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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.
Bone marrow is an important organ involved in hematopoiesis and immune regulation. It consists of a hematopoietic component containing hematopoietic stem and progenitor cells (HSPCs), and a stromal component containing non-hematopoietic progenitor cells that give rise to mesenchymal cells1. Two-thirds of hematopoietic activity is dedicated to the generation of myeloid cells2. In particular, a large number of neutrophils are produced in the bone marrow, with 1-2 x 1011 cells generated per day in a normal adult human2. Neutrophils are the first line of defense against microbial infections and are mostly reserved in the bone ma....
NOTE: All animal work was carried out under protocol #2615 approved by the University Health Network Institutional Animal Care and Use Committee.
1. Surgical Preparation of Mouse
Murine femoral bone marrow is successfully accessed using the WC to enable visualization of individual neutrophils and vascular networks. Figure 1 shows the WC instrument and describes the surgical procedure, which involves exposure of the femoral bone and thinning of cortical bone to gain optical access inside the bone. The surgery is well-tolerated in mice; they were assessed for adverse physical reactions, such as hind limb swelling, non-weight bearing on limb, self-mu.......
Real-time, serial imaging of the dynamic cellular processes in bone marrow provides information that is otherwise challenging to obtain using conventional techniques such as histology and total blood counts. The femur WC model described here provides unique opportunities to investigate cellular and structural alterations in the bone marrow over time. Although a femur WC model has been previously reported, our novel design provides a larger imaging field of view and smaller overall WC size, which are more compatible for u.......
The authors have no competing financial interests.
The authors would like to thank the Advanced Optical Microscopy Facility (www.aomf.ca) at the University Health Network for assistance with microscopy, and Mr. Jason Ellis from the Princess Margaret Cancer Center Machine Shop for manufacturing the WC and the imaging stage. We would also like to thank Dr. Iris Kulbatski for manuscript editing.
....Name | Company | Catalog Number | Comments |
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 |
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