이 작품은 부분적으로 손 파일럿 수상 및 NIH / NIAMS 1K01AR068386-01 (AEL에) 및 NIAMS / NIH P30AR061307의 수술에 대한 미국 사회에 의해 지원되었다.

로체스터 대학의 동물 연구에 대학위원회는 모든 동물 실험을 승인했다. 열-12 주 된 여성 C57BL은 / 6J 마우스를 사용 하였다. 굴근 힘줄 수술에 대한 동물의 1. 준비 (~ 15 분) <ol><li> 오토 클레이브 수술기구는 소독을 통해 멸균 장갑을 착용하고, 무균 조작 필드를 유지합니다. </li><li> 체중에 대응하는 양의 케타민 (80 ㎎ / ㎏)과 자일 라진 (10 ㎎ / ㎏)을 복강 내 주사 (IP)를 통해 마우스를 마취. 발가락 - 핀치 반사의 부재를 통해 진정의 깊이를 확인합니다. </li><li> 피하 주사를 통해 선제 진통제 (프레 노르 핀, 0.05-0.1 ㎎ / ㎏)을 관리 할 수​​ 있습니다. 건조에서 눈을 방지하기 위해 안과 연고를 적용합니다. </li><li> 전체 뒷다리에 모피를 클리핑하여 수술 부위를 준비합니다. 포비돈 요오드 다시 70 % 에탄올 다음 포비돈 요오드로 순차, 세정하여 피부를 소독하고. </li></ol><ol><li> 종아리의 내측 측면에서 표면적으로 보이는 굴곡 심지 longus (FDL) 건을 찾을 수 있습니다. 메스를 사용하여 건을 노출 마이크로 가위로 피부에 작은 0.5 cm의 절개를합니다. </li><li> 주변 조직에서 FDL 건을 분리하고 myotendinous 접합에 그것을 추적 집게를 사용합니다. 후방 경골 동맥 않도록주의하면서 그것을 풀어 봄 가위로이 접합에 힘줄을 잘라. </li><li> 5-0 나일론 봉합사로 피부를 닫습니다. 참고 : FDL의 절개 및 수리 (2.4 단계) 실체 현미경을 사용하여 수행해야합니다. </li><li> 봄, 마이크로 가위를 사용하여 뒷발의 외측면에 걸쳐 3mm의 절개를합니다. 부드럽게 조직 손상을 최소화하고, FDL 건을 확인해야하고, 집게로 주변의 연부 조직과 근육을 수축. <ol><li> 조심스럽게 FDL 건 올리고 완전히 마일을 사용하여 가로로 쪼개다CRO 가위. 주기적 식염수로 습윤함으로써 힘줄의 건조를 방지 8-0 봉합 변형 케슬 패턴 힘줄 함께 FDL의 단부 봉합사. </li><li> 힘줄을 통해 부드러운 조직과 근육을 교체 한 후 5-0 나일론 봉합사와 절개를 닫습니다. </li></ol></li><li> 마취에서 회복 될 때까지 체온을 유지하기 위해, 37 ° C에 슬라이드 따뜻한 설정에 마우스를 놓습니다. </li><li> 수술 후 증가 가창과 주름 장식 모피를 포함하여 손상이나 감염의 징후 마우스를 모니터링합니다. 마우스까지 매 12 시간이 더 이상 통증이나 고통의 흔적을 보여주지 진통제로 부 프레 노르 핀 (50 μL / 마우스)를 주입한다. </li><li> 마우스는 다음 단계를 계속하기 전에 십일 수술 후 기간 동안 치료를 할 수 있습니다. 참고 : 세포가 언제든지 포인트 수술 후 수확 할 수있다. 다음 10 일이 때 조직의 상대적으로 높은 세포 수를 소정 이러한 대표적인 실험을 위해 선택된다. </li></ol> 3. Labeli사이클링 세포 NG (~ 10 분) <ol><li> EDU (5- 에틴 2&#39;deoxyuridine) 용액을 준비한다 (멸균 인산 완충 식염수에 10 ㎎ / ㎖를 [PBS]은 + 1 % 디메틸 술폭 시드 [DMSO] 용해도를 증가시키기 위해). </li><li> IP 주입을 통해 희생하기 전에 24 시간을 100 ㎕를 EDU (50 ㎎ / ㎏)와 마우스를 주입한다. </li></ol> 세포학 원심 분리 4. 수확 셀 (2.5 시간 ~ 30 분 실습 시간) <ol><li> PBS에서 3 ㎎ / ㎖의 콜라게나 제 I을 준비합니다. </li><li> 이하 3 L / min의 유량으로 이산화탄소 질식 통해 마우스를 안락사 및 보조 수단으​​로 경추 탈구를 수행한다. </li><li> 위의 단계 2.4을 사용하여 수리 건을 찾은 마이크로 가위로 부상 사이트의 양쪽에 약 2 mm로 절단하여 힘줄 조직을 격리 할 것. <ol><li> 3 ㎎ / ㎖ 콜라게나 제 1 mL를 페트리 접시에 메스 말하다 조직은 다음 조직을 분쇄하기 위해 18 G 바늘을 여러 번을 통해 혼합물을 전달합니다. 1.5 ㎖의 원심 분리기 튜브로 혼합물을 수집합니다. </li&gt; <li> 진탕하면서 37 ℃에서 1 시간 동안 건 조직 다이제스트. </li><li> 스핀 다운 조직 (5 분 300 XG), 흡 콜라게나 후 수회 피펫을 상하로 PBS 500 ㎕의 3 % 소 혈청 알부민 (BSA)과 세포 / 조직을 재현 탁. </li><li> 70 μm의 셀 스트레이너를 통해 필터 세포 현탁액 파편과 힘줄의 큰 조각, 그리고 37 ° C 배양기에서 따로 설정을 제거합니다. </li><li> 또 다른 1.5 ㎖의 원심 분리기 튜브에 힘줄 조직을 배치하고 최대 피펫 팅, 1 ml의 콜라겐 용액으로 새로 고침 아래로 혼합. </li><li> 진탕 37 ° C에서 조직에게 또 다른 시간을 품어. </li><li> 소화 건 조직 (5 분 300 XG)를 스핀 다운, 다음, 최대 피펫 팅에 의해 여러 번 아래로 PBS 500 ㎕의 3 % BSA에 재현 탁. </li><li> , 70 μm의 셀 스트레이너를 통해 세포 현탁액을 필터 4.3.3에 고립 된 서스펜션과 결합, 다음 혈구와 세포를 계산합니다. </li><li> 다음 3-6로 재현 탁, PBS에서 3 % BSA와 함께 다시 한 번 세포를 씻으× 10 1백분의 4 μL. </li></ol></li><li> 양으로 하전 된 슬라이드에 세포 검사 깔때기를 연결하고 슬라이드 캐리어에 고정. 세포학 원심 분리기로 전체 장치를 삽입하고 깔때기에 100 ㎕의 세포 현탁액을 추가합니다. 5 분 300 XG에 원심 분리기는 슬라이드에 세포를 분산합니다. </li></ol> 5. 면역 세포 화학이 EDU를 감지하기 (2 시간 ~ 45 분 실습 시간) 주 : 모든 인큐베이션 단계는 형광체의 광표백을 제한하는 암에서 수행되어야한다. <ol><li> 단계에 따라 필요한 용액을 최소화 소수성 장벽 펜, 원형 셀, 즉시 실온에서 15 분간 PBS 150 ㎕의 3 % 파라 포름 알데히드 (RT)로 고정한다. <ol><li> PBS 150 ㎕의 3 % BSA 5 분 동안 두 번 씻으십시오. </li><li> RT에서 PBS 20 분에서 0.5 % 트리톤-X 100 Permeabilize 하시려면 세포. </li><li> 5.1.1에서와 같이 반복 세척 단계. </li></ol></li><li> 에듀 반응 cockt 준비괴롭히다 킷 지시하게 사용하기 전에 최소 30 이상에 따라. <ol><li> 각 슬라이드에 150 μl의 칵테일을 추가 RT에서 30 분을 품어. </li><li> 5.1.1에서와 같이 반복 세척 단계. </li></ol></li><li> RT에서 PBS 30 분에서 2000 : 핵 Counterstain과 Hoechst33342 1 희석 150 μL와 함께 품어. <ol><li> PBS 1 배 150 ㎕를 5 분 동안 두 번 씻으십시오. </li></ol></li><li> 천천히, 1-2 세포에 매체를 장착 안티 - 페이드 방울 거품을 방지하기 위해 낮은 커버 슬립을 추가합니다. <ol><li> 실온에서 어둠 속에서 밤새 치료 매체를 장착 할 수 있습니다. </li><li> 슬라이드에 커버 슬립을 밀봉하기 위해 명확한 매니큐어를 사용합니다. </li></ol></li><li> DAPI (Hoeschst33342)를 검출 할 수 여기 / 방출 필터가 장착 된 형광 현미경을 사용하여 40X 배율에서 이미지 슬라이드, 텍사스 레드 (알렉사 플 루어 594). </li></ol>

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The authors declare that they have no competing financial interests

완전 절개와 굴곡 심지 longus 건 수리의 쥐 모델에 대한 수술은이 연구에서 제시된다. 또한 세포학 원심 작은 세포 집단을 농축하는 새로운 애플리케이션은 굴근 힘줄 치유 동안에 셀룰러 환경의 면역 세포 화학적 정량 분석​​을 가능하게 입증된다. 굴근 힘줄이 수리 모델 녹아웃 모델을 사용하여 치료 과정에서 변경 또는 약리학 적 개입을 평가하기 위해 사용될 수있는 재현성 치유 반응을 보?...

굴근 팔뚝의 근육과 디지털 시스와 협력의 손 작업의 굴근 힘줄은 숫자의 굴곡과 손 잡고 기능을 활성화합니다. 굴근 힘줄은 손의 손바닥 측면을 따라 실행; 이 상대적으로 피상적 인 위치는 종종 손에 외상 동안 굴근 힘줄에 손상을 초래한다. 힘줄은 정상 힘줄 조직을 하나의 흉터 조직 응답보다는 재생을 통해 치유. 이 반흔 조직이 건에 연속성을 제공하지만, 기능은 건강한 건에 비해 감소 극적으로된다. 건 - 흉터 조직 복합체가 파열 할 가능성이 복구 된 힘줄을 렌더링, 장애인 기계적 특성 (1)을 특징으로한다. 또한 흉터 조직이 힘줄의 크기와 부피의 증가의 결과로, 고유 힘줄 콜라겐 섬유의 조직 구조 없다. 힘줄 - 시스 유닛의 해부학 적 제약, 건 크기도 완만 한 증가를 감안할 수 과감하게 빨간색활공 힘줄의 기능 및 동작과 손 기능에 따라서 숫자 범위를 UCE. 굴근 건에 1960의 부상, 손의 영역 II에서 특히 이전에 정기적으로 인해이 수리 2 일어나 치유의 심각한 합병증을 수리되지 않았다. 손이 지역은 &#39;어떤 사람의 땅&#39;3라고 하였다. 그러나, 수술 기법, 봉합 패턴과 물리 치료 재활 프로토콜의 개선은 크게 굴곡 건 수리 (2)의 결과를 개선했다. 이러한 발전에도 불구하고, 수리 40 % 손 함수 (4)를 방해하기에 충분한 유착 형성 될. 따라서, 생물학적 방법은 치료를 개선 할 필요가있다. 불행하게도, 약간은 세포 및 분자 수준에서 건 치료 과정에 대한 알려져있다. 따라서, 목표는 기본 understandin을 향상시키기 위해 사용될 수있는 쥐 모델을 개발했다치유를 개선하는 신규 한 치료 표적을 식별하기위한 수단으로서 굴근 힘줄 치유 및 흉터 형성 반응의 세포 및 분자 성분의 g. 큰 동물 모델은 굴근 건 치유 과정의 이해를 증진하는 수단이되고있다. 송곳니 토끼 연구는 굴근 힘줄 -5,6-의 고유 및 외부 치유 능력을 모두 증명 고정화 7 유착 형성에 대하여 최소화 초기 제어 수동 동작의 중요성뿐만 아니라, 치유 과정 8 가지 봉합 패턴의 영향 9. 게다가, 송곳니 치유 모델 (10)을 개선하기 위해 병진 조직 공학 방법을 테스트하는데 유용하고있다. 그러나, 상대적 비용, 뮤린 특정 시약의 이용 가능성을 포함하는 많은 동물 모델 쥐 모델 대하여 사용의 몇 가지 중요한 이점은, 글로벌 공사 생성의 용이성또는 조직 특이 삭제 / 과발현 구조 아웃 케이. 또한, 굴근 힘줄 (11)에 대한 인간과 쥐 사이의 기능적 유사성은 쥐 모델 개발에 잠재적 인 유틸리티를 나타냅니다. 굴근 건 절개 및 수리 모방 풍부한 흉터 조직 및 장애 기계적 특성의 형성을 포함하여 임상 치료의 여러 측면의 쥐 모델 개발. 여기에 설명 된 모델은 복구 사이트를 보호하기 때문에 myotendinous 접합부에서 FDL의 절개로 임상의 진정한 재현부 아니다. 복구가 발생하는 힘줄의 중앙 부분을 더 덮고 활막 시스가없는 더욱이,이 모델은 치유 응답 활막 세포 외피의 기여를 고려하지 않는다. 이러한 제한에도 불구하고,이 모델은 뮤린 모델이 더 클에서 입증 될 아직 움직임이 제한 유착의 발생 범위의 이점을 갖는다엘리는 임상 시나리오에 근접. 이 모델은 녹 - 아웃 마우스 모델 (12, 13)을 평가하고, 치료 14-17을 향상시키는 다른 약물 학적 방법을 테스트하기 위해 사용되어왔다. 조직 학적는 면역 조직 화학 염색을 사용하여,이 모델 분석 및 현장 하이브리드에, 치료 동안 키 유전자와 단백질의 현지화에 대한 중요한 통찰력을 제공 할 수 있습니다. 그러나, 조직학는 단면 공간 분석을 제공하고, 전체 조직에 걸쳐 정량화를 허용하지 않는다. 유세포보다 정량적 방법을 나타내지 만, 전지의 수가 매우 제한 마우스 모델에서 치료 힘줄 조직으로부터 단리 할 수​​ 있고,이 수는 상기 고정, permeabilization과 세정 단계 동안 감소된다. 계정에이 촬영, 세포 계측법으로 인해 요구되는 동물의 수에 실행할 수없는 접근을하게 흐른다. 다른 방법은 주문이 소세포 인구의 대부분을 보존 할 필요가있다상기 치료 환경을 특성화. 여기에 도시이를 달성하기 위해 사용 된 방법은, 면역 세포이어서 유리 슬라이드 상에 세포학 원심 분리를 통해 분리 된 세포의 농도를 포함한다. 본 연구 EDU (5- 에틴 2&#39;deoxyuridine, 티미 딘 유사체)에 통합 이후 라벨링 치료 부위에서 세포 증식의 상대적인 상태를 판정하는데 사용되었다. 이 방법은 세포 증식 유전자 녹아웃 또는 과발현에 약물 치료의 효능을 테스트하기 위해, 또는 다른 세포 집단을 식별 및 정량화에 적용 할 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="1047">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6J mice&#160;</td>
			<td>Jackson Laboratories</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine</td>
			<td>Hospira</td>
			<td>NDC# 0409-2051-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine</td>
			<td>Lloyd Inc.</td>
			<td>NDC# 61311-482-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenorphine</td>
			<td>Par Pharmaceutical Inc.</td>
			<td>NDC# 42023-179-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.9% sodium chloride irrigation</td>
			<td>Hospira</td>
			<td>NDC# 0409-6138-03</td>
			<td>For preparation of ketamine/xylazine and buprenorphine solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1ml syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30G needle</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone-Iodine solution</td>
			<td>Aplicare</td>
			<td>82-226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puralube vet opthalmic ointment</td>
			<td>Dechra Veterinary Products</td>
			<td>NDC# 17033-211-38</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Portable balance 200g</td>
			<td>Ohaus</td>
			<td>SP202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15124-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle holders</td>
			<td>Fine Science Tools</td>
			<td>91201-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15003-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro needle holders</td>
			<td>Fine Science Tools</td>
			<td>12061-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-0 nylon sutures</td>
			<td>Ethicon</td>
			<td>668G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8-0 microsurgery nylon sutures</td>
			<td>Ethicon</td>
			<td>2808G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lab-Line histology slide warmer</td>
			<td>Barnstead International</td>
			<td>26025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytospin method</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase Type I, lyophilized</td>
			<td>Life Technologies&#160;</td>
			<td>1700-017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>Cell Signaling Technologies</td>
			<td>9998S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X PBS</td>
			<td>Thermo Fisher</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytology funnels</td>
			<td>Fisher HealthCare</td>
			<td>10-354</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HistoBond+ microscope slides</td>
			<td>VWR</td>
			<td>16005-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytospin 2 centrifuge</td>
			<td>Shandon</td>
			<td>SH-CYTO2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunocytochemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide staining tray with black lid</td>
			<td>IHC World</td>
			<td>M920-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Click-iT Plus EdU Imaging Kit</td>
			<td>Life Technologies&#160;</td>
			<td>C10639</td>
			<td>Includes EdU and&#160; Hoeschst 33342</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immedge hydrophobic barrier pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Diamond mounting medium</td>
			<td>Thermo Fisher</td>
			<td>P36970</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass coverslips 24x50mm #1.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clear nail polish</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

murine flexor tendon injury and repair surgery

건 거의 몸 전체의 움직임을 촉진, 골격 근육과 뼈를 연결합니다. 손에, 굴근 힘줄 (FT들)은 손가락과 일반 손 기능의 굴곡을 할 수 있습니다. FT들에게 ​​부상은 공통이며, 만족스러운 치료는 종종 인해 과잉 흉터 조직 및 힘줄과 주변 조직 사이의 유착에 손상된다. 그러나, 거의 FT 수리의 분자 및 세포 성분에 대한 알려져있다. 이를 위해, 운동 장애를 포함한 다양한 인간 치료의 많은 부분을 되풀이되었습니다 및 기계적 특성을 저하 FT 수리의 뮤린 모델이 개발하고 이전에 설명되었다. 다음은이 수술에 대한 심층적 인 시위는 절개와 쥐의 뒷발의 굴곡 심지 longus (FDL) 힘줄의 후속 수리를 포함, 제공된다. 이 방법은 유전자 이득 또는 기능 상실의 영향을 평가하고 effi을 테스트하는 다른 세포 유형의 계통 분석을 수행하는데 사용될 수있다치유 과정의 약리학 적 개입 cacy. ⅰ) 절개 및 수리가 발생 쥐의 뒷발의 중간 부분에서 FDL 건을하는 활막 외피로 둘러싸여되지 않습니다 그러나,이 모델의 두 가지 주요 제한 사항이 있습니다. 따라서이 모델은 흉터 형성 공정에 시스의 잠재적 인 기여를 고려하지 않는다. ii) 상기 보수 사이트의 무결성을 보호하기 위해, FT는 가능성 증가 흉터 형성에 기여하고, 힘줄의 기계적 힘을 감소시키는 상기 myotendinous 교차점에서 해제된다. 유세포 분석을위한 치료 과정 동안 FT의 육아 조직의 충분한 세포의 분리는 어려운 입증되었다; 이들 세포를 집중 세포학 원심 분리에 사용되는 다른 방법이며, 면역 표지가 수행 될 수있는 세포 제제의 생성을 허용한다. 이 방법, FT 치유하는 동안 세포 또는 관심의 단백질의 정량이 가능하게된다.

굴근 심지 longus 종아리에있는 (FDL) 근육,의 myotendinous에서 근위 실행되는, (도 1a에 파란색으로 설명하고,도 2a에 조직 학적으로 표시) 굴근 건을 통해 마우스의 뒷발의 숫자를 플렉스 역할 접합 및 원위 지골의 종료. 상기 뒷발의 숫자 (빨간색 화살표, 그림 1A)에있는 분기에, 근위을 굴근 건 치유의이 모델에서, FDL 건은 횡단하고, 중간 ?...

Watch this Scientific Journal Video about Murine Flexor Tendon Injury and Repair Surgery at JoVE.com

Murine Flexor Tendon Injury and Repair Surgery

손에 굴근 힘줄은 일반적으로 손상된 손 기능에 이르는 부상하고 있습니다. 그러나, 흉터 조직의 치유 반응은 잘 특징되지 않습니다. 굴곡 건 치유의 쥐 모델은 여기에 설명된다. 이 모델은 치유 과정의 전체적인 이해를 향상시키고 치유를 개선하는 치료 적 접근 방식을 평가할 수있다.

쥐의 굴근 힘줄 손상 및 복구 수술

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers ...

murine-flexor-tendon-injury-and-repair-surgery

Research

JoVE Journal

Medicine

13.0K 조회수.  University of Rochester Medical Center.  손에 굴근 힘줄은 일반적으로 손상된 손 기능에 이르는 부상하고 있습니다. 그러나, 흉터 조직의 치유 반응은 잘 특징되지 않습니다. 굴곡 건 치유의 쥐 모델은 여기에 설명된다. 이 모델은 치유 과정의 전체적인 이해를 향상시키고 치유를 개선하는 치료 적 접근 방식을 평가할 수있다. 

비디오: Murine Flexor Tendon Injury and Repair Surgery

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

쥐의 척수를 다친 후에 급성 및 만성 촉각 감각 테스트

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

연구

의학

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

무한 호라이즌 임팩터을 사용하여 일방적 경부 척수의 부상의 Contusive 모델

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

혈관 동맥류 수리의 새로운 쥐과 모델

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

외상성 뇌 손상에 대한 제어 피질 충격 모델

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

흉터 상처 수리의 마우스 태아의 피부 모델

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

동맥 재 협착의 쥐 모델 : 대퇴 와이어 부상의 기술적 측면

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

창자 허혈 - 재관류 손상의 쥐 모델

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

쥐 모델에서 Infrarenal 복부 대동맥 클램핑에 의해 재현 동맥 발가 벗김 부상

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

쥐에서 양측 슬 개 골 힘 줄 부상 모델에서 줄기 세포 치료의 평가

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human Brown-S&#233;quard syndrome&#160;is a common type of incomplete SCI caused by a lesion to one half of the spinal cord which results in paralysis and loss of&#160;proprioception&#160;on the same (or ipsilesional) side as the injury, and loss of pain and temperature sensation on the opposite (or contralesional) side. Adequate methodologies for producing a spinal cord lateral hemisection (HX) and assessing neurological impairments are essential to establish a reliable animal model of Brown-S&#233;quard syndrome. Although lateral hemisection model plays a pivotal role in basic and translational research, standardized protocols for creating such a hemisection and assessing unilateralized function are lacking. The goal of this study is to describe step-by-step procedures to produce a rat spinal lateral HX at the 9th thoracic (T9) vertebral level. We, then, describe a combined behavior scale for HX (CBS-HX) that provides a simple and sensitive assessment of asymmetric neurological performance for unilateral SCI. The CBS-HX, ranging from 0 to 18, is composed of 4 individual assessments which include unilateral hindlimb stepping (UHS), coupling, contact placing, and grid walking. For CBS-HX, the ipsilateral and contralateral hindlimbs are assessed separately. We found that, after a T9 HX, the ipsilateral hindlimb showed impaired behavior function whereas the contralateral hindlimb showed substantial recovery. The CBS-HX effectively discriminated behavioral functions between ipsilateral and contralateral hindlimbs and detected temporal progression of recovery of the ipsilateral hindlimb. The CBS-HX components can be analyzed separately or in combination with other measures when needed. Although we only provided visual descriptions of the surgical procedures and behavioral assessments of a thoracic HX, the principle may be applied to other incomplete SCIs and at other levels of the injury.

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral injury.