이 연구는 2022 JST 기초 연구(일본 이식 학회)의 지원을 받았습니다.

실험 프로토콜은 교토 대학의 동물 실험 위원회의 승인을 받았습니다. 이 연구는 10주 이상 숙성되고 무게가 25g에서 30g 사이인 C57BL/6 마우스를 사용했으며, 상업용 출처에서 얻었습니다( 재료 표 참조). 모든 동물은 2.5% 이소플루란(기관에서 승인된 프로토콜에 따름)으로 마취하고, 특정 병원체가 없는 조건에서 유지하고, 모든 실험 절차는 교토 대학의 동물 실험에 관한 규정에 따라 수행되었습니다. 연구에 사용되는 적절한 도구는 재료 표 에 나열되어 있으며 그림 1 및 그림 2에 나와 있습니다.
1. 동물 선택
<ol><li>체중이 25-30g인 마우스를 사용하십시오. 참고: mouse liver allografts2 는 일반적으로 주요 조직적합성 복합체 장벽에서 받아들여지지만, 본 연구에서는 냉간허혈 재관류 손상의 상세한 메커니즘에 초점을 맞추기 위해 syngeneic MOLT 모델을 선택했다10. 무게가 25g 미만인 마우스는 얇은 담관에 내부 스텐트를 삽입하는 것이 불가능하기 때문에 권장하지 않습니다. 마찬가지로, 체중이 30g 이상인 쥐는 혈관 주변에 많은 양의 복강 내 지방이 존재하기 때문에 권장되지 않습니다.</li>
</ol>2. 커프 제작
<ol><li>PV 및 IHIVC를 위해 각각 20G 및 16G 정맥 카테터를 준비합니다.</li>
	<li>메스를 사용하여 카테터를 절단하여 커프를 만듭니다. 커프 본체의 길이는 2mm이고 확장 핸들은 1mm여야 합니다(그림 3A). 알림: 손잡이가 너무 크면 커프를 삽입하기 어려울 수 있습니다.</li>
	<li>커프의 표면에 메스 블레이드의 뒷면을 사용하여 실 고정을 위한 얕은 홈을 만듭니다. 알림: 메스 칼날의 뒷면을 커프에 적용할 때 칼날 끝이 약 60도 각도에 있는지 확인하십시오( 그림 3B 참조). 첫 번째 홈을 만든 후 메스 블레이드를 고정하고 커프를 회전시킵니다. 이상적으로는 두 개의 홈이 권장되지만 문제 없이 단일 홈으로 충분할 수 있습니다.</li>
</ol>3. 커프 부착
<ol><li>직사각형 플라스틱 용기에 작은 얼음 조각을 넣고 그 위에 작은 금속 컵(지름 6cm)을 놓습니다. 컵에 장기 보존 액체를 채우고( 재료 표 참조) 그 안에 합성 이식편을 놓습니다.</li>
	<li>면봉을 사용하여 합성 간 이식편(다른 기증자 마우스에서 추출)을 부드럽게 굴려 간장이 위쪽을 향하도록 합니다. 장기 보존액으로 IVC의 내강에 저장된 혈액을 철저히 제거합니다.</li>
	<li>예를 들어 비장 정맥에 부착된 실을 사용하여 커프를 통해 PV를 끼웁니다( 그림 4A 참조).</li>
	<li>커프 핸들이 12시 위치에 있는 상태에서 핸들과 커프를 모두 불독 cl로 고정합니다.amp ( 재료 표 참조), PV 가장자리에서 2-3mm 위치에 위치합니다.</li>
	<li>불독 cl을 고정amp 큰 혈관 집게와 고정 금형을 사용하여 작동 필드를 설정합니다( 그림 4B 참조).</li>
	<li>약 15-20x 배율로 PV 루멘을 주의 깊게 검사합니다. 필요한 경우 물방울을 떨어뜨리면 루멘의 가시성을 높일 수 있습니다.</li>
	<li>PV 루멘을 부드럽게 잡고 커프를 통해 외부화합니다.</li>
	<li>완전히 외부화되면 8-0을 사용하여 이중 결찰로 고정합니다. 커프의 홈을 따라 실크 실(그림 4C,D). 알림: 합자가 느슨하지 않은지 확인하십시오. 또한 합자 점이 너무 크지 않도록 주의하십시오. 커프 삽입을 방해할 수 있습니다. 합자 점은 어느 방향으로나 배치할 수 있습니다.</li>
	<li>PV에 사용된 것과 유사한 절차를 사용하여 커프를 IHIVC에 부착합니다(그림 4E). 알림: 클립을 할 때amp커프로 IHIVC를 끼우면 간 실질에 물지 마십시오. 넥이 PV보다 짧기 때문에 클램핑 위치를 신중하게 결정하십시오.</li>
	<li>IHIVC를 임시로 결찰하기 위해 IHIVC 바닥 주위에 실을 통과시켜 슬립매듭을 만듭니다(그림 5). 이 실은 재관류 후에 제거됩니다.</li>
</ol>4. 문맥 재건
<ol><li>게재문헌2에 기술된 바와 같이 이소플루란 2.5%로 마취를 유도하여 수용 마우스를 마취하고 0.6%로 줄인 후 본래의 간을 추출한다. Dins는 개복술 전에 요오드와 70% 에탄올을 번갈아 가며 수술 부위를 최소 3회 감염시킵니다.</li>
	<li>간엽이 올바른 위치에 있고 커프가 있는 PV에 꼬임과 비틀림이 없는지 확인하십시오.</li>
	<li>Pean 집게를 사용하여 수혜자의 PV 가장자리를 부드럽게 잡고 바이스로 고정합니다. 알림: 이 기사에서 바이스는 테이블에 Pean 집게를 고정하는 데 사용되는 기계 장치를 나타냅니다. 바이스에 대한 자세한 내용은 재료 테이블을 참조하십시오.</li>
	<li>혈관 클램프를 수혜자의 PV에 적용하고 8-0을 전달합니다. 그 주위에 실크 실.</li>
	<li>PV 가장자리에서 약 0.5mm 떨어진 곳에 1/4 원주 단면을 만듭니다(그림 6A). 특수 기구(끝이 잘리고 L자 모양으로 구부러진 27G 바늘)를 사용하여 내강을 통해 식염수를 통과시키면서 구멍을 확대합니다.</li>
	<li>직선 집게를 사용하여 커프 손잡이를 잡고 내강에 삽입합니다.</li>
	<li>완전히 삽입되면 커프를 8-0으로 고정합니다. 비단실. 단일 결찰로 고정에 충분합니다.</li>
	<li>혈관 클램프를 제거하고 간을 재관류합니다.</li>
	<li>Pean 집게를 조심스럽게 풀어주고 문맥 재건을 완료합니다. 전체 PV 재구성 프로세스는 약 5분 정도 소요됩니다.</li>
</ol>5. Infrahepatic 하대정맥 재건술
<ol><li>간엽을 적절하게 배치하고 수혜자 IHIVC 등쪽에 부착된 Pean 겸자를 이식편의 오른쪽 하엽으로 이동하여 바이스로 고정합니다.</li>
	<li>면봉으로 부드럽게 문질러 수혜자의 IVIHC 주변에 남아 있는 간 조직을 제거합니다.</li>
	<li>패스 8-0 수혜자의 IVIHC 주위에 실크 실.</li>
	<li>착용자의 IVIHC 가장자리에서 약 0.5mm 떨어진 곳에 1/4 원주 단면을 만듭니다(그림 6B).</li>
	<li>내강에 생리식염수를 주입한 후 커프를 내강에 삽입합니다. 알림: 목이 짧기 때문에 커프가 PV보다 더 얕게 삽입되는 경우가 많습니다.</li>
	<li>커프를 8-0으로 조심스럽게 고정합니다. 실크 실을 뽑은 다음 혈관 클램프와 Pean 집게를 제거합니다.</li>
</ol>6. 수술 후 관리
<ol><li>수술 후 회복을 용이하게 하기 위해 가열 패드와 보온 램프를 사용하십시오.</li>
	<li>혈압, 심박수, 호흡수, 체온, 음식 및 물 섭취량을 지속적으로 모니터링합니다.</li>
	<li>수술 후 통증을 완화하기 위해 카프로펜의 피하 주사(5mg/kg, 6시간마다 또는 필요할 때까지). 현지 기관 지침을 따르십시오. 참고: 생쥐의 상태가 악화되면 실험은 즉시 중단되고 생쥐는 이산화탄소 흡입으로 안락사됩니다.</li>
	<li>조직 샘플을 수집하려면 마우스를 다시 마취하고 IACUC 승인 방법으로 안락사시킵니다.</li>
</ol>

Strategies for efficient Vascular Anastomosis in Mouse Liver transplantation(마우스 간 이식에서 효율적인 혈관 문합을 위한 전략)

<ol>
	<li>Qian, S. G., Fung, J. J., Demetris, A. V., Ildstad, S. T., Starzl, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+the+mouse.">Orthotopic liver transplantation in the mouse.</a> Transplantation. 52 (3), 562-564 (1991).</li><li>Yokota, S., 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=Orthotopic+mouse+liver+transplantation+to+study+liver+biology+and+allograft+tolerance.">Orthotopic mouse liver transplantation to study liver biology and allograft tolerance.</a> Nat Protoc. 11 (7), 1163-1174 (2016).</li><li>Shen, X. D., 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=Inflammatory+responses+in+a+new+mouse+model+of+prolonged+hepatic+cold+ischemia+followed+by+arterialized+orthotopic+liver+transplantation.">Inflammatory responses in a new mouse model of prolonged hepatic cold ischemia followed by arterialized orthotopic liver transplantation.</a> Liver Transpl. 11 (10), 1273-1281 (2005).</li><li>Kageyama, S., 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=Ischemia-reperfusion+injury+in+allogeneic+liver+transplantation:+A+role+of+CD4+T+cells+in+early+allograft+injury.">Ischemia-reperfusion injury in allogeneic liver transplantation: A role of CD4 T cells in early allograft injury.</a> Transplantation. 105 (9), 1989-1997 (2021).</li><li>Li, T., Hu, Z., Wang, L., Lv, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Details+determining+the+success+in+establishing+a+mouse+orthotopic+liver+transplantation+model.">Details determining the success in establishing a mouse orthotopic liver transplantation model.</a> World J Gastroenterol. 26 (27), 3889-3898 (2020).</li><li>Tian, Y., R&#252;diger, H. A., Jochum, W., Clavien, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+arterialized+and+nonarterialized+orthotopic+liver+transplantation+in+mice:+Prowess+or+relevant+model.">Comparison of arterialized and nonarterialized orthotopic liver transplantation in mice: Prowess or relevant model.</a> Transplantation. 74 (9), 1242-1246 (2002).</li><li>Steger, U., Sawitzki, B., Gassel, A. M., Gassel, H. J., Wood, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+hepatic+rearterialization+on+reperfusion+injury+and+outcome+after+mouse+liver+transplantation.">Impact of hepatic rearterialization on reperfusion injury and outcome after mouse liver transplantation.</a> Transplantation. 76 (2), 327-332 (2003).</li><li>Chen, 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=A+review+of+various+techniques+of+mouse+liver+transplantation.">A review of various techniques of mouse liver transplantation.</a> Transplant Proc. 45 (6), 2517-2521 (2013).</li><li>Kojima, H., 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=T+cell+carcinoembryonic+antigen-related+cell+adhesion+molecule+1-T+cell+immunoglobulin+domain+and+mucin+domain-containing+protein+3+cross+talk+alleviates+liver+transplant+injury+in+mice+and+humans.">T cell carcinoembryonic antigen-related cell adhesion molecule 1-T cell immunoglobulin domain and mucin domain-containing protein 3 cross talk alleviates liver transplant injury in mice and humans.</a> Gastroenterology. 165 (5), 1233-1248 (2023).</li><li>Yokota, S., Yoshida, O., Ono, Y., Geller, D. A., Thomson, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+transplantation+in+the+mouse:+Insights+into+liver+immunobiology,+tissue+injury,+and+allograft+tolerance.">Liver transplantation in the mouse: Insights into liver immunobiology, tissue injury, and allograft tolerance.</a> Liver Transpl. 22 (4), 536-546 (2016).</li></ol>

어떤 저자도 공개해야 할 이해 상충이 없습니다.

혈관 재건술을 배우는 것은 성공적인 MOLT를 달성하는 데 가장 어려운 측면입니다. 커프의 품질은 마우스의 크기가 작다는 점을 감안할 때 재건의 어려움에 큰 영향을 미친다5. 이 문서에서는 커프 준비, 부착 및 재건에 대한 자세한 프로토콜을 제공합니다.
커프 준비 및 연결 2,5에 관한 이전 보고서와 큰 차이점은 ?...

생쥐 기형간 이식(MOLT)은 1991년에 처음 보고된 효과적인 실험 방법입니다1. 유전자 변형 마우스와 다양한 연구 시약을 활용한 이 실험 모델은 온열 및 냉간 허혈 및 재관류 손상을 조사하는 데 중추적인 역할을 했습니다. 그러나 이 모델의 높은 기술적 복잡성은 간 이식을 위한 기초 의학의 개발을 저해하고 있다2. MOLT는 (1) 기증 마우스에서 간을 채취하고, (2) 백테이블 수술, (3) 수혜자에게 간을 이식하는 세 가지 주요 단계를 포함합니다. 이러한 수혜자 시술 중 혈관 문합술이 가장 큰 도전입니다. 상하정맥 문합술은 일반적으로 수작업스티칭 2으로 완료되지만, IHIVC(infrahepatic inferior vena cava) 및 문맥(PV)은 손으로 꿰매는 봉합사 대신 플라스틱 커프를 사용하여 보다 효율적으로 재건할 수 있습니다.
무간주기는 수혜자의 본래 간이 제거된 시점과 이식편 이식 사이의 간격을 의미합니다. 일관된 결과를 보장하려면 무간 시간을 20분 미만으로 제한하는 것이 필수적입니다. 결과적으로, 이 모델을 채택한 연구는 특정 기관에 국한되어 있었다 3,4,5,6,7,8,9. MOLT의 다양한 단계 중에서 원활한 PV 및 IHIVC 재건을 달성하는 것은 무간 시간을 최소화하고 성공적인 이식을 보장하는 데 매우 중요합니다.
PV 및 IVC 재건술은 일반적으로 혈관 커프를 사용하여 수행되는데, 이는 커프 기술이 손으로 꿰매는 봉합사 2,5,8에 비해 혈관 문합을 단순화하기 때문입니다. 혈관 커프 준비 및 커프의 안전한 부착과 관련된 기술은 수혜자 혈관 재건의 복잡성에 상당한 영향을 미칩니다. 우리의 목표는 커프 방법과 관련된 수많은 기술 팁에 대한 자세한 시각적 지침을 제공하여 학습 곡선을 줄이는 것입니다. 이 비디오 클립은 이식자 수술 중 커프를 혈관에 부착하고 PV 및 IHIVC를 재구성하는 방법에 대한 명확한 이해를 제공합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16 G intravenous catheter</td>
			<td>TERUMO</td>
			<td>SR-FF2032</td>
			<td>IHIVC cuff</td>
		</tr>
		<tr>
			<td>20 G intravenous catheter</td>
			<td>TERUMO</td>
			<td>SR-FF1651</td>
			<td>PV cuff</td>
		</tr>
		<tr>
			<td>8-0 braid silk</td>
			<td>Natsume Seisakusho</td>
			<td>CR9-80B2</td>
			<td>8-0 silk</td>
		</tr>
		<tr>
			<td>Belzer UW Cold Storage Solution&#160;</td>
			<td>Astellas</td>
			<td></td>
			<td>Organ preservation fluid</td>
		</tr>
		<tr>
			<td>Bulldog clamp</td>
			<td>B BRAUN</td>
			<td>FB329R</td>
			<td>Bulldog clamp</td>
		</tr>
		<tr>
			<td>C57BL/6 mice&#160;&#160;</td>
			<td>Oriental Bio Service</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane inhalation solution</td>
			<td>Viatris</td>
			<td></td>
			<td>Anesthesic</td>
		</tr>
		<tr>
			<td>Micro Blunted Tips 0.1 mm x 0.06 mm&#160;</td>
			<td>F.S.T</td>
			<td>11253-20</td>
			<td>Straight microforceps</td>
		</tr>
		<tr>
			<td>Micro Serrefine Clamp Applicator with Lock</td>
			<td>F.S.T</td>
			<td>18056-14</td>
			<td>Vessel clip applicator</td>
		</tr>
		<tr>
			<td>Micro Serrefines&#160;</td>
			<td>F.S.T</td>
			<td>18055-4</td>
			<td>Vessel clip</td>
		</tr>
		<tr>
			<td>No.11 Spare Blades</td>
			<td>FEATHER Safety Razor</td>
			<td>11</td>
			<td>Blades</td>
		</tr>
		<tr>
			<td>Ophthalmic scissor, round handle</td>
			<td>B BRAUN</td>
			<td>FD103R</td>
			<td>Microscissor</td>
		</tr>
		<tr>
			<td>Plastic rectangular-shaped container&#160;&#160;</td>
			<td>Daiso</td>
			<td></td>
			<td>10 cm long, 15 cm wide and 6 cm high</td>
		</tr>
		<tr>
			<td>SuperGrip Tips</td>
			<td>F.S.T</td>
			<td>00649-11&#160;</td>
			<td>Curved microforceps</td>
		</tr>
		<tr>
			<td>SZX7</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td>Microscope</td>
		</tr>
	</tbody>
</table>

vascular reconstruction with the cuff technique in mouse orthotopic liver transplantation

마우스 orthotopic 간 이식은 간 허혈 및 재관류 손상의 근본적인 메커니즘을 조사하기 위한 효과적인 방법론입니다. 그러나 기술적 과제는 이 귀중한 실험 모델을 활용하고 이러한 기술을 다음 세대에 전달하는 데 장벽이 됩니다. 이 절차에서 가장 어려운 측면은 문맥(PV), 하대정맥(IHIVC) 및 상하정맥을 포함한 혈관 재건입니다. 봉합사 대신 플라스틱 커프를 사용하면 PV 및 IHIVC 재구성을 보다 원활하게 할 수 있습니다. 혈관은 정맥 카테터로 만든 커프를 이식 혈관의 끝에 부착하고 커프를 수용 혈관에 삽입하여 재건합니다. 가장 중요한 두 가지 측면은 혈관의 내부 내강을 적절하게 시각화하고 과도한 힘을 가하지 않는 것입니다. 우리의 목표는 수혜자 수술에서 커프 기술을 사용하여 혈관 재건에 대한 기술적 개요를 제공하는 것입니다. 커프 기술에 대한 이러한 기술 팁은 미세 외과 의사가 혈관 재건을 촉진하고 연구를 발전시키는 데 도움이 될 것으로 기대됩니다.

PV 재건술은 문맥을 풀어줄 때 비틀림이 없고 간이 균일하게 관류될 때 성공적입니다. 무반수소 시간은 25분을 초과하면 쥐 폐사 위험이 증가하므로 20분 미만이어야 합니다. IHIVC 재건술은 이식편에서 혈액 역류가 없는 경우 성공적인 것으로 간주됩니다.
장기 보존 용액을 사용하여 이식편을 저온에서 1시간 동안 보관하면 재관류 후 6시간에 혈청 알라닌 아미노전이효소 수치?...

Watch this Scientific Journal Video about Author Spotlight: Exploring Cold Ischemia and Warm Reperfusion Injury in Fatty Liver Transplantation at JoVE.com

저자 스포트라이트: 지방간 이식에서 한랭 허혈 및 온열 재관류 손상 탐구

이 프로토콜은 마우스 orthotopic 간 이식에서 커프 기술을 사용하여 혈관 재건을 위한 기술 정보를 제공합니다.

Mouse Orthotopic Liver Transplantation에서 Cuff Technique을 사용한 혈관 재건

Mouse orthotopic liver transplantation is an effective methodology for investigating the underlying mechanisms of liver ischemia and reperfusion injury. ...

우리는 간 이식, 특히 한랭 허혈 및 온열 재관류 손상에 대한 연구에 중점을 둡니다. 이를 위한 연구 도구로 마우스 간 이식 모델을 사용합니다. 이 기술은 어렵기 때문에 배우고 일관된 결과를 얻는 데 오랜 시간이 걸릴 수 있습니다.앞으로는 지방간 이식에 대한 연구를 진행하고 싶습니다. 이는 임상적 간 이식에서 여전히 극복해야 할 문제입니다.

vascular-reconstruction-with-cuff-technique-mouse-orthotopic-liver

Research

JoVE Journal

Medicine

Vascular Reconstruction with the Cuff Technique in Mouse Orthotopic Liver Transplantation

1.1K 조회수.  Kyoto University. 우리는 간 이식, 특히 한랭 허혈 및 온열 재관류 손상에 대한 연구에 중점을 둡니다. 이를 위한 연구 도구로 마우스 간 이식 모델을 사용합니다. 이 기술은 어렵기 때문에 배우고 일관된 결과를 얻는 데 오랜 시간이 걸릴 수 있습니다.앞으로는 지방간 이식에 대한 연구를 진행하고 싶습니다. 이는 임상적 간 이식에서 여전히 극복해야 할 문제입니다.

비디오: 저자 스포트라이트: 지방간 이식에서 한랭 허혈 및 온열 재관류 손상 탐구

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

쥐에게 Orthotopic 간 이식

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

연구

의학

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

혈관 질병의 연구에 대한 마우스의 Orthotopic 대동맥 이식

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

간 간선 재건과 부분 Orthotopic 간 이식의 쥐 모델에 대한 외과 절차

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

이소성 보조 쥐 간 이식 급성 간 장애에 포털 정맥 Arterialization를 유량 조절

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

마우스에서 소성을 뒷다리 이식

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

대동맥 이식-교육용 비디오의 마우스 모델에서 소매 기법을 사용 하 여

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

췌 장 이식 수정된 팔목 기술을 사용 하 여 마우스 모델

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

Orthotopic의 쥐 모델 간 이식 Suprahepatic 베 나 정 맥 재건에 대 한 새로운 자석 문 합 기술을 사용 하 여

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

마우스 자궁경부 이종 심장 이식 모델을 위한 수정된 커프 기술

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...

마우스의 간 이식 및 동종이식 개통성 분석

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

저자 스포트라이트: 쥐 모델에서 MicroCT 혈관 조영술을 통한 이식 연구 강화 

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

Microcomputed Tomography 혈관 조영술을 활용한 마우스의 성공적인 Orthotopic 간 이식

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...