The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.
Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.
骨髓是参与造血和免疫调节的重要器官。它由含有造血干细胞和祖细胞(骨髓干细胞)造血成分和包含于间充质细胞1产生非造血祖细胞的基质组分。造血活动的三分之二是专门为骨髓细胞2的产生。特别是,在骨髓中产生了大量的中性粒细胞,以在正常的成人每天2生成1-2×10 11个细胞。中性粒细胞是抗微生物感染的防御的第一线,在骨髓大多保留,直到触发器压力的动员,以补充外周血中性1,3。除了它们的抗微生物作用,最近的研究表明在癌症生物学中性粒细胞的重要作用,同时具有亲和抗肿瘤发生的表型取决于转化生长因子β(TGF-β)在肿瘤微环境4,5-信令。此外,研究表明,在原发肿瘤积聚的中性粒细胞产生通过抑制T细胞6,7-的细胞毒性作用的亲致瘤性和转移性的效果,而在循环嗜中性粒细胞产生细胞毒性,抗转移效应8。因此,造血细胞在骨髓中,特别是嗜中性粒细胞,调查是在免疫和肿瘤调节阐明它们的作用是至关重要的。
组织病理学和完整的外周血计数通常用于在骨髓9来评估细胞和结构改变。然而,这些方法仅提供了不同的细胞群或组织中微观结构的静态信息。纵向的体内成像可在结合使用的标准方法来评估多个蜂窝,血管和基质组分的动力学以及细胞到CELL相互作用以纵向方式。活体显微镜(IVM),定义为在显微镜的分辨率10活体动物成像,是对于相同的试样中随时间评估动态的细胞过程,减少了所需的实验动物的数量是特别有用的。 IVM通常与慢性移植窗口室(WC)数周的持续时间,以访问感兴趣的器官成像到几个月组合。颅骨和背皮褶WC车型有使用可以追溯到20世纪90年代中期的历史最长。最近,其他器官特异性厕所模型,例如那些乳腺脂肪垫和各种腹部器官已经开发11。
用于体内成像骨髓典型的方法有小鼠的颅盖,其中所述变薄的骨能以最少的手术干预12-14单个细胞的直接可视化的主要涉及曝光。然而,颅骨骨髓可以bE从其他骨骼,如长骨的不同,这表现在在颅盖的下数骨髓干细胞和缺氧细胞的,这表示降低骨髓干细胞15的维护和发展。因此,评估在长骨细胞成分的替代方法进行了研究。这些措施包括股骨骨髓16的直接暴露和在背皮褶WC 17分股骨移植异位。然而,前者是不允许蜂窝,结构和功能的改变的跟踪在更长的时间段的终端程序,后者可能是由于股骨移植向背侧皮褶厕所内异位位点扰乱正常骨髓的功能。使股骨骨髓原位串行成像随时间的另一种方法是在股骨骨使用WC的。一前一报告显示微循环的长期成像在股骨骨髓使用在小鼠18股骨WC。此外,作者证明肿瘤细胞在股骨的可视化,这表明它在监测骨髓转移工具。然而,这种厕所设计是由它的大尺寸(1.2厘米直径的)和相对小的成像区域(4毫米直径的),这是仅适用于大鼠(26-34克,3-6个月的年龄)的限制,从而使方法不切实际常规使用。
因此,一个新的WC具有较小的整体尺寸,以及较大的内部成像区域被设计为这项研究的目的。本研究的目标是提供用于在股骨骨髓成像各种细胞类型的方法。股骨WC模型内部开发的,并用于在3D血管网内可视化和跟踪中性粒细胞。利用该模型,骨髓IVM可以连续40天进行。这种方法可以适用于各种领域的阐明造血的方法中,免疫调节第二肿瘤的发展。
实时的,在骨髓动态细胞过程的串行成像提供以其它方式挑战,得到使用常规技术,如组织学和总血细胞计数信息。这里所描述的股骨厕所模型提供随时间调查在骨髓细胞和结构改变了独特的机会。虽然股骨WC模型先前已经报道,我们的新颖设计提供的视图和较小的整体WC尺寸较大的摄像视场,这是在成年小鼠使用更兼容。股骨厕所模型可在各种小鼠品系,包括免疫活性和/或转基因报告小鼠?…
The authors have nothing to disclose.
作者要感谢在大学健康网络高级光学显微镜基金(www.aomf.ca),与显微镜的帮助,并从玛嘉烈医院癌症中心的机械车间贾森·埃利斯先生制造WC和成像阶段。我们还要感谢光圈Kulbatski博士稿件编辑。
NRCNU-F athymic nude mice | Taconic | Ncr nude | 8-10 weeks old, female |
Saline | Baxter | JB1302P | |
Ketamine hydrochloride | Bioniche Animal Health Canada, Inc. | DIN 01989529 | |
Xylazine | Bayer HealthCare, Bayer Inc. | DIN 02169592 | |
Surgical drape | Proxima | DYNJP2405 | |
Electric heating pad | Life Brand | 57800827375 | |
Stereomicroscope | Leica | Leica M60 | |
Eye ointment (tear gel) | Novartis | T296/2 | |
7.5% betadine | Purdue Frederick Co | 67618-151-16 | |
70% isopropyl alcohol | GreenField | P010IP7P | |
10% betadine | Purdue Frederick Co | 67618-150-05 | |
Scalpel handle (#3) | Fine Science Tools | 10003-12 | |
Scalpel blade (#15) | VWR | 89176-368 | |
Spring Scissors curved | Fine science Tools | 15023-10 | |
Baby-Mixter Hemostat | Fine science Tools | 13013-14 | |
Fine Scissors | Fine science Tools | 14094-11 | |
Extra Fine Graefe Forceps | Fine science Tools | 11151-10 | |
Halsted-Mosquito Hemostats | Fine science Tools | 13008-12 | |
Micro-drill | Harvard Apparaus | 72-6065 | |
Micro-drill burrs | Fine Science Tools | 19007-14 | |
Femur window chamber | PMCC machine shop | custom design | 9.1mm- 8.5mm- 7.5 mm (outer to inner diameter), 2.16 mm (radius of two holes), 13.9mm (distance between two holes), 0.7mm (thickness) |
U-shaped bar | PMCC machine shop | custom design | 13.8mm (length), 1.6 mm (width), 3.7mm (height) |
Coverglass (8mm) | Warner Instruments | HBIO 64-0701 CS-8R | |
Retaining ring (8mm) | ACKLANDS GRAINGER | UNSPSC # 31163202 | |
Nuts (hexagon stainless steel) | Fastenal | 70701 | |
Dental cement | 3M | RelyX U200 | |
Suture (5-0 Monosof black) | Covioien | SN-5698 | |
Halsey needle holder | Fine Science Tools | 12501-13 | |
Buprenorphine (Temgesic) | Reckitt Benckiser | DIN 0281251 | |
Meloxicam (Metacam) | Boehringer Ingelheim | DIN 02240463 | |
Amoxicillin (Clamavox) | Pfizer | DIN 02027879 | |
FITC-Dextran | Sigma-Aldrich | FD2000S | |
APC- Anti-Mouse Ly-6G (Gr-1) | eBioscience | 17-9668 | |
Two-photon microscope LSM 710 | Carl Zeiss | Zeiss LSM 710 NLO | |
Imaging stage | PMCC machine shop | custom design | 15.9cm (length), 11cm (width), 0,9cm (height) |
Imaris software | Bitplane | Imaris 8.0 | Image analysis software described in Section 3 of the Protocol |
Zen 2012 | Zeiss | Zen 2012 | Image acqusition software described in Section 2 of the Protocol |