This work is supported by the SFB 824 subprojects C6 and C7 (DFG Sonderforschungsbereich 824), German Research Foundation, Bonn, Germany.

주 : 다음 단계는 우리 기관의 지침과 바바리아, 독일의 지방 정부에 따라 수행되고있다. 지역 동물 관리 및 사용위원회에 의해 결정으로이 프로토콜을 재현하는 모든 시도는 동물의 인도적인 처리를위한 로컬 가이드 라인 준수에해야합니다. 예를 들어, 많은 기관은 멸균 장갑 설치류 수술 중 착용 할 것을 요구하고 있습니다. 또한, 바이러스와 작품은 개인과 환경 안전을위한주의하면서 현지 규정에 따라 수행해야합니다. 관리가 제대로 오염 된 폐기물 처분하고 (고온 가압 멸균 및 제조업체의 지침에 따라 적절한 항 바이러스 소독제로 청소하여, 예) 장비와 작업 공간을 청소주의해야한다. 시작 수술 전에 1. 준비 <ol><li> (고압 증기 멸균에 의해, 즉) 수술기구를 소독. </li><li> appropr에 바이러스를 희석사용되는 특정 바이러스의 독성 래트 균주의 민감성에 따라 달라질 늦었 농도. 주 : 수컷 버팔로 쥐 포성 구내염 바이러스를 사용하는 경우 예를 들어, 우리는 일반적으로 그 모델에서의 최대 허용 투여 량 인 1 ml의 부피에서 107 PFU를 주입. <ol><li> 1 ml를 주사기에 (무균 기술을 사용하여) 바이러스를 미리 기입하고, 주사기와 바늘에서 공기를 제거하기 위해주의하면서 30 G 바늘을 연결합니다. </li></ol></li><li> 필요한 모든 재료와 장비와 수술 공간을 준비하고 소독하는 에탄올로 영역을 스프레이. 거즈 면봉과 면봉 어플리케이터를 가습에 대한 멸균 생리 식염수 (0.9 % NaCl을)이있는 작은 용기를 입력합니다. </li></ol> 쥐 2. 준비 <ol><li> (로컬 동물 관리 및 사용위원회에 의해 지정된대로) 적절한 용량을 30 분에서 전에 수술 절차를 시작하기에, 진통제를 관리 할 수​​ 있습니다. 예를 들면,설피 린의 높은 용량 (100 ~ 200 ㎎ / ㎏)을 관리 할 수​​ 있습니다. </li><li> 흡입 (이소 플루 란) 또는 주사 (즉, 또한 MMF로 알려진 메데 토미의 혼합물 (0.15 ㎎ / ㎏), 미다 졸람 (2 ㎎ / ㎏)과 펜타닐 (0.005 ㎎ / ㎏)) 마취를 사용하여 쥐를 마취. 1-3%에 증발기를 통해 isolflurane 관리 정상 호흡 및 심박수를 유지하기 위해 필요한만큼 조정한다. <ol><li> 진행하기 전에, 쥐가 완전히 진정과 발 패드를 곤란하게하고 반응 관찰하여 통증을 느낄 수없는되어 있는지 확인합니다. </li><li> 마취 동안 건조를 방지하기 위해 동물의 눈 연고를 적용합니다. </li></ol></li><li> 단지 작은 동물 머리 깎기를 사용하여 성기 위 아래로 흉골에서 복부 영역을 면도. 피부를 부상없이 밀착 면도를 달성하기 위해주의하십시오. </li><li> 흡입 마취를 사용하여 적절한 크기의 코 콘에 주둥이를 배치하면, 앞장서고와 뒷다리에 테이프를 사용하여 수술 침대에 쥐를 수정합니다. 가열 패드를 배치래트 아래 체온을 유지한다. 방부제 페인트 알코올 린스 및 응용 하였다 외과 등급의 살균제를 사용하여 복부를 소독. </li></ol> 3. 개복술 <ol><li> 예 # 15로, 작은 만곡 메스를 사용하여, 직접 정중선을 따라 스킨 층의 작은 수직 절개를 만든다. xyphoid 과정까지 절개를 확장합니다. 다음 부드럽게 메스 절개를하기 Adson 집게 쌍으로 올려 근육층을 절개하고 수술 용 가위로 xyphoid 공정까지 절개를 연장한다. 복강의 장기를 천공하거나 xyphoid 프로세스 양쪽의 작은 혈관 손상되지 않도록주의한다. </li><li> 근육과 피부를 확산하고, 전체 복부를 노출 복부에 걸쳐 트랙터를 놓습니다. </li><li> 멸균 생리 식염수와 함께이 7.5 X 7.5 cm 2 거즈 면봉을 적셔 수술의 상단에 하나를 확산도 2a에 도시 된 바와 같이, 개방 및 하단에 하나. </li></ol> 간장 동맥 4. 분리 <ol><li> 부드럽게 복강 밖으로 장과 맹장을 들어 올려 하부 거즈 면봉에 배치, 멸균 생리 식염수에 적신이 젖은면 도포 면봉을 사용. 촉촉한 장기를 유지하고 길에서 그들을 유지하기 위에 거즈를 접으십시오. </li><li> 두 적신면 도포 면봉을 사용하여 부드럽게이 단계에서보기에 가장 지배적 인 로브 될 것입니다 왼쪽 측면 간 엽을 올립니다. 로브가 완전히 위쪽으로 이성을 상실하고 이전에 배치 거즈 면봉의 상단에 휴식을 할 수 있도록 로브의 밑면에있는 섬유 멤브레인을 통해 잘라 봄 가위의 작은 쌍을 사용하여 위쪽 로브 플립합니다. 제자리에 고정하기 위해 엽 위에 거즈 면봉을 접습니다. </li><li> 지금보기에서 가장 전엽 인 다음 로브 (전방 미상)를 계속합니다. 번째로해부 현미경 미세 겸자 E 보조제,이를 분리하고 자유롭게 위쪽으로 대칭되도록하기 위하여 로브를 둘러싼 박막을 해부. 왼쪽 측면 로브와 함께 거즈 면봉 아래 로브를 놓습니다. </li><li> 지금 볼 수 있습니다 후방 꼬리가 로브를 관찰합니다. 완전히 후방 꼬리가 로브를 해부 위쪽과 거즈 안에 함께 이전에 배치 간 로브와 그것을 뒤집어 단계를 반복 4.3. </li><li> 그냥 꼬리가 로브의 원래 위치 아래에, 지금 눈에 보이는 쉽고 강력한 맥동에 의해 인식 될 것입니다 일반적인 간동맥을 찾을 수 있습니다. 해부학 오른쪽에있는 동맥을 이어보기에서 십이지장과 적절한 간 동맥을 가리는 두꺼운 멤브레인 재료를 관찰합니다. 미세 집게를 사용하여이 물질을 들어 올려 조심스럽게이 지역에있는 작은 혈관을 피하기 위해주의하면서 그것을 찢어. 발생할 수있는 출혈을 멈추게하는면 도포 면봉을 사용합니다. <ol><li> ㄱ을 찾습니다멤브레인이 제대로 해부하면 해부 현미경의 도움을 명확히 볼 수 rterial 분기. </li></ol></li><li> 이 좋은, 무 외상성 집게를 사용하여 조심스럽게 일반적인 간 동맥, 위 십이지장 동맥, 그들은 주변의 막 (그림 2B)에서 해방되도록 적절한 간 동맥을 해부하다. </li></ol> 주입을위한 동맥 5. 준비 <ol><li> 십이지장 동맥 아래에 7-0 prolene의 봉합을 전달, 그냥 분기 (적절한 배치를위한 그림 1 참조) 이상, 선단에 영구적 인 끈을 묶어 마이크로 바늘 홀더를 사용합니다. 끝 부분에 남아있는 봉합 재료의 약 2 인치를 허용하고도 2c에서와 같이 가르쳐 동맥을 당겨 바늘 홀더를 고정. </li><li> 일시적으로 혈액의 흐름 (그림 2C)를 차단하는 일반적인 간 동맥에 작은 클램프를 놓습니다. 참고 :이 꼬마 도깨비입니다바늘이 혈관으로부터 제거되면 ortant 다량 출혈을 방지한다. 또한, 동맥 혈류의 차단은 느린 바이러스 관리 및 더 나은 전달 효율성을 수 있습니다. <ol><li> 중요 혈액 흐름이 정지되는 시간 제한 신속하게 작업 - 공통 간동맥 클램핑 5 분 이내에 동맥 주입 이후 결찰을 수행한다. </li></ol></li><li> 가능하다면, 위 십이지장 동맥 날카로운 초점이 정확하게 30 G 바늘을 삽입하기에 충분히 큰 표시되도록, 해부 현미경의 배율을 높일 수 있습니다. 십이지장 동맥의 내강에 주입 주사기의 바늘을 놓고 플런저를 밀어 천천히. 참고 : 바늘의 적절한 배치는 적절한 간동맥을 통해 간으로 직접 흐르는 주입 된 용액에 발생합니다. <ol><li> 전달 효율을 최대화하는 것이 매우 천천히 주입을 수행한다. </li></ol></li> &lt;리&gt; 전체 주입량을 투여 한 후, 천천히 동맥 바늘 후퇴. 참고 : 주사 부위로부터 출혈이 없어야합니다. 출혈이 발생 않는 경우에는, 상기 클램프가 적절하게 배치되지 않고 신속하게 조정되어야 함을 나타낸다. </li></ol> 6. 결산 <ol><li> 십이지장 동맥 아래 두 번째 7-0 prolene의 봉합을 전달하고, 주사 부위 위에 제 2 영구 합자 넥타이. </li><li> 일반적인 간동맥에서 클램프를 제거합니다. 간으로 적절한 간 동맥을 통해 혈액의 흐름을 확인하는 복원되었습니다. <ol><li> 합자의 느슨한 끝을 잘라. </li></ol></li><li> 복강에 창자와 간을 반환하기 전에 더 출혈이 없음을 확인합니다. 부록은 원래의 방향으로 반환주의하십시오. </li><li> 곡선 바늘로 4-0 vicryl 봉합사 재료를 사용하여 별도의 레이어에서 근육과 피부를 봉합. 장소 continuou의 봉합은 약 1mm 떨어져 단단히 잡아 당깁니다. <ol><li> 봉합 단계에서, 진통제 등의 부 프레 노르 핀 등 (0.05 ㎎ / ㎏)을 관리 할 수​​ 있습니다. </li></ol></li><li> 마취에서 쥐를 제거합니다. 이소 플루 란이 사용 된 경우, 래트는 몇 분 안에 의식을 회복한다. MMF는 주사 마취로 사용 된 경우, 마취 효과를 반전되는, Atipamezole (0.75 ㎎ / ㎏), 플루 마제 닐 (0.2 ㎎ / ㎏) 및 날록손 (9.12 ㎎ / ㎏)의 피하 주사로 길항 과 동물이 20 분 안에 의식을 회복 할 수 있습니다. 초기 각성 단계에서 적외선 온난화 램프와 따뜻한 쥐를 유지합니다. <ol><li> 이 흉골 드러 누움을 유지하기 위해 충분한 의식을 회복 할 때까지 무인 동물을 두지 마십시오. </li><li> 완전히 회복 될 때까지 다른 동물의 회사에 수술을 한 동물을 반환하지 않습니다. </li></ol></li><li> 고통의 병적 징후가 수술 후 쥐를 모니터링하고 follow-진통제와 로컬 동물 관리 및 사용위원회가 필요. 예를 들면, 부 프레 노르 핀은 / 0.05 mg을 투여 12 시간마다 kg을 할 수있다. </li></ol>

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The authors have nothing to disclose.

Although direct intratumoral injection is undoubtedly the simplest method to result in efficient tumor transduction of a single tumor nodule, hepatic arterial infusion represents an ideal administration route to target multifocal, orthotopic HCC. This method has proven to be both safe and effective for treating HCC in immune competent rats with oncolytic viruses. Furthermore, since HCC patients are routinely treated by transarterial application of chemoembolization, the method described here is readily translatable to ...

간세포 암 (HCC)은 전 세계적으로 다섯 번째 가장 일반적인 암, 그리고 중요한 건강 문제 1,2하고 암 관련 사망의 세 번째 주요 원인이다. 색전술 (TAE) 또는 화학 색전술 (TACE)을 포함하는 종양 절제술, 또는 그 기다리고 간 이식, 국소 치료를받을 수없는 환자의 표준 완화 의료 3,4으로 적용됩니다. 주변 간 간문맥 5,6-로부터 혈액 공급의 대부분을 수신하면서 이들 치료법은 종양 간 동맥 혈류가 거의 독점적으로 공급되고, 이에 의해 간에서 이중 혈액 공급의 고유 기능을 이용한다. HCC에 대한 설립 치료의 매우 제한된 효능으로 인해 종양 살상 바이러스는 유망한 대체 치료제로 등장했다. JX-594는 최근 Pexa-VEC 이름을 변경하는 티미 딘 키나제 삭제 된 우두 벡터, 과립구-m로 무장이다HCC 7의 II 상 임상 시험이 완료된 acrophage 콜로니 자극 인자 (GM-CSF). 최근 인간 인터페론 - 베타를 발현하는 재조합 수 포성 구내염 바이러스 벡터 (VSV)는 sorafenib-내화 HCC (NCT01628640)에 대한 상 임상 시험을 체결했다. 종양 살상 바이러스가 가까운 환자에서 간세포 암종에 대한 임상 응용 프로그램에 대한 승인을 얻기로 이동함에 따라, 효과적인 투여 경로에 대한 필요성은 다 초점 질병이 분명하다 목표로한다. 전신적 전달로 인해 비효율적 종양 전달에 크게 효과가 있지만, 애플리케이션은 종양 질환 진행에 민감 uninjectable 미세한 병변을 떠나는 주입 종양 치료의 효능을 제한 할 수있다. 우리는 소성을 HCC를 대상으로하는 국소 방식으로 콜리 틱 바이러스 치료를 관리하는 쥐의 간 동맥을 분리하는 방법을 설립했다. 우리는 증명이 안전하고 효과적인 광고에서이 투여 경로 결과면역 능력이 쥐 8-10에서 중요한 생존 연장의 결과로, 다 초점 HCC 결절의 전달을했습니다. 여기서는, 액세스 해부 및 쥐의 간동맥에 주입하는 방법을 설명한다. 절차의 방식은도 1에 도시된다 (이전 9 공개)

<table border="0" cellpadding="0" cellspacing="0" width="597">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:144px;">Veterinary clippers</td>
			<td style="width:157px;">Aesculap</td>
			<td style="width:88px;">GT415</td>
			<td style="width:208px;">Small, cordless trimmer ideal for removing fur from surgical area</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:144px;">Stereomicroscope</td>
			<td style="width:157px;">Zeiss</td>
			<td style="width:88px;">Stemi SV6</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:144px;">30G Needles</td>
			<td style="width:157px;">Braun</td>
			<td align="right" style="width:88px;">4656300</td>
			<td style="width:208px;">30G x &#189;&#8221;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:144px;">1ml syringes</td>
			<td style="width:157px;">Braun</td>
			<td style="width:88px;">9161406V</td>
			<td style="width:208px;">Tuberculin syringe</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:144px;">Disposable scalpel</td>
			<td style="width:157px;">Feather</td>
			<td style="width:88px;">2975#15</td>
			<td style="width:208px;">#15 blade&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Standard surgical scissors</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">14001-13&#160;</td>
			<td style="width:208px;">Sharp/blunt, for opening skin and muscle</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Adson forcep</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">1101-12</td>
			<td style="width:208px;">With teeth, for grasping skin and muscle</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:144px;">Alm retractor</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">17008-07</td>
			<td style="width:208px;">With blunt teeth, for spreading abdominal cavity open during surgery</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Gauze swabs</td>
			<td style="width:157px;">Lohmann &#38; Rauscher</td>
			<td align="right" style="width:88px;">18504</td>
			<td style="width:208px;">7.5 x 7.5 cm, should be autoclaved prior to use</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Cotton-tipped applicator swabs</td>
			<td style="width:157px;">Lohmann &#38; Rauscher</td>
			<td align="right" style="width:88px;">11970</td>
			<td style="width:208px;">Sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Fine-tipped foreceps</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">11063-07</td>
			<td style="width:208px;">0.4mm, angled tip, for dissecting hepatic artery</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Vannas spring scissors</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">91500-09</td>
			<td style="width:208px;">For delicate cutting</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Micro-needle holder</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">12076-12</td>
			<td style="width:208px;">For ligating gastroduodenal artery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:144px;">Needle holder</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">12005-15</td>
			<td style="width:208px;">Tungsten carbide jaws</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:144px;">7-0 Prolene sutures</td>
			<td style="width:157px;">Ethicon</td>
			<td style="width:88px;">8648H</td>
			<td style="width:208px;">Polypropylene suture with curved needle, for ligating gastroduodenal artery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:144px;">4-0 Vicryl sutures</td>
			<td style="width:157px;">Ethicon</td>
			<td style="width:88px;">V3040H</td>
			<td style="width:208px;">With curved needle attached</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Infrared warming lamp</td>
			<td style="width:157px;">Beurer</td>
			<td style="width:88px;">IL11</td>
			<td style="width:208px;">For maintaining body temperature post-operatively</td>
		</tr>
	</tbody>
</table>

transarterial administration of oncolytic viruses for locoregional therapy of orthotopic hcc in rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

경험 (동맥 성공적 해부 주입 한 동물이 수술을 살아되었음을 의미 함)이 거의 100 % 성공률을 달성 할 수있다. 그러나, (기타 종양 부담 정도 기본적인 간 기능) 수술 전에 래트의 상태 분명 수술 개입의 결과에 중요한 역할을한다. 수술 후, 래트를 단독 버퍼해도 바이러스없이 주입하고, 일시적인 체중 감량과 간 효소의 약간의 일시적인 상승이 발생할 것으로 예상 ?...

Watch this Scientific Journal Video about Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats at JoVE.com

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

쥐에서 동소 이식 간세포 암의 국소 치료에 대한 종양 살상 바이러스의 간동맥 관리

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis.  In recent years, oncolytic virotherapies have proven ...

transarterial-administration-oncolytic-viruses-for-locoregional

Research

JoVE Journal

Medicine

9.4K 조회수.  Klinikum rechts der Isar der TU München. Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

비디오: Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Oncolytic 바이러스 살아있는 조직의 전의 VIVO 감염

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

연구

의학

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

구강 편평 세포 암종의 Orthotopic 마우스 모델에서 종양 세포 침략의 다중 광자 이미징

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

암의 생산 및 반응 산소 종의 감지 (ROS)

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

쥐 Orthotopic 작은 창자 이식

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

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 ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

태아 마우스 모델에서 Bioactive 에이전트의 Intratracheal 관리를위한 소설 외과 접근

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

골육종 세포의 광 역학 치료의 세포 독성 효능<em&gt; 체외</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

종양 살상 바이러스의 전 임상 평가에 대한 연구에면 쥐의 취급

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Design, Fabrication, and Administration of the Hand Active Sensation Test (HASTe)

The concept of personalizing neurologic rehabilitation, based on individual impairments, has experienced a recent surge. In parallel, the number of outcome measures of upper extremity motor performance has grown. However, clinicians and researchers lack practical, quantitative measures of the hand&#8217;s natural role as a receptor of the environment. The Hand Active Sensation Test (HASTe), developed by Williams and colleagues in 2006, is a valid and reliable measure of haptic performance. Though not available commercially, the HASTe can be fabricated from inexpensive materials, and it has been used successfully to identify impairments in haptic touch in individuals with stroke. (Williams, 2006). This paper presents the methods of design and fabrication of the HASTe testing kit, as well as a visual screen to be used during administration, and instructions for the tests administration and scoring.

Design, Fabrication, and Administration of the Hand Active Sensation Test HASTe

The Hand Active Sensation Test (HASTe) is a valid and reliable measure of haptic performance, which has been used successfully to identify impaired haptic touch in individuals with stroke. The purpose of this paper is to describe the design, fabrication and administration of the HASTe.

디자인, 및 관리 손의 제작 활동 감각 테스트 (가속)

The concept of personalizing neurologic rehabilitation, based on individual impairments, has experienced a recent surge. In parallel, the number of ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

가속도계를 사용하여 일상 생활에서 상지 성능을 정량화하는 방법

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...