Name: intravital microscopy imaging of the liver following leishmania infection: an assessment of hepatic hemodynamics
Uploaded: 2015-07-28T13:00:00.000+00:00
Duration: 10 min 48 s
Description: Watch this Scientific Journal Video about Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics at JoVE.com

이 연구는 INSERM, 엑스 - 마르세유 대학과 CL 포레스에 의해 얻어진 HFSPO에서 경력 개발의 수상에 의해 지원되었다.

윤리 문 : 모든 동물 연구가 엑스 - 마르세유 Université, 프랑스의 기관 동물 케어 및 사용위원회에 의해 승인되었습니다 가이드 라인 및 프로토콜에 따라 수행되었다. 8에서 여성 C57BL / 6 마​​우스 - 오래된 10 주 상업적 Décret N ° 8 87-848 년 10 월 19 일 1987 년 파리의 규칙에 따라 획득 및 처리 하였다. L.를 사용하는 모든 실험 donovani LD1S 기생충은 프랑스와 유럽 연합 (EU) 법률에서 바이오 안전성 규정에 따라 실시 하였다. L. 1. 마우스 감염 donovani Promastigote 기생충 <ol><li> L.의 유지 보수를 위해 시험 관내 배양 배지를 준비 donovani의 promastigote 기생충, LD1S (MHOM / SD / 62 / 1S-CL2D). <ol><li> M199 매체를 사용합니다. (30 분 동안 56 ℃에서 열 - 불 활성화)를 10 % 소 태아 혈청 M199 배지를 보충하는 25mM HEPES pH가 6.9, 12 mM의 NaHCO3를, 1 밀리미터 GLUtamine, RPMI 16,040 비타민 믹스, 10mM의 엽산, 100 mM의 아데노신, 7.6 mM의 헤민, 50 U / ㎖ 페니실린, 50 ㎎ / ㎖ 스트렙토 마이신. </li></ol></li><li> 문화는 25cm 2 통풍이 플라스크에 26 ° C에서 완전한 M199 배지 10ml에 기생충을 LD1S. </li><li> 연속 차동 원심 분리 단계를 사용하여, metacyclic 형태로 충실 promastigote 기생충의 인구를 준비합니다. <ol><li> 중반 로그 위상 기생충 문화를 수확. hemocytomer에 계산합니다. 완전한 M199 배지 10ml에 5 × 5 기생충 / ㎖를 재현 탁. 그들은 (약 5-6일에서) 말 고정상에 도달 할 때까지 혐기성 조건에서 기생충 문화를 성장. </li><li> 비 metacyclic 기생충 펠렛 뜨는을 복구하고 10 분 동안 26 ° C에서 2,500 XG에 다시 스핀 26 ° C에서 10 분 동안 1,200 XG에서 LD1S 문화를 스핀. 참고 :이 최종 펠렛은 매우 metacyclic promastigote 기생충에 충실. </li><li> 1의 펠렛을 재현 탁PBS의 00 μL. V 및 혈구를 사용하여 기생충을 계산 : 25 % 글루 타르 알데히드와 1/100 V를 고정을위한 작은 나누어지는을 제거합니다. </li></ol></li><li> PBS에 1 × 10 1백분의 7 μL로 농축-metacyclic 기생충 인구를 희석. 마취 된 쥐의 꼬리 정맥에 기생충 현탁액을 주사 30 G 바늘로 1 ML의 주사기를 사용하여 (아래 참조). 대조군의 경우, 꼬리 정맥에 100 ㎕의 PBS를 주입. </li></ol> 2. 외과 적 치료 <ol><li> 30 분 동안 70 % 에탄올로 살균 소독제 분무 작업 공간과 모든 수술기구를 소독. </li><li> 125 ㎎ / ㎏의 케타민 12.5 밀리그램 / PBS에 희석 kg 자일 라진을 함유하는, 동물의 중량에 따라 마취 용액을 준비한다. </li><li> 마우스를 달아 복강 30 G 바늘 1 ML의 주사기를 사용하여 마취의 적절한 투여 량을 주입. </li><li> 따뜻한 마우스를 유지하고 마우스가 완전히 anesthe 있는지 확인진행하기 전에 발 패드를 집어 HSV 공간. 60 분마다 마우스의 마취 용액 반 도즈를 재 주입. </li><li> 마우스의 복부를 면도하고 Vetedine로 청소. 복부의 왼쪽에있는 흉부 케이지 아래의 피부와 근육의 작은 절개를합니다. </li><li> 다만 열린 영역 아래 복부에 촉촉한 거즈 조각을 놓습니다. 간을 노출하고 거즈에 놓습니다. 간 위의 촉촉한 거즈의 다른 조각을 놓습니다. </li><li> 장소는 24 X 60mm 2-150 μm의 두께 coverslip에, 시아 노 아크릴 레이트 결합 현미경 시각화를위한 간에, 금속 프레임에. </li><li> 젖은 거즈로 마우스의 눈을 보호합니다. </li><li> 촬상 세션 후, 경부 탈구에 의해 동물의 마취를 안락사. </li></ol> 간 아키텍처 3. 생체 내에 현미경 이미징 <ol><li> 에 장착 반전 촛점 현미경 생체 내에 현미경 실험을 수행hermostatic 실, 아포 크로매틱 63X 오일 글리세린 침지 목표 (NA 1.4), 아르곤 (488 ㎚)와 헬륨 네온 (543 nm의, 633 nm의) 레이저 및 청색 다이오드 (405 나노 미터)을 제어. </li><li> 목적에 간 얼굴을 덮고있는 커버 슬립과 현미경의 무대에 마우스를 놓습니다. 29 ° C까지 챔버의 온도를 설정합니다. 사인 곡선의 시각화를 허용하는 간 자기 형광을 사용하여 초점을 조절합니다. </li><li> 혈관을 시각화하기 위해 각 마우스 PBS 100 ㎕에 희석 500 μg의 BSA - 알렉사 647의 솔루션을 준비합니다. 30 G 바늘을 1 mL를 주사기를 사용하여 마취시킨 쥐의 꼬리 정맥에 정맥 내 용액을 주입한다. </li><li> 간세포 핵을 시각화하기 위해 PBS에 훽스트 33342의 용액을 제조 하였다. 복강 30 G 바늘 1 ML의 주사기를 사용하여 마우스의 8 ㎎ / ㎏에서이 솔루션을 주입한다. </li><li> 이미지 간 400 Hz에서 일반 연속 모드, 63X 침지 객관적이고 스캐너를 사용하여세포 핵 및 간 혈관. 훽스트 여기에 대한 405 nm의 청색 다이오드와 BSA - 알렉사 647 여기의 633 nm의 레이저를 켭니다. 아르곤 488 nm의 레이저를 켜고 간자가 형광을 시각화하기 위해 대규모 인수 대역을 정의합니다. </li></ol> 혈류 속도 측정을위한 간 4. 생체 내에 현미경 이미징 <ol><li> 현미경 (LAS-AF 뷰어 버전 3.1.0 빌드 8587)의 LAS 소프트웨어를 엽니 다. 현미경 소프트웨어를 열 때 공진 스캐너 모드를 선택합니다. </li><li> 하드웨어를 설정하려면 구성 탭을 클릭합니다. 레이저를 클릭하고 헬륨 네온 레이저 (633)를 선택합니다. 설정을 클릭하고 12 비트 해상도를 선택합니다. </li><li> 취득 메뉴를 클릭합니다. 빔 경로 설정 창에서, 목적 63X를 선택하고 100 %로 633 레이저 파워를 설정합니다. 신호 이득과 PM1-3 드롭 다운 메뉴에서 알렉사-633을 선택하여 설정 매개 변수 오프를 활성화합니다. </li><li> 획득 탭에서 &#39;xyt&#39;인수를 선택모드. 1,024 X 1,024에서 설정 서식의 폭. 8,000 Hz에서 속도를 선택합니다. 3 에어리 단위의 핀홀을 선택합니다. 양방향 모드를 선택하지 마십시오 3의 줌 배율을 선택합니다. </li></ol> RBC 속도 5. 정량 분석​​ xyt 이미지를 사용 (방법 1) <ol><li> 라이브 모드에 혈액의 흐름을 시각화하는 &#39;라이브&#39;아이콘을 클릭합니다. 관심 영역을 선택하고 분석을 위해 특정 혈관을 선택한다. USB 제어판 &#39;X&#39;, &#39;Y&#39;및 스캔 필드 회전 좌표를 조정하여 수평 위치에 관심 용기를 설정한다. </li><li> 관심 용기가 설정되면 &#39;라이브&#39;모드를 중지. 선택, 창, 선 평균 = 1, 프레임 평균 = 1을 설정 획득 모드에서 1,024 X 256 포맷 설정 폭을 변경 이미지를 얻기위한 &#39;시작&#39;에 150 클릭 &quot;스택&quot;. </li><li> 사용 RBC 속도의 정량적 분석을위한 &quot;xyt&quot;이미지는 ImageJ에 소프트웨어를 엽니 다. &#39;파일&#39;을 선택ImageJ에있는 탭을 누른 다음 원하는 파일을 선택 &quot;열기&quot;를 클릭합니다. </li><li> ImageJ에있는 &#39;플러그인&#39;메뉴로 이동 궤적 및 바이오 형식 옵션을 가져 오기를 선택합니다. 이 창에서 &#39;hyperstack&#39;로 매개 변수를 볼 수 스택을 선택합니다. &quot;xyzct&quot;스택 순서 옵션에서 옵션을 선택합니다. 참고 :이 인수의 모든 일련의 창을 엽니 다. </li><li> ImageJ에 메뉴에서 &#39;플러그인&#39;을 클릭하고 MTrackJ 옵션을 선택합니다. 작은 창에 &#39;추가&#39;아이콘을 클릭하고 추적하기 위해 적혈구를 클릭합니다. 각각의 다음 프레임에서 같은 RBC 클릭하고 속도를 측정하는 &#39;측정&#39;아이콘을 클릭합니다. 참고 : .exl 형식으로 저장할 수 있습니다 생성 된 파일을. </li><li> 용기 당 다섯 적혈구 최소 트랙과 동일한 크기의 세 개의 개개의 용기의 최소 분석. </li></ol> XT 라인의 이미지를 사용하여 RBC 속도 6. 정량 분석​​ (방법 2) <ol><li> 혈액 F를 시각화하기 위해 &#39;라이브&#39;를 클릭라이브 모드에서 낮은. 분석 용 특정 혈관을 선택한다. 수평 위치에 관심있는 용기를 설정합니다. 관심 용기가 설정되면 &#39;라이브&#39;모드를 중지. </li><li> &#39;XT&#39;스캔 모드를 선택하고 라인 스캔 따라 선택된 선박의 중앙 루멘을 설정합니다. 설정 형식 폭 1024 X 512, 속도 = 8,000Hz, 라인 평균 = 32, &#39;시작&#39;과 XT 라인 이미지를 수집에 시간 = 512 클릭에서. </li><li> .lif 파일을 열고 관심있는 XT 라인 이미지를 열어 RBC 속도의 정량 분석​​을위한 줄무늬를 생성합니다. LAS-AF 소프트웨어에서, &quot;실험&quot;을 선택 .lif를 열고 이미지를 선택합니다. 참고 : 자동으로 kymograph을 엽니 다. </li><li> 이 kymograph에 (X 축 상 얻어진 D) 소정 거리를 이동하기 (어두운 반사 표시) 입자 연속 요구 (t, Y 축 상에 얻음) 시간을 측정한다. 도구 &quot;선을 그립니다&quot;하고 행진에 수평으로 선을 그릴을 선택합니다. 거리에 유의μm의 이미지 하단의 결과 탭 발생 시간 (초 단위). </li><li> kymograph에서 얻은 값을 사용하여 V = 거리 / 시간과 속도를 계산합니다. </li><li> XT 이미지 당 다섯 입자 줄무늬의 최소값과 동일한 크기의 세 개의 개개의 용기의 최소 정량화. </li></ol>

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The authors have nothing to disclose.

마우스 간 생체 내에 현미경의 최근 발전은 생체 내에서 실시간으로 5,9,10 감염에 대한 생리적 반응의 조사에 대한 새로운 가능성을 엽니 다. 장기 혈액의 흐름은 종종 많은 질병에서 변경되는 중요한 생리적 파라미터이다. 그러나 생리 학적 감염성 조건 하에서 간 혈류의 상태를 제대로 탐구 영역 남아있다. 본 연구에서는 이전에 비장 및 종양 맥관 연구를위한 적응시켰다 IVM 기반 ...

장기 별 혈역학는 포유류의 기관의 중요한 생리적 기능입니다. 혈액의 흐름에 이상이 염증의 결과 및 장기 부전 1의 표시 일 수있다. 따라서, 혈류 조직의 구조와 기능은 병리 생리 학적 조건 하에서 분석을위한 임계 파라미터로서 나타난다. 일반적으로 특정 기관에 혈액 흐름을 분석하기 위해 사용 된 기술은 기술 자체의 분해능 한계 등 여러 한계를 포함 (예를 들어 혈류 도플러 촬상) 당 혈액 만 (양의 절대 혈류 측정을위한 용량 기관) (예를 들어, 빛 간섭 단층 촬영)와 혈관 2,3의 크고 이질적인 인구에서 속도의 평균 변화의 측정을 제공하는 장치. 간의 순환 시스템은 크기, 구조와 기능에 이질적인 다른 선박의 하위 유형을 연결합니다. 에이 연구, 생체 내에 현미경 (IVM) 이미징 기술은 고해상도 실시간으로 생체 내 간 혈류 역학을 평가하기 위해 적용되고 평행 간 기관을 구성하는 개별 혈관의 특성을 밝히기 위해서. 이 강력한 광학 이미징 기술의 최근 발전은 연구자가 높은 공간 및 시간 해상도에서 살아있는 동물에서 동적 데이터를 수집 할 수있다. 직접적인 시각화 및 생체 내에서 특이적이고 신속한 생물학적 과정의 실시간 모니터링을 허용함으로써 IVM 이미지 개별 혈관 연구원 수있는 독특한 기회를 제공하고, 측정하고 특별히 선택된 내의 단일 적혈구의 속도 (RBC)를 정량화 간 선박. 본 연구에서 우리는 간에서 혈역학 hepatotropic 슈만 편모충 기생충에 의한 감염 마우스의 영향을 조사하기 위하여 마우스의 간에서 IVM 기법을 구현 하였다. L. donovani내장 리 슈만 편모충 증, 급성 온 만성 염증 반응을 특징으로하는 질환 및 중증 간 및 비장 등의 여러 기관에 존재하는 병변을 담당 에이전트이다. 내장 슈만 편모충 증의 실험 마우스 모델에서 간 감염 자체 해결 비장 감염 4 진보적 인 반면이다. 아직 완전히 이해되지 않은 개별 기관에 대하여 리 슈만 편모충 감염이 결과. 병적 인 상태에서 간 및 비장 혈역학의 조사는 호스트 기생충 상호 작용과 질병 발병 새롭게 조명한다. 우리의 실험 모델 시스템은 노광 및 간 intravasculature의 표지 특정 형광 염료의 정맥 주사를받은 마취 된 마우스의 간을 이미징에 기초한다. 간은 내 생명 현미경 유리한 기관이다. 작은 incisio을 수행 한 후n은 복부, 간은 부드럽게 외부화 된 후 심장 박동과 호흡으로 인해 어떤 모션 아티팩트를 감소시키는 것을 목표로 커버 슬립에 젖은 거즈에 배치합니다. 간 후, 현미경 렌즈의 시야 내에 배치된다. IVM 연구를위한 두 개의 광자 현미경의 사용을 필요로 비장과 림프절에 비해 간암의 이점은 최대 침투 깊이와, 종래의 공 초점 현미경의 사용을 허용 그 균질 3D 구조 / 해부학에 놓여 약 50 μm의, 생체 내에 현미경 영상 5-8합니다. 본 연구는 개개 혈관 RBC 속도와 혈류 속도의 정량적 측정을위한 두 개의 독립적 인 촬상 방법을 설명한다. 첫번째 방법에서, 간 혈류량은 시간 차원 XY 양방향 모드를 사용하여 획득된다. 그 결과 xyt 데이터는 individ의 추적을 허용 무료 ImageJ에 소프트웨어의 MtrackJ 플러그인을 사용하여 분석시간이 지남에 따라 연간 적혈구. 두번째 방법에서, 하나의 혈관이 선택되고, 그 대응하는 혈류는 공 초점 레이저 주사 현미경의 라인 주사 빠른 획득 모드를 사용하여 분석된다. 관심의 용기 축선을 통해 중심 축을 따라 높은 주파수에서 스캔됩니다. 혈액 유속이어서 표지 된 어두운 적혈구와 혈장 사이의 형광 표지 된 콘트라스트의 차이에 기초하여 정량화된다. 스캔 라인을 따라 얻은 적혈구와 혈장의 형광 강도가 줄무늬를 얻는 시간에 대해 도시되고, 각도가있는 개별 RBC의 속도에 비례한다. 이 문서의 목적은 간 개별 혈관 내 이미징을위한 간단하고 재현 가능한 방법 및 측정 혈류 속도를 제공하고, 마우스 수술, IVM 그리고 속도의 정량 분석​​의 성공적인 성능에 대한 기본적인 도구를 사용할 수 있도록하는 것이다 개별 적혈구. 티그의 접근 방식은 연구자가 병적 인 상태에서 혈액 속도에 대한 새로운 통찰력을 얻을 수 있습니다.

<table><tbody><tr><td>Hoechst 33342</td><td>Sigma Aldrich</td><td>B2261</td><td></td></tr><tr><td>BSA-Alexa 647</td><td>lifetechnologies</td><td>A34785</td><td></td></tr><tr><td>Dextran-FITC 500 mol wt</td><td>SIGMA</td><td>46947</td><td></td></tr><tr><td>Ketamine</td><td>PanPharma</td><td>20434</td><td></td></tr><tr><td>Xylazine</td><td>Bayer</td><td>KP07KEU</td><td></td></tr><tr><td>Vetedine</td><td>Pharma Animal</td><td>6869029</td><td></td></tr><tr><td>Cyanoacrylate liquid</td><td>Cyanolit</td><td>5833300005</td><td></td></tr><tr><td>Coverslip frame: Membrane slide for microdissection part N&amp;deg;: 5013</td><td>Molecular machines</td><td>50103</td><td></td></tr><tr><td>Coverslip DiaPath 24x60 ep: 1.6 mm</td><td>DiaPath</td><td>61061</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica&amp;nbsp;</td><td>TCS-SP5</td><td></td></tr><tr><td>LAS-AF viewer&amp;nbsp;</td><td>Leica software</td><td>Version 3.1.0 buid 8587</td><td></td></tr></tbody></table>

intravital microscopy imaging of the liver following leishmania infection: an assessment of hepatic hemodynamics

생체 내에 현미경 (IVM)을 실시간으로 라이브 동물의 다양한 생물학적 이벤트의 시각화, 모니터링 및 정량을 가능하게 한 강력한 광학 영상 기술이다. 이 기술은 크게 생리적 프로세스 및 특정 기관의 병원체 매개 현상에 대한 우리의 이해를 전진했다. 이 연구에서, IVM은 마우스 간인가하고, 프로토콜 설계 상으로 생체 내 간 순환계를 개별 용기 간 적혈구 (RBC)의 속도를 측정한다. 간 장기 특성을 다른 용기 아형을 시각화 및 혈액 유속 측정을 수행하기 위해 C57BL / 6 마​​우스에게 정맥 ​​내 간 - 관련 맥관 라벨 형광 플라즈마 시약으로 주입된다. IVM은 관심의 특정 용기 생체 실시간, RBC 속도의 측정에 가능하게한다. 이 방법을 설정하면하는 것이 가능하게됩니다생리적, 병리 적 조건에서 간 혈류 역학 조사. 궁극적으로,이 촬상 계 방법론 L.의 영향을 연구를 위해 중요한 것 간 혈류 역학에 donovani 감염. 이 방법은 다른 감염성 모델 마우스 기관에 적용될 수있다 또한 혈류에 미치는 영향을 정량함으로써 염증에 대한 약물의 효과의 임상 테스트를 미리하도록 확장 될 수있다.

간에서 정현파의 구체적인 건축 조직 시각이 기관 (도 1, 패널 B와 C, 녹색), 간세포 핵 라벨링 훽스트의 복강 내 주사의 autofluorescent 속성에 기초 할 수있다 (도 1B, 블루) 그리고 간 순환 시스템 (그림 1C, 적색)의 염색에 대한 형광 BSA의 정맥 주사. 직경 40-700 μm의 2 (D)에서 300 μm의 2 이상 μm의 2, 대형 선박의 D 40 ~ 80 사이의 간은에 이르기?...

Watch this Scientific Journal Video about Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics at JoVE.com

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

다음 간 생체 내에 현미경 이미징<em&gt; 슈만 편모충</em&gt; 감염 : 간 혈류의 평가

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

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JoVE Journal

Immunology and Infection

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

11.5K 조회수.  University of Aix-Marseille. This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

비디오: Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

라이브 호스트에 트렌스 제닉 Leishmania의 기생충의 생체내 이미징에서

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

연구

면역학 및 감염병학

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

사타구니 림프 노드의 Intravital 현미경

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

비장의 Intravital 현미경 : 기생충 이동성 및 혈류의 정량 분석

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

세포에 붙어 Antileishmanial 검사에 대한 기생충 구조 및 변화 분석<em&gt; Leishmania donovani</emTHP1 인간 급성 백혈병 Monocytic 셀 라인에서&gt; Amastigotes

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

CXCR6.Gfp 기자 마우스를 사용하여 건강하고 병에 걸린 간에서 면역 세포의 장기 생체 내에 다중 광자 현미경 이미징

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Lipopolysaccharide 취급 마우스 간에 면역 반응 동안 백혈구의 2 광자 Intravital 이미징

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

대 식 세포 안의 세포내 성장의 정량화는 빠르고 Leishmania 기생충의 독성을 평가 하기 위한 신뢰할 수 있는 방법

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to visceral lesions. Currently, 12 million people are estimated to be infected with Leishmania worldwide and over 1 billion people live at the risk of infection. Leishmania amazonensis is endemic in Central and South America and usually leads to the cutaneous form of the disease, which can be directly visualized in an animal model. Therefore, L. amazonensis strains are good models for cutaneous leishmaniasis studies because they are also easily cultivated in vitro. C57BL/6 mice mimic the L. amazonensis-driven disease progression observed in humans and are considered one of the best mice strains model for cutaneous leishmaniasis. In the vertebrate host, these parasites inhabit macrophages despite the defense mechanisms of these cells. Several studies use in vitro macrophage infection assays to evaluate the parasite infectivity under different conditions. However, the in vitro approach is limited to an isolated cell system that disregards the organism&#39;s response. Here, we compile an in vivo murine infection method that provides a systemic physiological overview of the host-parasite interaction. The detailed protocol for the in vivo infection of C57BL/6 mice with L. amazonensis comprises parasite differentiation into infective amastigotes, mice footpad cutaneous inoculation, lesion development, and parasite load determination. We propose this well-established method as the most adequate method for physiological studies of the host immune and metabolic responses to cutaneous leishmaniasis.

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Here, we present a compiled protocol to evaluate the cutaneous infection of mice with Leishmania amazonensis. This is a reliable method for studying parasite virulence, allowing a systemic view of the vertebrate host response to the infection.

리슈마니아 아마존과 생체 감염에서 마우스에서 기생충 독성을 평가하기 위해

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to ...

Intravital Microscopy to Study Platelet-Leukocyte-Endothelial Interactions in the Mouse Liver

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the end pathway for both inflammation and thrombosis, it is not capable of showing the temporal changes that occur throughout the time course of these processes. Intravital microscopy (IVM) is the use of live-animal imaging to gain temporal insight into physiologic processes in vivo. This method is particularly powerful when assessing cellular and protein interactions within the circulation due to the rapid and sequential events that are often necessary for these interactions to occur. While IVM is an extremely powerful imaging methodology capable of viewing complex processes in vivo, there are a number of methodological factors that are important to consider when planning an IVM study. This paper outlines the process of conducting intravital imaging of the liver, identifying important considerations and potential pitfalls that may arise. Thus, this paper describes the use of IVM to study platelet-leukocyte-endothelial interactions in liver sinusoids to study the relative contributions of each in different models of acute liver injury.

Intravital microscopy is a powerful tool that provides insight into both the temporal and spatial relationships of rapid and/or sequential processes. Herein, we describe a protocol to assess both protein-protein interactions and platelet-neutrophil-endothelial interactions in liver sinusoids in a murine model of experimental sepsis (endotoxemia).

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the ...

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In Vivo Imaging of Leishmania infantum-infected Hamsters by Gingival Inoculation of Axenic Amastigotes Expressing Luciferase

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if not treated; the drugs used in treatment are toxic, and there are cases of resistance. Preclinical tests can represent a bottleneck in discovering new medicines for treatment, depending on the animal model, the strain used, and the inoculum route. The golden hamster stands out for its high susceptibility to subgenera Viannia and Leishmania species, displaying many of the clinical and immunopathological processes observed in human disease.
By hamster anatomy, which has a short tail and limbs, the intracardiac route is usually the choice for intravenous injection of Leishmania. However, it is an inoculum that can lead to bleeding and eventually to animal death. Thus, we standardized an alternative intravenous inoculation route for infection at the gingival vein, which is minimally invasive, allows easy venous access, and causes few local and systemic injuries to the animal. Therefore, hamsters infected by the intraperitoneal (IP) or intragingival (IG) route with Leishmania Infantum expressing luciferase (Luc) were followed up for 22 days by the bioluminescence imaging system and 50 days and 8 months post infection by PCR.
After gingival inoculation of both axenic amastigotes and promastigotes of L. infantum-Luc, bioluminescence was restricted for at least 2 weeks at the site of injection, which is an indicator of infection in the tissues around the gingival plexus. Hamsters infected intraperitoneally with L. infantum-Luc displayed bioluminescence dispersed throughout the abdomen, as expected. However, by the bioluminescence imaging system infection declined until the 50th dpi and was only detectable by PCR.&#160;Axenic amastigotes showed better infection than promastigotes, as evaluated by PCR. Indeed, 8 months after infection, parasites were detected by PCR in the liver of animals inoculated with axenic amastigotes by the intravenous route, which can be a characteristic of the reference strain of L. infantum MHOM/BR/1974/PP75, whose infection progresses slowly and display low parasite burden, below the bioluminescent imaging resolution. Thus, axenic amastigotes can be a better choice for infection and follow-up than promastigotes, and the gingival inoculum is a feasible route for intravenous injection of Leishmania and other pathogens.

Here we present a longitudinal evaluation of golden hamsters infected intraperitoneally (IP) or via intragingival (IG) with L. infantum-Luc by bioluminescent imaging and by PCR. Hamsters were evaluated 1 day post infection (1 dpi), 1 week after infection (8 dpi), and 3 weeks after infection (22 dpi) and euthanized at the 50th dpi and 8 months post infection.

생체 내 루시페라아제를 발현하는 Axenic Amastigotes의 치은 접종에 의한 Leishmania infantum-infected Hamsters의 이미징

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if ...

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