하는 데 필요한 수술 일 수에 따라 동물 중요 한 병 적 상태와 사망률 진행으로 경험할 수 있습니다. 조직 상해 및 담 즙 괴 사 정도 간 질병의 비정상적인 간 실질 및 기준선에서 기능을 설명 하는 몇 가지 유전자 변형 마우스 모델에서 작업할 때 더 악화 됩니다. CBDL에 비?...

급성 간 손상 및 수리, 부분 hepatectomy 등 일반적인 담 관 결 찰 (cBDL)의 외과 모델 간 재생 pathobiology1,2를 공부 하 고 설치류에 수십 년 동안 사용 되었습니다. CBDL에서 일반적인 담 관 (간에 서 생산 하는 모든 담 즙을 빼낸)는 출혈, 폐쇄성 cholestasis, 염증과 섬유 증 수술, 간 경 변3최종 진행 후 첫 2-3 주에 결과. 담 즙 덕트 확산, transdifferentiation, 및 주민 간 조상 세포의 유도 또한 cBDL4,5의 기능. 비록 cBDL 보통 잘 재현할 결과 야생-타입 마우스와 정상적인 간 은6,,78그 긴장 특징이 있는 폐쇄성 cholangiopathy의 특정 메커니즘에 절차는 높은 사망률 및 사망 시간이 지남에 담 즙 방해의 예상 보다 더 많은 동물을 요구 하는 간 질환의 일부 유전자 변형 마우스 모델에 기술적으로 요구 될 수 있습니다. 이 기술은 또한 유전자 변형 생쥐 기본 간 질병, 그렇지 않으면 장기간된 시간 과정 실험 cBDL 다음의 스트레스를 용납 하지 않을 수 있습니다 공부에 대 한 도움이 됩니다.
pBDL는 이러한 문제 들을 극복할 수 있도록 적당 한 대안을 제공할 수 있습니다. 초기 pBDL 로컬 행동 및 조직의 염증9의 중재자를 구별 하는 목적으로 야생-타입 쥐에 있는 학문. PBDL에서 일반적인 담 관 위에 좌우 간 담 관의 담 즙 주 합류는 hilum에서 신중 하 게 시각 이다 고만 왼쪽된 간 담 즙 덕트 지역화 된 폐쇄성 cholestasis 생성 하 출혈. pBDL은 cBDL에 여러 이점을 제공합니다. PBDL에서 unligated 오른쪽 엽 합자 왼쪽된 엽, 영양 상태 등 요소를 순환 같은 조직의 효과를 받게에 대 한 동일 하 게 일치 하는 내부 통제 역할을 합니다. 조직 프로-염증 성 중재자 존재 했지만, 오 사와 외 적은 섬유 증 및 unligated 돌출부에 괴 사를 관찰 하지만 염증 성 세포와 변형 성장 인자-β 표현의 합자 엽9증가.
더 최근에, pBDL는 유지 담 즙10간 parenchymal 효과 연구 재정의 되었다. 내부 제어 (unligated "가짜") 및 실험적인 그룹 같은 동물을 사용 하 여 pBDL는 효과적으로 차례 차례로 더 비용 효율적인 실험 당 필요한 동물의 수를 반. 이상적인 시간 과정 실험 (≥2 주) 합자 엽에서 달성 될 수 있다 그래야 쥐 훨씬, pBDL의 효과 용납. 가장 중요 한 것은, pBDL의 폐쇄성 cholestasis 직접 intrahepatic 효과 구분할 수 있습니다 하 고 합자 엽, unligated 제어 엽에 비해 담 즙 구성 요소를 유지. 전반적으로, pBDL 간에서 지역화 cholestasis의 엽 특정 효과 공부 하는 매력적인 방법입니다.

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			<td height="21" style="height:21px;width:343px;">Microscope-Leica Wild M650, 6&#8211;40&#215; magnification</td>
			<td style="width:336px;">Leica Microsystems</td>
			<td style="width:195px;">n/a</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tec 3 isoflurane funnel fill vaporizer</td>
			<td>General Anesthetic Services</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stevens Tenotomy Scissors</td>
			<td>Accurate Surgical &#38; Scientific Instruments Corporation</td>
			<td>ASSI.9193</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adson Forceps</td>
			<td>Fine science tools</td>
			<td>11006-12</td>
			<td></td>
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			<td height="21" style="height:21px;">Mosquito classic delicate hemostatic forceps</td>
			<td>Codman</td>
			<td>30-4472</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Micro-retractor</td>
			<td>Murdock; Roboz Surgical Instrument</td>
			<td>RS-6550</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Nonwoven gauze sponges</td>
			<td>Fisherband</td>
			<td>870-PC-DBL</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Puritan 6&#8221; Small Cotton Swab w/Wooden Handle</td>
			<td>Puritan Medical Products Company</td>
			<td>868-WCS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont SS Forceps - Standard Tips/Straight/Inox/13.5cm</td>
			<td>Fine science tools</td>
			<td>11203-23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont SS Forceps - Standard Tips/Angled 45&#176;/Inox/13.5cm</td>
			<td>Fine science tools</td>
			<td>11203-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas Spring Scissors - 3mm Cutting Edge</td>
			<td>Fine science tools</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microneedle holder</td>
			<td>Aesculap Surgical Instruments</td>
			<td>FD231R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro AROSuture, sterile 10-0 nylon suture, 70 &#181;m, TAP points</td>
			<td>AROSurgical Instruments Corporation</td>
			<td>T4A10N07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Vicryl</td>
			<td>Ethicon</td>
			<td>J662H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-ml Syringe</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Falcon tissue culture dish, 60 &#215; 15 mm</td>
			<td>Corning</td>
			<td>353002</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:343px;">Isoflurane, United States Pharmacopeia (USP) liquid for inhalation, 250 ml</td>
			<td>Piramal Healthcare</td>
			<td>NDC 66794-017-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenex injectable (buprenorphine hydrochloride)</td>
			<td>Reckitt Benckiser Pharmaceuticals</td>
			<td>NDC 12496-0757-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cefazolin for injection , USP</td>
			<td>APP Pharmaceuticals</td>
			<td>NDC 63323-238-61</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVP scrub solution (povidone&#8211;iodine 7.5%)</td>
			<td>Medline</td>
			<td>NDC 12496-0757-1</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Phosphate Buffered Saline Without Calcium or Magnesium</td>
			<td style="width:336px;">LONZA</td>
			<td style="width:195px;">17-516F</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sirius Red</td>
			<td style="width:336px;">Sigma</td>
			<td style="width:195px;">365548</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Hematolxylin</td>
			<td style="width:336px;">Fisher (Richard-Allan Scientific)</td>
			<td style="width:195px;">22-050-111 (7211)</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Eosin</td>
			<td style="width:336px;">Anatech (Fisher)</td>
			<td style="width:195px;">832 (NC9686037)</td>
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			<td height="21" style="height:21px;width:343px;">Formalin</td>
			<td style="width:336px;">Fisher</td>
			<td style="width:195px;">SF100-20</td>
			<td></td>
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partial bile duct ligation in the mouse: a controlled model of localized obstructive cholestasis

설치류, 일반적인 담 관의 완전 한 담 관 결 찰 (cBDL)에서는 폐쇄성 cholestasis 및 담 즙 덕트 확산 연구 설립된 수술 방법입니다. 그러나, 장기 실험 증가 병 적 상태와 사망률 이어질 수 있습니다. 선택 마우스 기본 간 질환과 긴장, 의미 있는 비교 동물 손실 및 비용을 줄일 수 있는 간, 단일 엽의 결 찰으로도 만들 수 있습니다. 여기, 우리 왼쪽된 엽만 하지 나머지 돌출부에 담 즙 방해를 일으키는 부분 담 관 결 찰 (pBDL)는 간 담 즙 덕트 왼쪽된만 출혈은, 마우스에서 설명 합니다. 주의 microsurgical 기법, pBDL 실험 수 비용, 같은 동물에 동일한 조건을 복종 때 unligated 엽 합자 돌출부를 내부 통제 역할 때문. CBDL, 달리 별도 운영 하는 가짜 제어 그룹 필요 하지 않습니다. pBDL은 cholestasis 및 기타 유지 담 즙 구성 요소 조직의 효과 대 지역화 직접 비교에 매우 유용 합니다. pBDL 또한 메커니즘 약물 및 세포 마이그레이션에 관련 된 조사를 새로운 방법으로 repurposed 수 있습니다.

이 전형적인 실험에 대 한 pBDL (A1AT) 알파-1 antitrypsin 결핍에서 발견 인간의 간 질환 정리는 triplicate 피 즈 유전자 변형 생쥐에서 수행 되었다. 피 즈 마우스 overexpresses transgene 조각의 여러 genomic DNA의 돌연변이 인간 A1AT 유전자, introns 및 업스트림 및 다운스트림 영역14, 측면의 ~ 2 kilobases 발기인의 코딩 영역을 포함 하는 구성 된 15....

Watch this Scientific Journal Video about Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis at JoVE.com

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

여기, 선물이 간 부상 및 설치류에서 중생의 외과 모델로 부분 담 관 결 찰.

마우스에 부분적 담 관 결 찰: 지역화 된 폐쇄성 Cholestasis의 제어 모델

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

partial-bile-duct-ligation-mouse-controlled-model-localized

Research

JoVE Journal

Biology

15.4K 조회수.  Jichi Medical University. 여기, 선물이 간 부상 및 설치류에서 중생의 외과 모델로 부분 담 관 결 찰.

비디오: Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

Microfluidic 프로브 : 운영 및 현지화된 표면 처리에 사용

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

연구

생물학

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

내사 - 결합 : 심근 경색의 Murine 모델

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal model of the condition and an important correlate for human Meniere's disease. In 1965, Robert Kimura and Harold Schuknecht first described an intradural approach for obstruction of the endolymphatic duct (Kimura 1965). Although effective, this technique, which requires penetration of the brain's protective covering, incurred an undesirable level of morbidity and mortality in the animal subjects. Consequently, Andrews and Bohmer developed an extradural approach, which predictably produces fewer of the complications associated with central nervous system (CNS) penetration.(Andrews and Bohmer 1989) 
The extradural approach described here first requires a midline incision in the region of the occiput to expose the underlying muscular layer. We operate only on the right side. After appropriate retraction of the overlying tissue, a horizontal incision is made into the musculature of the right occiput to expose the right temporo-occipital suture line. The bone immediately inferio-lateral the suture line (Fig 1) is then drilled with an otologic drill until the sigmoid sinus becomes visible. Medial retraction of the sigmoid sinus reveals the operculum of the endolymphatic duct, which houses the endolymphatic sac. Drilling medial to the operculum into the area of the endolymphatic sac reveals the endolymphatic duct, which is then packed with bone wax to produce obstruction and ultimately ELH. 
In the following weeks, the animal will demonstrate the progressive, fluctuating hearing loss and histologic evidence of ELH.

This video shows how to surgically obstruct the guinea pig's endolymphatic duct to produce endolymphatic hydrops.

Endolymphatic 덕트의 말살로 Endolymphatic의 Hydrops의 외과 유도

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

생체내 이미징 및 난소암의 Orthotopic 마우스 모델에서 치료 트리 트먼트

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

포유류의 세포에서 Endoplasmic 소포체 현지화 mRNAs의 시각화

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

마우스 유선 화 된 약물 전달을위한 췌관 주입

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

사용<em&gt; 예쁜 꼬마 선충</em&gt; 다세포 생물의 단백질 항상성을 연구하는 모델 시스템으로

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

마우스 모델의 유효성 검사는 세포 고유의 방식으로 LINC 단지를 방해

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

장 부분 방해의 마우스 모델

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

체외에서 담즙 관내 형성에서 세포골격의 역할을 연구하기 위해 1 차 마우스 간세포의 격리 및 3D 콜라겐 샌드위치 문화

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...