Summary

마우스 골수 단핵구를 추적<em> 생체</em

Published: February 27, 2015
doi:

Summary

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Abstract

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Introduction

The bone marrow plays a central role in hematopoiesis and represents the main reservoir of monocytes that constitutively recirculate between the blood and the medullar parenchyma, renew the pool of circulating monocytes with a short life span 2,3 and participate in the reconstitution of the steady state tissue-macrophages and dendritic cells 4. During inflammation or after transient aplasia, monocytes are actively mobilized from either the bone marrow or the spleen 5, 6, 7 and colonize inflamed organs. Several chemoattractant axis have been involved in the process of myeloid cell mobilization from the bone marrow 8, 5, 6,9. Beyond the myeloid compartment the bone marrow is also an important site of T lymphocyte priming 10 and a niche of immunological memory 11,12. Thus, this tissue is central for numerous investigations in the field of hematology and immunology. Our knowledge on the structural organization of medullar myeloid cells mainly arises from the analysis of histological section of fixed tissues 13. This static view does not allow for a study of the cellular exchange dynamic between the different compartments of the bone marrow, which is the basis of its functional activity.

Intravital imaging constitutes an important biological input in the study of cell mobility, cell adherence and cell-to-cell interactions, which were previously described only from in vitro systems. Technical challenges for proper intravital imaging include the ability to reach the tissue of interest in an optical point of view, and to maximize its isolation from physiological (breath, muscle or peristaltic contractions) or mechanical drifts (tissue disruption and extension following surgery, and exposure to microscope objective as well as temperature and vascular/oxygenation perturbations). Microscopic drifts may limit the ability to keep the focus a long time and could introduce artifacts in the quantification of cell motility. One alternative, validated for several tissues to reduce these technical difficulties, is to work on explanted tissue incubated in a thermostated and oxygenated medium; however, complete disruption of the lymphatic and vascular circulation may be problematic. Intravital imaging of skull bone marrow has several advantages concerning these issues. Firstly, it requires minimal surgical action. Secondly, thickness of the bone in this region allows direct visualization of bone marrow niches without abrasion, thus reducing physiological perturbations. The medullar network can be imaged in the parasagittal region of the bone; however the sinusoids are more visible in the fronto-parietal area where the bone matrix is thinner12,14.

Intravital imaging relies on the availability of the most accurate fluorescent reporter tagging the population of interest. In vitro labelling of purified cell population before adoptive transfer led to important characterization of hematopoietic stem cell niches 15 or bone marrow endothelial microdomains favouring tumor engraftment 16, and provided several fundamental inputs on key concepts in immunology 17 . However, this approach usually requires hundreds of thousands of cells to get a chance to detect them afterwards in vivo. This could be explained by the high mortality rate following staining, the dilution in the whole body and the change in the activation state, which might lead to biased homing. Endogenous tagging from transgenic mouse system greatly overcomes these limitations and has allowed to image the behaviour of endogenous osteoblast 8, megakaryocytes 18 or myeloid-lineage subsets 6 . Nevertheless, one has to be cautious when considering the specificity of the fluorescent reporter among the studied subset.

The Csf1r-Gal4VP16/UAS-ECFP, called MacBlue mouse 1, is a valuable transgenic system to study medullar monocytes with real time imaging 6. Intravenous injection of high molecular weight rhodamin-dextran distinguishes the medullar parenchyma from the vascular sinusoid network of the bone marrow. Using this approach, it is possible to track the monocyte behaviour in the different medullar compartments in a specific physiopathological context of interest. Furthermore, we propose an additional strategy to compare monocyte dynamics with that of neutrophils through in vivo labelling using a specific antibody.

Protocol

참고 : 모든 실험 프로토콜 프랑스 동물 실험 윤리위원회의 승인 번호 A-75-2065에 "서비스 보호 등 상테 짐승 같은, ENVIRONNEMENT"에 의해 검증되었다. 샘플 크기는 실험의 재현성을 보장하기 위해 선택, 동물 윤리 규정의 3R에 따라 있습니다. 마우스 1. 준비 마취 촬상의 짧은 기간 (1 시간 미만)를 들면, 케타민 (100 ㎎ / kg) 및 크 실라 진 (10 ㎎ / kg)을 함유?…

Representative Results

마우스 두개골 구조는 생체 내에 영상에 의해 골수 생리학을 연구 할 수있는 좋은 기회를 제공합니다. 프런트 정수리 주변 얇게 뼈, 그것은 뼈의 마모없이 medullar 틈새에 접근 할 수있다. 1 MacBlue 트랜스 제닉 마우스의 두개골 넓은 2D 필드를 나타내는 도표. 뼈 매트릭스는 주로 SHG (19)에 의해 쉽게 검출 I 콜라겐으로 구성되어있다. rhodamin – 덱스 트란의 주입은 골수?…

Discussion

생체 내 영상화 방법의 중요한 점은 영상의 지속 시간을 최대화하고 염증 세포의 역학에 영향을 줄 수있는 세균 오염 및 염증의 위험을 최소화하기 위해 초점 안정성을 보장한다. 수술은 골수에 접근하는 것은 최소한의 수행으로 두개골 골수의 영상은 이러한 목표를 다음과 같습니다. 멸균 물질 및 방부제의 사용은 세포 항상성 교란을 유발할 수있는 감염의 위험을 제한하기 위해 필?…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

저자는 편집 지원 앤 다론 (Daron)과 피에르 루이 Loyher을 감사드립니다, 두 광자 현미경 및 지원을 사육 쥐에 대한 동물 시설 "NAC"와 카밀 Baudesson에 대한 지원은 Plateforme Imagerie Pitié-Salpêtrière (PICPS). 부여 계약에 따라 이러한 결과로 이어지는 연구는 유럽 공동체의 일곱 번째 프레임 워크 프로그램에서 자금 지원을받은 (FP7 / 2007-2013) N 304810 ° – 공습을, n은 대학 학자 피에르 등의 마리 퀴리에서 INSERM에서 241,440 – Endostem을, ° "등장 "라에서" "에서,"리그 contre 르 암 협회는 라 공들인 쉬르 르 암을 부어 "과에서"직원은 국립 드 라 공들인 "프로그램의 출현 2012 (ANR-EMMA-050). PH는 라 "리그 contre 르 암"에 의해 지원되었다.

Materials

Name of Material/ Equipment Company Catalog Number Comments/Description
Ketamin Merial 100mg/mL, anesthetic
Xylazin Bayer HealthCare 10mg/mL, anesthetic
Isofluran Baxter 2.5%, anesthetic
O2/NO2 70/30 mixture, anesthetic
Rhodamin-Dextran Invitrogen 2MDa, 10mg/mL, Vascular staining
Ly6G-PE Becton-Dickinson clone 1A8, neutrophils staining
Stereotactic holder Home made surgery
Ethanol 70% surgery
Sterile scissors and nippers surgery
Rubber ring 18mm diameter, surgery
Glubran 2 Queryo Medical Surgical Glue, rubber ring fixation
Small gauge needles Terumo surgery
Zeiss LSM 710 NLO multiphoton microscope  Carl Zeiss Microscope
Ti:Sapphire crystal laser  Coherent Chameleon Ultra 140fs pulses of NIR light
Zen 2010 Carl Zeiss Acquistion Software
Imaris Bitplane  Bitplane Analysis Software, 3D automatic tracking
PBS 1X D. Dutscher surgery
Thermostated chamber Carl Zeiss intravital imaging

Referenzen

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Hamon, P., Rodero, M. P., Combadière, C., Boissonnas, A. Tracking Mouse Bone Marrow Monocytes In Vivo. J. Vis. Exp. (96), e52476, doi:10.3791/52476 (2015).

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