저자는 편집 지원을위한 마이크로 CT 수술 지원 영상, 에드워드 TH 예치, MD, 레베카 바토, 박사와의 기술 지원을위한 MD 앤더슨 작은 동물 이미징 시설의 키스 미셸에게 감사의 말씀을 전합니다. 이 작품은 미국 심장 협회 (American Heart Association)에 의해 부분적으로 지원되었다.

1. 프라이밍 삼투 펌프 <ol><li> 펌프를 준비 할 때 무균 기법 (예 : 장갑, 층류 후드)를 사용합니다. 포장의 펌프와 유량 사회자를 제거합니다. 빈 펌프를 달아 무게 (MG)를 기록합니다. </li><li> 작은 1CC 주사기와 뭉툭한 팁 27G 충전 튜브를 사용하여 공기 방울을 만드는 피하기 위해 천천히 펌프를 채우십시오. 솔루션은 펌프의 상단에 도달하면 충전 중지합니다. </li><li> 과량의 용액을 닦아 채워진 펌프를 무게. 대부분의 수성 솔루션, MG의 무게 μL의 볼륨과 동일합니다. 빈 대 채워진 무게의 차이는 지정된 평균 충전량의 90 % 이상이어야한다. 그렇지 않은 경우, 주사기와 리필 펌프를 대피. 당신이 기술적 인 어려움을 경험하는 경우하시기 바랍니다 http://www.alzet.com/products/guide_to_use/filling.html에서 펌프 설명서의 문제 해결 단원을 참조하십시오 </li><li> 흐름 moderat에서 흰색 플랜지를 깰 포셉 한 켤레를 사용또는, 관 구부리거나 분쇄하지 않도록주의해야합니다. 3-4mm에 대해 취재 운영자의 한쪽 끝을 P50 카테터를 연결합니다. 카테터 및 연결된 운영자를 작성하고 카테터의 원위부에 부착 된 주사기를 남길 수있는 주사기를 사용합니다. </li><li> 카테터 펌프 표면에 닿을 때까지 펌프에 흐름 사회자를 삽입합니다. 그런 다음, 카테터의 말단부에서 주사기를 제거합니다. 프라임 6 시간의 최소 (바람직 O / N) 마우스에 그것을 주입하기 전에 펌프에 대해 37 ° C 배양기에서 무균 0.9 %의 식염수 또는 PBS 용액에 펌프를 놓습니다. 펌프 전달 속도는 모델 번호 (예 : 모델 1002 = 14 일 동안 0.25 μL / HR).에 따라 달라집니다 </li></ol> 2. 마우스의 일방적 인 뒷다리의 허혈 만들기 <ol><li> 0.4 L / min의 유량에서 100 % O 2에있는 2-4 % 범위 isoflurane을 세트로 쥐를 마취하고 depilato와 뒷다리 다시 영역 (가급적 수술 전 날) 머리를 제거스피 크림. </li><li> 수술 전에 수술 도구를 소독. 유도 마취 챔버에 마우스를 놓습니다. 2.1 절에서 설명 된 것과 동일한 마취 설정을 사용합니다. </li><li> 마우스가 외부 자극에 비 반응하고 isoflurane을의 연속 흐름 (1.5 ~ 2.0 %)을 제공하는 장착 노즈 콘 수술대에 발생하기 쉬운 위치에 배치 될 때까지 기다립니다. </li><li> 절개 사이트에서, 마취의 권장 용량 (0.25 % (&lt;1 ML / kg) 부피 바카 인)를, 주사 70 % 에탄올 다음 포비돈 - 요오드 용액으로 피부를 취급합니다. 다시 (척추의 측면에서, 견갑골 지역)에 4-6 밀리미터 절개를합니다. 미세 집게로 피하 막 분리 한 후 앙와위로 마우스를 켜 70 % 에탄올 다음과 내측 허벅지쪽으로 무릎 위의 마이크로 수술 가위로 0.8-1 cm의 절개를 포비돈 - 요오드 용액으로 피부를 치료, 대퇴 동맥의 경로를 따라. </li><li> 최대 터널뒷면에 절개 내측 허벅지에서 작은 칼집 튜브 (6 프랑스어)와 몸통의 측면. </li><li> 쉬운 위치에 마우스를 놓습니다. PBS 용액에서 준비하는 삼투 펌프 / 카테터를 제거하고 허벅지, 칼집 관을 통해 카테터를 안내합니다. </li><li> 안으로 펌프를 배치하고 6-0 prolene의 봉합 (70 % 에탄올 다음 포비돈 - 요오드 용액으로 피부를 치료)를 다시 절개를 닫습니다. 앙와위에서 마우스를 다시 놓고, 허벅지 절개를 통해 노출 카테터를 떠나, 칼집 튜브를 제거합니다. 외과 테이프로 다리를 고정합니다. </li><li> 혈관을 나타 내기 위해 피하 지방 조직을 분리합니다. 더 나은 혈관을 시각화하기 위해 절개를 열고 견인기를 사용합니다. </li><li> 혈관 번들을 액세스하는 미세 뾰족한 집게를 사용하여 부드럽게 피어스 막 대퇴 칼집. 해부 사타구니 근처의 대퇴 정맥 및 신경에서 대퇴 동맥을 분리 일으키는 원인이되지 않도록주의 예과도한 출혈. </li><li> 장소 두 8-0 prolene의 봉합 인접 대퇴 동맥에게 대퇴 분기 (profunda 대퇴 및 epigastrica 동맥의 기원)에 인접 2-3밀리미터을 중단 할 수 있습니다. </li><li> 적당한 길이로 카테터를 잘라 물질의 타겟 배달을 보장하기 위해 표면 근막에 6-0 prolene의 봉합으로 고정, 허벅지의 외측 영역으로 끝을 놓으십시오. 6-0 실행 봉합과 다리 절개를 닫습니다. 진통제의 최종 피하 주사 (부 프레 놀핀, 0.1 ㎎ / ㎏)를 관리, 70 % 에탄올 다음 포비돈 - 요오드 용액으로 피부를 치료하고 새장에 마우스를 반환합니다. 복구하는 동안 가열 램프를 사용합니다. </li><li> 30 분 회복 기간 후, 뒷다리 허혈의 생성을 확인하는 레이저 도플러 이미징 마우스를 준비합니다. </li><li> 매일 두 번 수술 후 5 일을위한 동물을 모니터링 할 수 있습니다. 동물의 이동성은 고통을 분석하는 관찰 될 것이다 케이지 폐기물 인도어 될 것입니다먹는 ATOR. 동물이 통증이나 불편을 경험하는 경우 또한 모니터 기간 동안 확인하​​고 하루에 두 번 부 프레 놀핀 (0.1mg/kg)의 피하 주사를 제공합니다. </li></ol> 3. 이미징 표본 준비 <ol><li> 2.1 절에 명시된 바와 같이 마우스를 마취. 앙와위에서 운영 테이블 위에 마우스를 전송합니다. 피하 혈관에 응고 방지하기 위해 헤파린 1,000 IU를 주입한다. 마우스가 노즈콘에 연결되어있는 동안, 흉강 및 낮은 복부를 노출하기에 충분히 큰 절개를합니다. infrarenal 복부 대동맥 (IAA)를 노출 동물의 왼쪽 복부 장기를 이동합니다. 열등 정맥 정맥에서 대동맥을 조심스럽게 분리합니다. </li><li> 카테터 삽입시 출혈을 줄이기 위해 인접 IAA를 결찰. 그런 다음, IAA에 작은 절개를합니다. IAA에 ½ G 무딘 바늘 (P10 카테터에 부착) 27를 삽입하고 6-0 봉합사로 고정. </li><li> 기입 두헤파린 식염수와 10 % 중성 20CC 주사기 (NBF) 포르말린. 대비 방사선 불 투과성 실리콘 고무 화합물 5CC 주사기를 채우십시오. MV-희석제 : 1:1.25:0.5의 작업 비율의 MV-경화제 MV-화합물의 세 가지 재료를 혼합. 에이전트가 중합하기 전에 혼합물 (Microfil)은 단지 20 분의 작업 시간을 가지고 있기 때문에, 그것은 혈관에 주입 직전까지 MV-경화제를 추가하지 않는 것이 좋습니다. </li><li> 오른쪽 심방에 상처를 확인하고 안락사를 유도하는 과잉 액체의 배수 수 있도록 isoflurane을의 과다 복용을 관리 할 수​​ 있습니다. 바늘 끝을 식염수로 채워진 주사기를 연결하고 5 분 동안 혈관을 세척하기 시작합니다. 모든 혈액이 혈관 밖으로 플러시되어 있는지 확인합니다, isoflurane을을 해제하고 세척이 시작되면 노즈콘을 분리합니다. 다음으로, 위에 설명 카테터 (3.2)과 추가로 5 분간 압력 붓는다에 포르말린으로 채워진 주사기를 연결합니다. 마지막으로, 콘트라스트, 마지막 주사기를 붓는다천천히 5 분 마우스로 에이전트. </li><li> 선택 사항 : 사진 카메라 혈관에 Microfil은 혈관 구조가 관류되어 있는지 확인합니다. </li><li> 4에서 10 % NBF 및 저장 ° CO / 조영제 중합 N 가득 용기에 마우스를 놓습니다. 24 시간 후, 해부 도구를 사용하여 몸통의 아래쪽 절반 (복부 대동맥 결찰 원위부)에 피부를 제거 10 % NBF를 새로하고, 실온에서 4 일 이상 용기에 다시 마우스를 놓습니다. </li><li> 반대측과 허혈성 뒷다리를 해부하고, 뼈의 탈회를 촉진하기 위해 48 시간 동안 개미의 산성 용액 (칼 - 예 II)의 표본을 배치합니다. 그런 다음, 1 시간 및 10 % NBF에 반환 흐르는 물에서 표본을 씻어 Faxitron (X-레이 시스템 골이 탈회되어 있는지 확인하는 데 사용)와 마이크로-CT 영상과 함께 진행합니다. </li></ol>

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저자는 이익에 대한 갈등을 선언하지 않습니다. 모든 동물의 절차는 실험 동물의 관리 및 사용에 대한 지침에 따라 수행되었다 휴스턴 텍사스 건강 과학 센터의 대학에서 기관 애니멀 케어 및 사용위원회 (IACUC)에 의해 승인되었다.

여기에서 우리는 뒷다리 허혈의 마우스 모델에서 삼투압 약물 / 물질 전달을위한 방법을 제시한다. 또한, 우리는 우리가 혈관 네트워크 분석을위한 3D 재구성을 생산하는 Microfil을 사용하고있는 주조 기술을 설명합니다. 허혈의 수준이나 정도는 동맥 결찰술 / 절단이 이루어진 위치에 따라 다릅니다. 우리는 profunda 대퇴 및 epigastrica 동맥의 기원에 인접 일반적으로 대퇴 동맥?...

말초 동맥 질환 (PAD)는 다리 4 불충분 한 혈액 공급을 일으키는 동맥 경화성 질환입니다. 그것은 8-12000000 미국인에 영향을 미치는, 그리고 현재의 의료 치료는 제한된 구제 5,6을 제공합니다. 다리에 혈액 순환을 개선하는 새로운 치료제는 질병의 진행을 억제뿐만 아니라 삶의 질을 향상하지 않을 것입니다. 종종 노인 환자에서 볼 수 감소 신장과 간 기능이 약의 대사를 감소시키고 조직 관리와 부작용을 증가시킬 수 있기 때문에 PAD의 발생 50 세 이상의 사람에서 더 높은, 그래서 지역 약물 치료는 더 바람직한 치료 방법이다. 따라서, 우리는 로컬에서 관리 에이전트가 혈관 및 미세 혈관의 리모델링을 촉진하여 뒷다리 허혈을 완화 여부를 검사하는 PAD의 마우스 모델을 만들었습니다. 특히, 우리는 지속적으로 치료제를 제공 할 catheterized 삼투 펌프를 사용쥐의 허혈성 허벅지 근육. 우리의 배달 시스템을 사용하여, 우리는 로컬 환경에서 약물의 최적 농도를 유지할 수 있었다;이 방법은 적절한 약물 생리 활성을 허용 가능한 전신 부작용을 피하고, 전신 관리와 관련된 제한 지역 마약 액세스의 단점을 극복한다. 또한, 로컬 관리 에이전트가 시술을 촉진 여부를 평가하기 위해, 우리는 진보 된 주조와 미세 혈관의 변화를 정량화를 가능하게 고화질 영상 기법을 사용했다. 종합적으로,이 비디오 문서에서 사용되는 방법론의 조합은 PAD 환자에 7-9 약리학 유도 시술을 이해하는 데 도움이되는 전임상 연구에 유용합니다.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent/Material</td>
		 </tr>
		 <tr>
			<td>Surgical tools</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Puritan sterile cotton swabs</td>
			<td>Fisher Scientific</td>
			<td>22-029-499</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Betadine (povidone-iodine)</td>
			<td>Fisher Scientific</td>
			<td>19-065534</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>70% Alcohol pads</td>
			<td>Fisher Scientific</td>
			<td>NC9926371</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Phosphate buffered saline</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>6-0 prolene suture</td>
			<td>Cardinal Health</td>
			<td>8709</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>8-0 prolene suture</td>
			<td>Cardinal Health</td>
			<td>2775</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Depilatory cream</td>
			<td>Nair</td>
			<td></td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Osmotic pump</td>
			<td>ALZET</td>
			<td>1002</td>
			<td>Type: Tool, 14 day release</td>
		 </tr>
		 <tr>
			<td>Vinyl catheter</td>
			<td>ALZET</td>
			<td>7760</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Heparinized saline (0.9%)</td>
			<td>Baxter</td>
			<td>2B0944</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Neutral buffered formalin</td>
			<td>Richard-Allan Scientific</td>
			<td>5705</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Microfil (silicone rubber contrast agent)</td>
			<td>Flowtech</td>
			<td>MV-112</td>
			<td>Type Reagent, Microfil White</td>
		 </tr>
		 <tr>
			<td>Cal-Ex II (formic acid solution)</td>
			<td>Fisher Scientific</td>
			<td>CS511-1D</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Buprenex</td>
			<td>CIII</td>
			<td>7571</td>
			<td>Type: Analgesic</td>
		 </tr>
		 <tr>
			<td>Bupivicaine</td>
			<td>Hospira, Inc.</td>
			<td>381</td>
			<td>Type: Analgesic</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>Dissecting microscope</td>
			<td>Carl Zeiss Microimaging</td>
			<td>Zeiss Stemi 2000-C</td>
			<td>Type:Equipment</td>
		 </tr>
		 <tr>
			<td>Laser Doppler perfusion imager</td>
			<td>Perimed Inc.</td>
			<td>Periscan PIM3 </td>
			<td>Type:Equipment</td>
		 </tr>
		 <tr>
			<td>Micro-CT imaging system</td>
			<td>GE Healthcare</td>
			<td>Explore Locus SP </td>
			<td>Type:Equipment</td>
		 </tr>
		</tbody>
 </table>

osmotic drug delivery to ischemic hindlimbs and perfusion of vasculature with microfil for micro-computed tomography imaging

말초 동맥 질환의 동물 모델에서 전임상 연구는 미세 혈관을 자극하도록 설계 치료제의 효능을 테스트하는 중요한 역할을합니다. 투여 경로 뿌리깊은 이러한 에이전트 1,2의 생리 활성과 효능에 영향을주기 때문에 이러한 에이전트에 대한 배송 방법의 선택이 중요합니다. 이 문서에서는, 우리는 로컬 catheterized 삼투 펌프를 사용하여 허혈성 뒷다리에있는 물질을 관리하는 방법을 보여줍니다. 이 펌프는 시간이 할당 된 기간 동안 지속적으로 수용액의 정량을 제공 할 수 있습니다. 우리는 또한 왼쪽 뒷다리에 profunda 대퇴 및 epigastrica 동맥의 기원에 인접 일반적으로 대퇴 동맥의 결찰에 의한 일방적 인 뒷다리 허혈 우리의 마우스 모델을 제시한다. 마지막으로, 우리는 Microfil, 실리콘 방사선 불 투과성 캐스팅 에이전트 infrarenal 복부 대동맥과 뒷다리의 혈관의 관류의 생체 도관 및 결찰에 대해 설명합니다. Microfil이 붓는다 및 채우기 전체 혈관 침대 (동맥과 정맥), 우리는 출구의 주요 혈관 도관 결찰했기 때문에 에이전트는 작은 표본 마이크로 CT 3의 사용과 향후 생체 이미징 혈관에서 유지 될 수있다 .

애니메이션은 프로토콜에 설명 된 삼투 펌프 삽입 및 뒷다리 허혈 수술을 보여줍니다. 그림 1은 허혈을 확인, 뒷다리의 관류의 레이저 도플러 영상을 보여줍니다. Microfil와 혈관 캐스팅 후 혈관 네트워크의 3D 마이크로 CT 이미지 Microfil (그림 2A) 효과적으로 혈관을 채울 수 있음을 보여줍니다하지만 불연속 (예를 들면, 기포의 부족으로 여러 절차 요인으로 인해 <stron...

Watch this Scientific Journal Video about Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging at JoVE.com

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

우리는 여기서 보여<em&gt; 생체 내</em일정한 지역 약물 전달 및 마우스 모델에서 뒷다리 허혈의 생성을위한 삼투 펌프&gt; 삽입. 또한, 뒷다리의 혈관은 미세 전산화 단층 촬영 (마이크로 CT) 영상을 준비하기 위해, Microfil, 실리콘 방사선 불 투과성 에이전트와 관류 있습니다.

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 ...

osmotic-drug-delivery-to-ischemic-hindlimbs-perfusion-vasculature

Research

JoVE Journal

Medicine

12.2K 조회수.  The Texas Heart Institute at St. Luke's Episcopal Hospital. 우리는 여기서 보여 생체 내 삽입. 또한, 뒷다리의 혈관은 미세 전산화 단층 촬영 (마이크로 CT) 영상을 준비하기 위해, Microfil, 실리콘 방사선 불 투과성 에이전트와 관류 있습니다.

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

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

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

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 ...

연구

의학

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 ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

마우스 동정맥루 내피에 관내 약물 전달

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

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 ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

쥐 간 문부 클램프 모델을 사용하여 직접 분절 인트라 간 납품 방법

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Ivacaftor, 그것의 주요 대사 산물, 및 생물 학적 체액의 낭 성 섬유 증 환자에서 Lumacaftor의 임상 샘플의 높은 처리량 분석에 대 한 최적화 된 LC-MS/MS 방법

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

생체 외에서 혈액-뇌 장벽 돼지 뇌 내 피 세포를 기반으로 하는 모델의 설립에 대 한 방법 개선

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

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.