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><tbody><tr><td colspan="4">&lt;strong&gt;Reagent/Material&lt;/strong&gt;</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><a href="http://www.alzet.com" target="_blank"><img src="/img/new_window.png" /> ALZET</a></td><td>1002</td><td>Type: Tool, 14 day release</td></tr><tr><td>Vinyl catheter</td><td><a href="http://www.alzet.com" target="_blank"><img src="/img/new_window.png" /> ALZET</a></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 colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</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

12.3K 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

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

MicroCT를 사용하여 대비 향상된 베셀 영상

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

연구

의학

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 ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

Intravital 현미경 검사 법 및 대비 향상 된 초음파 마우스 Hindlimb의 인슐린 유도 된 혈관과 근육 관류 결합

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain.
Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

혈액-뇌 장벽 투과성을 높이기 위해 집중된 초음파 및 마이크로 버블 치료를 시각화하는 실시간 인트라바이탈 다광원 현미경 검사법

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

생체공학

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Β-aminopropionitrile-유도 대동맥 동맥 및 해 부의 마우스 모델에서 Aortopathy의 양적 마이크로 CT 분석

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

힌들보 리 허 혈의 뮤린 모델에서 치료 혈관 신생 평가

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

마우스 힌드림에 있는 레이저 도플러 관류 화상 진찰

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

허혈/재관류의 돼지 모델에서 척수 미세 퍼퍼퓨전의 실시간 평가

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperfusion 기술은 쥐 장간막에서 미세 혈관 투과성의 규정을 조사하기 위해

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

생물학

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 ...