NIH R01 CA128919 및 NIH R01 HL092923 :이 작품은 에바 Sevick에 다음 보조금에 의해 지원되었다.

모든 동물 연구는 텍사스 건강 과학 센터 (휴스턴, TX), 비교 의학 부, 검토 및 해당 기관 동물 케어하여 프로토콜의 승인 후 분자 이미징 센터 대학의 기준에 따라 수행하고위원회를 사용했다 (IACUC) 또는 동물 복지위원회 (AWC). 1. 이미징에 앞서 24 시간 동물의 작성 림프 영상이 일어나기 전에 아래의 단계 일 (필요에 따라)을 수행해야합니다. <ol><li> 장소 유도 상자에 동물과 isoflurane과 진정. </li><li> 동물은 깊이 마취 (발가락 - 핀치 연습과 모니터링), isoflurane 가스에 연결된 코 콘에서 기저귀 / 보풀 패드 및 위치 코 곳 진정제 동물의 상태에되면. </li><li> 이미징 될 수있는 영역이 주위 머리카락 / 털을 (해당되는 경우) 클립. </li><li> 클립 영역으로 탈모 에이전트 (NAIR)을 적용하고 O 남겨N 최대 3 분까지의 피부. </li><li> 부드럽게 따뜻하고 축축한 거즈 나 종이 타월로 모든 탈모 에이전트를 닦아. </li><li> 부드럽게 따뜻한 물로 피부를 씻어 부드럽게 거즈 나 종이 타월로 영역을 건조. </li><li> 동물 가열 패드 또는 열 램프 아래의 복구, 그들의 케이지로 돌아갈 수 있습니다. </li></ol> 2. 이미징의 날 <ol><li> 멸균 물 Reconstitute 이미징 에이전트 다음 cABD-IRDye800을위한 ICG 또는 200 μM (6.8 μg/10 μl)를 645 μM을 (5 μg/10 μl) 달성하기 위해 멸균, 정상 (0.85 %) 염분을 사용하여 희석. 어둠 속에서 솔루션을 유지 조건과 reconstitution 6 시간 내에 사용. </li><li> 장소 유도 상자에 동물과 isoflurane과 진정. </li><li> 동물은 깊이 마취 (발가락 - 핀치 연습과 모니터링), isoflurane 가스에 연결된 코 콘에서 기저귀 / 보풀 패드 및 위치 코의 측면에 위치 진정제 동물의 상태에되면. </li><li> (그래서 방은 불을 끄고어두운). 필요한 경우, 작은 책상 할로겐 빛이 주사를 볼 수 빛의 작은 양에 사용할 수 있습니다. </li><li> 31 게이지 바늘로 인슐린 주사기를 사용에 따라 각 뒷다리 발 /의 등 부분이나 꼬리의 기지의 왼쪽과 오른쪽에있는 ICG 또는 cABD - IRDye800의 50 μl에 ID 5 μl를 주입 관심 영역 (토론 참조). 각 주입 투여 량은 0.083에서 1.25 밀리그램 / kg (ICG) 또는 0.113-1.7 밀리그램 / kg (cABD-IRDye800)로 다양 할 수 있습니다. 사출 볼륨 동물 스트레인, 사출 사이트와 다를 수 있습니다. athymic 마우스의 경우, 사출의 볼륨은 5 μl (뒷다리 앞발로 땅을 차다) 또는 10 μl (꼬리베이스)이 될 수 있습니다. 동물 주입 (들)에 대한 이미징 시스템에서없는 경우, 바로 뒤에 이미징 시스템에서 동물을 배치 주사 (들). </li><li> 더 염료 이해가 lymphatics에서 볼 수없는 경우 동물의 프로토콜에 따라 필요한 단계 2.5 반복해야합니다. </li><li> 일단 lymphatics이 보이는되며, 검은 색 전기 테이프 나 검은 색 아빠와 주사 사이트를 포함당. </li><li> 최대 사용 1 시간에 대한 림프 이미지를 취득 V + + 소프트웨어와 작은 동물, NIRF 이미징 시스템. (동물 isoflurane으로 마취하고 이미지가 인수하는 동안 호흡을 모니터링됩니다.) 작은 동물 있지만, NIRF의 imagers가 상업적으로 사용할 수 있습니다, 우리는 785-nm의 레이저 다이오드로 구성된 사용자 정의, 작은 동물 NIRF 이미징 시스템 (1천5~9밀리미터-78503을 이용 , 강렬한, 노스 비구면 렌즈 (C24TME​​-B, Thorlabs, 뉴턴, NJ), 디퓨저 (ED1-C20, Thorlabs)와 복 브런 즈윅, 뉴저지) 및 필터 (LD01-785/10-25, Semrock, 로체스터, NY ) cm2 당 10 개씩 미만 1.4 MW의 사고 fluence 속도에서 동물을 비춰 균일 여기 필드를 만들 수 있습니다. 전자는 두 830-nm의 필터 (AND11333, 앤도버 공사, 세일럼, NH)와 28 mm Nikkor 렌즈 (1992, 니콘과 카메라 시스템 부과 - 커플 링 장치 (EMCCD, PhotonMax512, 프린스턴 악기, 트 렌튼, NJ) 곱한 멜빌, NY)이 통합 팀과 림프 이미지를 캡처하는 데 사용됩니다정적 이미지 5 동적 이미징 및 800 밀리 초 200 밀리 초의 에스. 시스템 구성, 각 구성 요소의 추가 내용은 표, 키 영상 특성에 대한 간략한 논의에 대한 토론을위한 그림 1을 참조하십시오. </li><li> 동물 가열 패드 또는 열 램프 아래의 복구와 케이지로 돌아 오거나 안락사시켜야 할 수 있습니다. </li><li> ImageJ 또는 MATLAB 소프트웨어를 사용하여 이미지를 분석합니다. 그림 6을 참조하십시오. </li></ol>

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저자는 공개 할 필요가 없어하지만 일부 저자는 특허에 기재되어 있습니다.

우리는 쥐이라는 림프 혈관의 이미지를 캡처하는 사용자 정의, 작은 동물 NIRF 이미징 시스템을 사용합니다. 림프 운동의 영화를 구성하는 300 개 이상의 이미지를 수집하고 있습니다. 영화에서 lymphatics의 기능 분석을 위해, 두 개 이상의 ROIs를 수동 림프 혈관을 따라 그려 있습니다. ROIs의 크기는 각 선박에 대한 지속적인 유지와 약 혈관의 직경 있습니다. 전체 동물 공간적 해상도가 100 마이크론 이...

<table border="1"><tbody><tr><td> 솔루션, 시약 및 장비 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> Indocyanine 녹색 (ICG) </td><td> Patheon 이탈리아 SPA </td><td> NDC 25431-424-02 </td><td> 645 μM (5 μg/10 μL)에 Reconstitute </td></tr><tr><td> 순환 알부민 바인딩 도메인 (cABD) </td><td> Bachem </td><td> 관습 </td><td> 200 μM (6.8 μg/10 μL)에 Reconstitute </td></tr><tr><td> IRDye800 </td><td> 리튬 COR </td><td> IRDye 800CW </td><td> 제조의 지시에 따라 Reconstitute는, equilmolar 농도에 cABD와 어원이 같은 말 </td></tr><tr><td> 멸균 물 </td><td> Hospira, 주식회사, 레이크 포레스트, IL </td><td> NDC 0409-4887-10 </td><td></td></tr><tr><td> NAIR </td><td> 교회 &amp; 드와이트 (주)주식회사 </td><td> 지역 상점 </td><td> <a href="http://www.nairlikeneverbefore.com" target="_blank">www.nairlikeneverbefore.com</a> </td></tr><tr><td> 이미징 시스템 (구성 요소 아래) </td><td> 분자 이미징 센터 </td><td> N / A </td><td> 우리 실험실에서 사용자 지정 - 내장. </td></tr><tr><td> 전자 - 곱 전하 결합 장치 (EMCCD) 카메라 </td><td> 프린스턴 악기, 트 렌튼, NJ </td><td> 광자 최대 512 </td><td></td></tr><tr><td> 니콘 카메라 렌즈 </td><td> 니콘 주식회사, 멜빌, NY </td><td> 모델 번호 1992 Nikkor 28mm </td><td></td></tr><tr><td> 광학 필터 </td><td> 앤도버 공사, 세일럼, NH </td><td> ANDV11333 </td><td> 두 830.0/10.0 나노 미터 대역 통과 필터는 렌즈 앞에 사용됩니다 </td></tr><tr><td> 785 나노 미터 레이저 다이오드 </td><td> 강렬한 (주), 노스 브런 즈윅, 뉴저지 </td><td> 1005-9 밀리-78503 </td><td> 광 출력 500 MW </td></tr&gt; <tr><td> Collimating 광학 </td><td> Thorlabs, 뉴턴, NJ </td><td> C240TME-B </td><td> 이전 정화 필터 Collimates 레이저 출력 </td></tr><tr><td> 청소 필터 </td><td> Semrock 주식회사, 로체스터, NY </td><td> LD01-785/10-25 </td><td> 형광 밴드에서 레이저 방출을 제거 </td></tr><tr><td> 광 확산 </td><td> Thorlabs, 뉴턴, NJ </td><td> ED1-C20 </td><td> 동물을 통해 레이저를 Diffuses </td></tr><tr><td> V + + </td><td> 디지털 광학, 브라운 베이, 오클랜드, 뉴질랜드 </td><td> 버전 5.0 </td><td> 소프트웨어는 카메라 시스템을 제어하고 컴퓨터에 이미지를 저장하는 데 사용됩니다. <a href="http://digitaloptics.net/" target="_blank">http://digitaloptics.net/은</a> </td></tr><tr><td></td><td></td><td></td><td> 다음과 같은 소프트웨어 패키지의 어느 분석 소프트웨어는 이미지 분석에 사용하실 수 있습니다 </td></tr><tr><td> ImageJ </td><td> 국립 Insti건강, 베데스다, MD의 tutes </td><td> 사용 가능한 최신 버전 </td><td> 에서 프리웨어 가능 <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> </td></tr><tr><td> MATLAB </td><td> MathWorks, Natic​​k, MA </td><td> 버전 2008a 이상 </td><td> <a href="http://www.mathworks.com/" target="_blank">http://www.mathworks.com/</a> </td></tr></tbody></table>

non-invasive optical imaging of the lymphatic vasculature of a mouse

림프 혈관 시스템은 유체 항상성을 유지 배에 면역 감시 및 mediates 지방 흡수를 제공하는 순환 시스템의 중요한 구성 요소입니다. 그러나 그 중요한 기능에도 불구하고 림프 체계는 건강과 질병 1이 기능을 제공하기 위해 적응하는 방법에 비교적 작은 이해가 있습니다. 최근, 우리는 세대 III는 -, 동적​​ 이미지 림프 건축과 림프 등의 추적에 가까운 적외선 형광 (NIRF) 염료의 관리 및 사용자 정의를 사용하여 림프 장애를 앓고 명에서뿐만 아니라 정상적인 인간의 과목에서 &#39;펌핑&#39;작업 할 수있는 기능을 증명 한 이미징 시스템 2-4 강화. NIRF 영상은 인간의 질병과 림프 아키텍처 및 기능에 극적인 변화를 보여 주었다. 그것은 이러한 변경 사항이 발생하여 새로운 동물 모델의 유전자와 분자 기초를 명료하게하다하기 위해 개발되는지는 확실치 남아 있습니다. 이 프로토콜에서는, 우리는 NIRF의 림프, S를 제공쇼핑몰 동물 영상 5,6 녹색 (ICG), 인간 7 50 년 동안 사용 된 염료, 그리고 우선적으로 마우스와 인간의 알부민 8 바인딩 NIRF 염료 표시 순환 알부민 결합 도메인 (cABD-IRDye800) 펩타이드를 indocyanine 사용 . ICG보다 약 5.5 배 더 밝아, cABD - IRDye800은 비슷한 림프 통관 프로필을 보유하고 있으며 영상 8 충분한 NIRF 신호를 달성하기 위해 ICG보다 작은 회에 걸쳐 주입 할 수 있습니다. 중간 공간 8 알부민에 모두 cABD - IRDye800과 ICG 구속, 그들은 모두에 그리고 lymphatics에서 활성 단백질 수송을 묘사 할 수 있습니다 때문입니다. Intradermal (ID) ICG의 주사 (5-50 μl의) (645 μM) 또는 생리에 cABD-IRDye800 (200 μM)는 각 뒷다리 앞발로 땅을 차다 및 / 또는의 기초의 왼쪽과 오른쪽의 등 부분에 관리되는 isoflurane - anesthetized 마우스의 꼬리. 동물의 결과 염료 농도는 83-1,250 μg / ICG에 대한 kg 또는 113-1,700 μg / kg을위한 것입니다cABD - IRDye800. 즉시 주사 후, 기능 림프 이미징은 사용자 정의, 작은 동물 NIRF 이미징 시스템을 사용하여 최대 1 시간에 진행됩니다. 전체 동물 공간적 해상도는 깊이 3cm 9 취득 할 수에 구조의 형광 림프 100 마이크론 이하의 선박, 이미지를 묘사 할 수 있습니다. 이미지는 V + + 소프트웨어를 사용하여 인수 ImageJ 또는 MATLAB 소프트웨어를 사용하여 분석하고 있습니다. 분석하는 동안, 전체 선박 직경을 포함한 관심 (ROIs)의 연속 영역은 주어진 림프 혈관을 따라 그려 있습니다. 각 투자 수익 (ROI)에 대한 크기가 양적 림프가 혈관을 통해 이동의 &quot;패킷&quot;을 평가하기 위해 각 투자 수익 (ROI)에 대한 측정 주어진 선박 및 NIRF 강도에 대한 지속적인 유지하고있다.

마우스의 NIRF의 림프 영상의 예 ICG 또는 cABD-IRDye800는 보통 마우스의 꼬리의 기지에서 ID를 주입되면 꼬리의 기지에서 주입 사이트와 사타구니 림프절 (후면) 사이의 림프 vasculature은 즉시 시각화해야합니다. 얼마 주입 후 (몇 초 분), 사타구니 에선과 겨드랑이 에선 사이의 림프 배는 그림 2에서 볼 시각화해야합니다. 그들은 인간처럼 마우스의 lymphatics 동물에서 동?...

Watch this Scientific Journal Video about Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse at JoVE.com

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

가까운 적외선 형광 (NIRF)를 사용하여 최근 개발 된 이미징 기술은 림프 시스템의 암 전이에서 활약 역할, 면역 반응, 상처 수리 및 기타 림프 - 관련 질병을 명료하게하다하는 데 도움이 될 수 있습니다.

마우스의 림프 Vasculature의 비 침습적 광학 이미징

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

non-invasive-optical-imaging-of-the-lymphatic-vasculature-of-a-mouse

Research

JoVE Journal

Immunology and Infection

16.0K 조회수.  University of Texas Health Science Center-Houston. 가까운 적외선 형광 (NIRF)를 사용하여 최근 개발 된 이미징 기술은 림프 시스템의 암 전이에서 활약 역할, 면역 반응, 상처 수리 및 기타 림프 - 관련 질병을 명료하게하다하는 데 도움이 될 수 있습니다.

비디오: Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

백혈구 호밍 및 마이 그 레이션의 비침습 영상 생체내에</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

연구

면역학 및 감염병학

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

실험 면역 Hypophysitis의 유도를위한 마우스 뇌하수체 Immunogen의 준비

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

동적 라이브 셀 이미징에 대한 호스트 병원체 상호 작용 연구를위한 광학 트랩의 사용

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

배아 마우스에 Vasculature을 라벨링하는 방법

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

마우스 오금 림프절의 Intravital 이미징

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Zebrafish 애벌레의 분해해 칸디다 증의 비침습 이미징

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic의 비 침습 모델<em&gt; 대장균</em&gt; 신생아 쥐에 감염

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

비 침습적 기관 내 삽관 성인 쥐에서 전체 흉선

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

의 Zebrafish의 애벌레 모델의 선천성 면역 반응의 비 침습적 영상<em&gt; 연쇄상 구균 iniae</em&gt; 감염

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

마우스의 실험 세균성 폐렴의 유도 및 표현형에 대한 비 침습적 및 기술적 비 집약적 인 방법

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...