우리는 프로토콜 박사 마이클 Simon 씨의, 닥터 킵의 Hauch, 그리고 일반적인 토론을위한 그들의 실험실의 두 구성원들의 초기 실험 용 박사 켈리 스티븐스 감사드립니다. 이 작품은 NIH 보조금 HL087513 및 P01 HL094374에 의해 지원입니다.

1. 시작하기 전에 준비 <ol><li> 각각 PBS에서 Vasodilator 버퍼 (4mg / L Papaverine + 1g / L은 PBS에 아데노신) 또는 4 % Paraformaldehyde (PFA)로 압력 재관류 장치의 각 측면을 채웁니다. </li><li> 1:100 헤파린의 0.1 ML (5000U/ml 주식)로 작성하고 최대 베벨과 함께 ~ 120도 각도로 바늘을 벤딩하여 1/2cc 인슐린 주사기를 (영구적으로 연결된 29G ½ &quot;바늘로) 준비합니다. 수행 0.3 ML 포화 KCl 용액으로 가득 한 ML의 주사기 (26G ½ &quot;바늘로)와 동일. </li></ol> 2. 가슴을 노출하고 대동맥을 cannulating <ol><li> 선택의 마취제를 사용하여 마우스를 마취. (우리는 케타민을 / Xylazine 혼합물의 과다 사용합니다. 130 밀리그램 / kg 케타민을와 염분의 8.8 밀리그램 / kg Xylazine의 IP 주사) </li><li> 해부 트레이, 복부 측면까지에 anesthetized 마우스를 핀. 중간선 절개로 복강을 열고 피부를 철회장기를 노출합니다. 후부 베나 카바 (PVC)의 영역을 폭로 한쪽으로 창자를 이동합니다. </li><li> PVC로 헤파린 용액을 주사. 당신은 바늘을 추출로 누출되는 것을 방지하고 PVC 벽의 혈전 및 밀봉 때까지 몇 초 동안 누르고있는 솜 스쳐지나 작은 주걱과 바늘 구멍을 포괄합니다. 마우스 순환에 걸쳐 분산하기 위해 헤파린을위한 2-3분 기다립니다. </li><li> 당신이 고동 치는 심장을 관찰할 수 있도록 격막과 흉곽 해부. 천천히 심장 체포 때까지 PVC에 KCl 용액을 주입. </li><li> 피임기구의 지역 앞부분은 그대로두고, 격막 및 소비세 마우스 후부 부분 아래의 모든 장기를 제거합니다. 심장에 근위 부분은 후속 단계에서 찾을 수 있도록 쉽고 피임기구 근처 PVC 이발하는데 신중하다고, 횡경막을 제거합니다. </li><li> 대동맥의 컷 끝을 찾습니다. 대동맥 아래 앞부분 몇 mm 아프로 6-0 꼰 실크 봉합사 중 하나 긴 길이를 놓으십시오m 봉합이 다시 자체 배로되는 등 컷 끝. 대동맥 아래에서 봉합사의 2 가지가되도록 반으로이 더 이상 봉합 잘라. 대동맥 (그림 1A, B)의 절단 끝에 angiocatheter를 넣고 제자리에 angiocatheter 잡고 밖으로 새어에서 대동맥 내의 백 압력을 방지하기 위해 이중 매듭으로 각 봉합을 묶어. </li></ol> 3. 관류 및 Microfil 분사 <ol><li> 압력 재관류 장치 (그림 2)에 angiocatheter를 연결하고 100-110 mmHg의 운전 압력으로 관류 장치를 펌핑하여 vasodilator 버퍼 (그림 1C)와 혈관을 perfusing 시작합니다. 버퍼가 액체를 확보하여 coronaries 통해 perfusing되었는지 확인 더블은 PVC에서 종료됩니다. 최소 3 분 동안 perfuse 계속 또는 PVC를 종료 유체는 분명까지. (perfusing하면서 다음 단계를 계속합니다.) </li><li> 갈비와 핀 뒤로 (또는 제거) 가슴을 노출하는 ribcage 해부. 일단 노출, CA 수심장이 거즈의 버퍼 젖은 부분에서 마음에 버퍼의 방울을 쥐어 의해 말리면 않도록 reful. 대동맥 아치를 폭로 thymus을 치워. 액체를 보장하기 위해 6-0 꼰 실크 봉합사를 사용하여 세 가지 주요 대동맥 가지를 Ligate 것은 오히려 이러한 큰, 낮은 저항 혈관 (그림의 1D)를 통해보다 coronaries 통해 우회합니다. </li><li> 15 분동안 정착액과 함께 마음을 Perfuse 후 최소한 2 분 동안 Vasodilation 버퍼와 린스. 한편, 주사 후 심장 (그림 1E) 밖으로 유출 Microfil을 방지하기 위해 양쪽 앞부분 베나 Cavae을 ligate. 장소 PVC 및 대동맥 주위 봉합하지만, 충전 후 때까지 그들을 조여하지 않습니다. </li><li> Microfil을 준비합니다 (시약의 테이블에서 지정한)와 1 ML의 주사기에 그 파일을로드합니다. 카테터를 (Microfil 주사기로 관류 관에서 전환할 때 기포의 도입을 방지하기 위해있는만큼) 충당하기 위해 충분한 물을 절개 트레이를 채웁니다. 재관류 AP의 연결을 해제카테터의 paratus하고 준비된 Microfil에 주사기를 연결합니다. </li><li> 관상 동맥의 좋은 충전합니다 (무화과 1 층, 3A) 분명 때까지 대동맥으로 Microfil을 주사 : 동맥 먼저 작성되며, 다음 Microfil는 Microfil의 색상과 조직 플러시으로서 모세 혈관에 &quot;누설&quot;합니다. 일단 정맥쪽에, Microfil의 소수성 자연은 작은 혈관에서 나온다로서 초기에는 독립된 분야로 표시하기 위하여 그것을 일으 킵니다. 연속 열이 혈관을 가득 채우고 때까지 Microfil을 주입 계속합니다. Microfil이 혈관 내에서 지속되면 완전 충전이 분명있을 것이고, 그것은 PVC로 종료됩니다. </li><li> 충전이 완료되면 신속하게 이전에 혈관 밖으로 Microfil을 짜면 심장 조직의 탄성 특성을 방지하기 위해 PVC와 대동맥 주위에 배치했습니다 봉합을 조입니다. </li><li> (해부 트레이에서 물에 젖었 서부 유럽 표준시 거즈로 마음을 커버)은 밖으로 건조를 방지하고, Microfil가 polymerized 때까지 그것이 실온에서 약 1 시간 동안 앉아 알려주세요. 같은 리프팅이나 심장의 뒤쪽에 채워 선박의 초기보기를 얻기 위해 시도에 마음을 돌려 같은 중합 과정 동안 마음에 어떤 외부 압력을 피하십시오. 이것은 Microfil에서 봐야 원인, 심장보다 탄성 영역으로 일부 선박에서 Microfil를 당겨 수 있습니다. </li><li> 마음을 제거하고 4에서 하룻밤 4% PFA에 게시 - 수리 ° C. 그렇다면 4 ° C.에 70 % 에탄올에 저장 심장 vasculature 이제 μCT 이미징을위한 준비가되었습니다. </li></ol> 4. 대표 결과 효과적 Microfil 의해 perfused되는 선박은 선박에 걸쳐 지속 깨지지 Microfil (그림 3A)를해야합니다. 관상 혈관의 작성의 범위는 눈으로에서 재판받을 수 있으며, 정맥은 epicardially 18에 위치하고 있으며, 쉽게 (그림 3A, 화살촉) 관찰 수 있으며, 동맥,이것은 18보다 intramyocardial있는 심장의 표면 (그림 3A, 화살표)를 통해도 볼 수 있습니다. 심장 조직은 모세 혈관의 매우 높은 밀도를 가지고로서 모세관 충전은 또한 분명하다, 따라서, 언제 모세 혈관 채우기는 심장 조직 Microfil의 색상 (그림 3A, 별표)로 플러시됩니다. 따라서 작성하는 데 실패하는 혈관 네트워크는 Microfil의 부족 (그림 3B, C)로 인해 눈에 띄게 될 것이다. Microfil의 소수성 자연이 그 자체로 계약 가득한 혈관 내에서 &quot;휴식&quot;을 일으키는 원인이되므로 Microfil (그림 3B 관련 별표)에 불연속은 종종 나타납니다. 혈관 내의 압력이 심장에서 혈관 출구 포인트의 타이 오프 적정을 통해 유지하는 경우이 &quot;휴식&quot;이 감소될 수있다. 다른 불연속은 microfil 내의 기포로 인해 발생할 수 있습니다. 재관류 AP에서 전환할 때 공기의 도입을 방지하기 위해 angiocatheter이 완전히 물에 빠져들되어 있는지 확인<html> Microfil 주사기로 paratus. 공기 방울이 도입되면, 그것은 종종 거품이 관상 동맥 혈관의 통해 밀고 나가는 때까지 Microfil 재관류를 계속하여 간단하게 제거할 수 있습니다. 혈관 침대 부분 (그림 3B, 화살표)가 차단되는 경우 혈관 네트워크는 완전히 채울 수 없습니다. 헤파린은 혈전의 형성을 억제하지만, 가끔 막혀 여전히 이전 절차, 기타 알려지지 않은 요인으로 인해를 시작으로 불완전하게 헤파린 재관류로 인해 발생할 수 있습니다. 차단이 발생하면 혈관 채우기를 완료하기 위해 차단을 dislodging위한 방법은, 우리의 지식,이 없습니다. 너무 작은 압력 Microfil 모든 혈관 침대와 모세관 네트워크 (그림 3C)에 강제되지 않습니다 충전 중에 사용하는 경우 불완전한 충전도 발생할 수 있습니다. 반대로, 너무 많은 압력은 모세 혈관은 (그림 3D) 주변 조직에 Microfil을 터뜨릴하고 extravasate시킬 수 있습니다. files/ftp_upload/3740/3740fig1.jpg &quot;고도 =&quot;그림 1 &quot;/&gt; 1 그림. Microfil 재관류 제도의 개요. () 대동맥과 PVC는 다이어프램의 약 수준에서 잘라 있습니다. (B) 오름차순 대동맥은 angiocatheter로 cannulated있다. (D) 대동맥 아치 오프 세 가지 주요 지점은 출혈도 잡았하는 동안 (C) Vasodilation 버퍼는 압력 재관류 장치 (사진에 없음)에 의한 혈관,를 통해 perfused됩니다. 모두 앞쪽에 베나의 Cavas은 출혈도 잡았하는 동안 (E) 4% PFA는 coronaries 통해 perfused됩니다. 그것은 PVC에서 종료 관찰되기 전까지 (F) 주사기를 사용하여 Microfil는 coronaries 통해 perfused됩니다. <img src="/files/ftp_upload/3740/3740fig2.jpg" alt="그림 2"/> 그림 2. 관류 장치가. 두 Erlenmeyer의 flasks, Vasodilation 버퍼 또는 4퍼센트 PFA 하나 가득 각각은 입사하고 sidearms에 연결된 튜브를 통해 압력됩니다. 시스템의 펌핑 매뉴얼을 통해 가압전구 및 압력 게이지는 모니터링 및 압력의 유지를 허용하도록 flasks 중 하나에 연결됩니다. 작은 튜브가 고무 stoppers 통해 아래 각 플라스크에 액체로 확대. 압력이 작은 튜브 밖으로 각 플라스크에서 sidearms 펌프 유체으로 ​​들어갔다. 튜브 그러면 한 번에 하나의 플라스크에서 흘러 유체 있습니다 꼭지에 병합. <img src="/files/ftp_upload/3740/3740fig3.jpg" alt="그림 3"/> 그림 3. Microfilled 하트를 드셔보세요. () 잘 채워진다 선박은 (있는 경우) Microfil에서 봐야을 몇 가지게 될 것이며, 심장 조직이 채워진 모세관 (별표 및 C 비교)에 의한 Microfil의 물들어 색이 될 것입니다. 두 동맥 (화살표 - 왼쪽 앞부분 내림차순 동맥)과 정맥 (화살촉 - 왼쪽 관상 정맥)은 심장 표면을 통해 볼 수 있습니다. (B) 완전한 마이크를 방해 일부 선박의 microfil에서 봐야 (별표)뿐만 아니라 막혀있는 마음침투력을 rofil. 차단된 혈관은 혈액이 재관류 과정에서 밖으로 플러시되지 않았습니다으로, 빨강 (화살표) 남아 있습니다. 불완전하게 가득 차 있었다 혈관과 (C) 심장. 조직은 Microfil가 모세 혈관으로 침투하지 않은 나타내는 Microfil의 노란 색깔에 찍은되지 않았습니다납니다. (D) 마음이 어디 Microfil가 주변 조직 (화살표)으로 누출 원인, 채우는 동안 모세관 파열.

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마우스는, 워싱턴 대학의 기관 동물 케어 및 사용위원회에 의해 건강의 미국 국립 연구소 (NIH 발행 번호 85-23으로 출판 실험실 동물의 관리 및 이용 안내에 따라 승인된 방법으로 처리되었습니다 ) 1996 개정.

심장 조직은 매우 높은 신진 대사 수요를 가지고 있으며, 따라서 관상 vasculature에 의해 전달되는 혈액의 영양분과 산소의 지속적인 공급이 필요합니다. 선박 협착과 막힘으로 인해 관상 동맥 기능을 감소 관상 혈관, 환자는 조직 hypoxia와 국소 빈혈을 초래할, 그리고 심근 경색과 심장 근육에 치유할 수없는 손상에 대한 위험에 영향을받는 환자를 넣을 수 있습니다. 이러한 혈관의 질병 상태에 대한...

<html><table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 한 ML의 주사기 </td><td> <a href="http://www.bd.com/" target="_blank">Becton 디킨슨</a> </td><td> BD-309602 </td><td></td></tr><tr><td> 영구적으로 연결된 29G ½ &#39;바늘과 1/2cc 인슐린 주사기 </td><td> <a href="http://www.bd.com/" target="_blank">Becton 디킨슨</a> </td><td> BD-309306 </td><td></td></tr><tr><td> 2 &quot;X 2&quot;거즈 패드 </td><td> Med101store.com </td><td> SKU 2,208 </td><td></td></tr><tr><td> 24G 잡았다 &quot;Angiocath IV 카테터 </td><td> <a href="http://www.bd.com/" target="_blank">Becton 디킨슨</a> </td><td> BD-381112 </td><td></td></tr><tr><td> 26G ½ &quot;게이지 바늘 </td><td>여윈 &quot;&gt; Becton 디킨슨 </td><td> BD-305111 </td><td></td></tr><tr><td> 아데노신 </td><td> <a href="http://www.sigmaaldrich.com/" target="_blank">시그마</a> </td><td> A9251 </td><td> 1g / Vasodilation 버퍼를위한 PBS에서 L (Papaverine 포함) </td></tr><tr><td> 각도 Graefe의 집게 </td><td> <a href="http://www.finescience.com/" target="_blank">파인 과학 도구</a> </td><td> 11052-10 </td><td></td></tr><tr><td> 면과 스쳐 applicators : 6 &quot;이외의 멸균 </td><td> <a href="http://www.cardinal.com/" target="_blank">추기경 건강</a> </td><td> C15055-006 </td><td></td></tr><tr><td> 곡선 외과 가위 </td><td> <a href="http://www.finescience.com/" target="_blank">파인 과학 도구</a> </td><td> 14085-09 </td><td></td></tr><tr><td> 해부 입체경 및 광원 </td><td> <a href="http://www.nikon.com/" target="_blank">니콘</a> </td><td> NA </td><td> NA </td></tr><tr><td> 해부 트레이, 11.5 X 7.5 인치 </td><td> <a href="http://www.coleparmer.com/" target="_blank">콜 - Parmer</a> </td><td> 요 - 10915-12 </td><td> 마우스를 아래로 달아 위해 타르로 가득 </td></tr><tr><td> 파인 곡선 집게 </td><td> <a href="http://www.aesculapusa.com/" target="_blank">Aesculap</a> </td><td> FD281R </td><td> 게 2 </td></tr><tr><td> 헤파린 5000 U / ML 주식 </td><td> <a href="http://www.apppharma.com/" target="_blank">APP 제약 LLC</a> </td><td> NDC 63323-047-10 </td><td> 물에 1:100 희석 </td></tr><tr><td> KCl </td><td> <a href="http://www.fishersci.com/" target="_blank">어부</a> </td><td> P217 </td><td> H2O의 포화 용액 </td></tr><tr><td> Ketamin (Ketaset), 100 밀리그램 / ML 주식 </td><td> 포트 닷지, 오버랜드 파크, 캔사스, 미국 </td><td> NDC 0856-2013-01 </td><td> 0.9 % s에 Xylazine과 함께, 130 밀리그램 / kg 체중으로 혼합일직선으로하다 </td></tr><tr><td> Microfil </td><td> <a href="http://www.flowtech-inc.com/" target="_blank">흐름 테크</a> </td><td> MV-122 (노랑). 기타 색 옵션도 사용할 수 있습니다. </td><td> 경화 요원의 부피에 의해 10 %, 중량으로 1:1 섞는다. 그것이 잘 혼합한지 단지 분사하고, 소용돌이 전에 준비 </td></tr><tr><td> 비 멸균 봉합해 : 6 대 0으로 꼰 명주실 </td><td> <a href="http://www.harvardapparatus.com/" target="_blank">하버드 장치</a> </td><td> 723,287 </td><td></td></tr><tr><td> Papaverine </td><td> <a href="http://www.americanregent.com/" target="_blank">미국 리젠트 주식 회사</a> </td><td> NDC 0517-4010-01 </td><td> 4mg / Vasodilation 버퍼를위한 PBS에서 L (아데노신 포함) </td></tr><tr><td> Paraformaldehyde </td><td> <a href="http://www.sigmaaldrich.com/" target="_blank">시그마</a> </td><td> P6148 </td><td> 4 % 용액의 준비 </td></tr><tr><td> 관류 장치 </td><td></td><td></td><td> 그림 2 참조 </td></tr><tr><td> 봄 가위 </td><td> <a href="http://www.finescience.com/" target="_blank">파인 과학 도구</a> </td><td> 15018-10 </td><td></td></tr><tr><td> Xylazine (Anased), 20 밀리그램 / GL 주식 </td><td> <a href="http://www.lloydlab.com/" target="_blank">로이드 실험실</a> </td><td> 나다 # 139-236 </td><td> 0.9 %의 식염수에 Ketamin과 8.8 밀리그램 / kg 체중으로 혼합 </td></tr></tbody></table>

retrograde perfusion and filling of mouse coronary vasculature as preparation for micro computed tomography imaging

vasculature의 시각화는 여러 질병 상태를 이해를 위해 점점 더 중요 해지고 있습니다. 여러 기법 이미징 vasculature을 위해 존재하지만, 몇몇 작은 혈관의 1,2를 포함 해상도로 확대하면서 전체적으로 혈관 네트워크를 시각화 할 수 있습니다. 또한 많은 혈관 주조 기법 예제 3-5의 자세한 분석을 방지, 주변 조직을 파괴. 이러한 문제를 circumvents 한 가지 방법은 마이크로 계산된 Tomography (μCT)입니다. μCT 영상은 해상도 &lt;10 미크론에서 스캔 혈관 네트워크의 3 차원 reconstructions를 생산 할 수 있으며 (예 : 조직학 및 morphometry) 후속 분석을 위해 그대로 6-11을 조직을 떠날 수 있습니다. 그러나 전직 생체내 μCT 방법에 의한 이미징 혈관 혈관이 radiopaque 화합물로 가득해야합니다. 따라서 μCT 이미징 제작한 vasculature의 정확한 표현이 조건으로하고있다혈관의 신뢰성과 완벽한 충전. 이 프로토콜에서는 μCT 이미징을위한 준비 마우스 관상 동맥 혈관을 작성을위한 기법을 설명합니다. 두 predominate 기술은 관상 vasculature 가득 채우는 것은 존재 : Langendorff 재관류 시스템을 통해 15-17 cannulation과 역행 대동맥의 관류 (또는 대동맥 아치 해제 지점) 12-14, 또는 전직 생체내 통해 생체내 인치 여기에서 우리는 특별히 모든 선박에 대해 작성하도록 설계되었습니다 생체내의 대동맥 cannulation 방법에 대해 설명합니다. 우리 모두는 모세 혈관뿐만 아니라, 혈관 네트워크의 동맥과 정맥의 양쪽 모두를 채우기 위해 작은 혈관을 통해 perfuse 수 Microfil라는 낮은 점도의 radiopaque 화합물을 사용합니다. 혈관이 압력 관류 시스템을 사용하여 버퍼로 perfused 후 Microfil으로 가득합니다. 그 Microfil가 작은 높은 저항 혈관을 채우고 확보하기 위해 대규모 지점 emanatin ligatecoronaries로 Microfil을 산란 대동맥에서 g. 일단 충전 일부 표본 Microfil을 짜면 심장 조직의 탄성 특성을 방지하기 위해, 완료 즉시 충전 후 접근이 주요 혈관 출구 포인트를 ligate. 따라서, 우리의 기술은 완전한 관상 혈관 네트워크의 시각화을 사용, 충전 대리인의 완전한 충전과 최대 보존에 최적화되어 있습니다 - 동맥, 모세 혈관과 정맥이 비슷하다.

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Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

관상 혈관의 시각화는 심장 혈관 질병의 우리의 이해를 증진하기 위해 중요합니다. 여기에서 우리는 마이크로 계산된 Tomography (μCT) 이미징을위한 준비, radiopaque 실리콘 고무 (Microfil)로 murine 관상 vasculature을 perfusing위한 방법을 설명합니다.

마이크로 계산된 Tomography 이미징을위한 준비로서 역행 관류 및 마우스 관상 Vasculature의 작성

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

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Research

JoVE Journal

Medicine

32.3K 조회수.  University of Washington. 관상 혈관의 시각화는 심장 혈관 질병의 우리의 이해를 증진하기 위해 중요합니다. 여기에서 우리는 마이크로 계산된 Tomography (μCT) 이미징을위한 준비, radiopaque 실리콘 고무 (Microfil)로 murine 관상 vasculature을 perfusing위한 방법을 설명합니다.

비디오: Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

세분화 및 Murine 비만 모델에서 지방 볼륨 측정 X 선 계산된 Tomography를 사용하여

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

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Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

마이크로 컴퓨터 단층 촬영 영상에 대한 Microfil와 허혈성 뒷발과 혈관의 관류에 삼투의 약물 전달 (Drug Delivery)

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

마이크로 컴퓨터 단층 촬영과 누드 마우스에 인간의 지방 주입의 생존 능력의 평가

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

종양 개발의 평가를 위해 마이크로 컴퓨터 단층 촬영을 사용하고 후속 치료에 대한 반응의를 폐암의 마우스 모델에서

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

직접 대뇌 Thromboemboli 시각화를위한 결합 된 근적외선 형광 이미징 및 마이크로 계산 된 단층 촬영

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Identifying Coronary Artery Calcification on Non-gated Computed Tomography Scans

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed tomography (CT) scans with a high correlation with gated cardiac CT scans. This standardized protocol takes a step-wise approach to not only optimizing an image for the identification of calcification but also to distinguishing CAC from other common causes of calcification in the cardiac silhouette. Recognition of CAC on non-gated CT scans helps to identify a very powerful prognostic factor that can influence therapeutic interventions or downstream diagnostic testing without requiring a gated cardiac scan. These non-gated CT scans are often acquired as part of the routine care of the patient, and this data is readily available without another dose of&#160;ionizing radiation. This protocol allows for the precise and accurate extraction of this data for the purposes of retrospective data analysis in clinical research studies, but also in the clinical evaluation and management of patients.

Here, we present a protocol to reliably and systematically identify coronary artery calcification (CAC) on non-gated computed tomography (CT) scans of the chest or abdomen. CAC provides an objective measure of coronary artery disease for both research and clinical purposes.

단층 촬영 검사 계산 문이 비에 관상 동맥 석 회화를 식별

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed ...

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([18F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Here a protocol for continuous blood sampling during PET/CT imaging of rats to measure the arterial input function (AIF) is described. The catheterization, the calibration and setup of the system and the data analysis of the blood radioactivity are demonstrated. The generated data provide input parameters for subsequent bio-kinetic modeling.

작은 동물 양전자 방출 단층 촬영 / 컴퓨터 단층 촬영에서 지속적인 혈액 샘플링은 동맥 입력 기능의 측정을 가능하게

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

만성 질환을 앓고 있는 대형 포유류 심장 모델의 콘트라스트 강화 마이크로 컴퓨터 단층 촬영 이미징을 위한 조직 준비 기술

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

덕트: 3D 간 분석을 위한 마이크로 컴퓨팅 단층 촬영 다음에 이중 수지 주조

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

Here, we describe a visualization and quantification method for murine hind-limb vessels using micro-X-ray computed tomography.

Murine 힌드사지 선박의 계산 단층 촬영 기반 3차원 시각화를 위한 샘플 준비

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ ...