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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

  1. Prior to surgery, sterilize all surgical instruments and the window chamber (WC) by autoclaving. Supplement the drinking water with 50 mg/kg body weight of amoxicillin 2 days prior to surgery. Inject the mouse with 0.1 mg/kg body weight of buprenorphine intraperitoneally 4 hr prior to surgery.
  2. Anesthetize the athymic nude mouse by intraperitoneal injection of 350-400 μl saline solution containing 80 mg/kg ketamine and 13 mg/kg xylazine (Note: This was ap....

Representative Results

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.......

Discussion

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.......

Disclosures

The authors have no competing financial interests.

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
NRCNU-F athymic nude miceTaconicNcr nude8-10 weeks old, female
SalineBaxterJB1302P
Ketamine hydrochlorideBioniche Animal Health Canada, Inc. DIN 01989529
XylazineBayer HealthCare, Bayer Inc.DIN 02169592
Surgical drapeProximaDYNJP2405
Electric heating padLife Brand57800827375
StereomicroscopeLeicaLeica M60
Eye ointment (tear gel)Novartis T296/2
7.5% betadinePurdue Frederick Co67618-151-16
70% isopropyl alcoholGreenFieldP010IP7P
10% betadinePurdue Frederick Co67618-150-05
Scalpel handle (#3)Fine Science Tools10003-12
Scalpel blade (#15)VWR89176-368
Spring Scissors curvedFine science Tools15023-10
Baby-Mixter HemostatFine science Tools13013-14
Fine ScissorsFine science Tools14094-11
Extra Fine Graefe ForcepsFine science Tools11151-10
Halsted-Mosquito HemostatsFine science Tools13008-12
Micro-drillHarvard Apparaus72-6065
Micro-drill burrsFine Science Tools19007-14
Femur window chamberPMCC machine shopcustom design9.1mm- 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 barPMCC machine shopcustom design13.8mm (length), 1.6 mm (width), 3.7mm (height)
Coverglass (8mm)Warner Instruments HBIO 64-0701 CS-8R
Retaining ring (8mm)ACKLANDS GRAINGERUNSPSC # 31163202
Nuts (hexagon stainless steel)Fastenal70701
Dental cement3MRelyX U200
Suture (5-0 Monosof black)CovioienSN-5698
Halsey needle holderFine Science Tools12501-13
Buprenorphine (Temgesic)Reckitt BenckiserDIN 0281251
Meloxicam (Metacam)Boehringer IngelheimDIN 02240463
Amoxicillin (Clamavox)PfizerDIN 02027879
FITC-DextranSigma-AldrichFD2000S
APC- Anti-Mouse Ly-6G (Gr-1) eBioscience17-9668
Two-photon microscope LSM 710Carl ZeissZeiss LSM 710 NLO
Imaging stagePMCC machine shopcustom design15.9cm (length), 11cm (width), 0,9cm (height)
Imaris softwareBitplaneImaris 8.0Image analysis software described in Section 3 of the Protocol 
Zen 2012ZeissZen 2012Image acqusition software described in Section 2 of the Protocol 

References

  1. Zhao, E., et al. Bone marrow and the control of immunity. Cell Mol Immunol. 9 (1), 11-19 (2012).
  2. Borregaard, N. Neutrophils, from marrow to microbes. Immunity. 33 (5), 657-670 (2010).
  3. Boxio, R., Bossenmeyer-Pourie, C., St....

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Femur Window ChamberIn Vivo Cell TrackingMurine Bone MarrowNeutrophil ImagingVascular NetworkHematopoiesisImmunologyOncologyTumorigenesisTumor MetastasisAthymic Nude MouseSurgical ProcedureMicroscopyMultiphotonConfocal

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