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 marrow until stress triggers their mobilization to supplement peripheral neutrophils1,3. In addition to their anti-microbial effects, recent studies suggest an important role of neutrophils in cancer biology, having both pro- and anti-tumorigenic phenotypes depending on transforming growth factor beta (TGF-β) signaling in the tumor microenvironment4,5. Moreover, studies demonstrated that neutrophils that accumulate in primary tumors exert pro-tumorigenic and metastatic effects by suppressing the cytotoxic function of T cells6,7, while neutrophils in circulation exert a cytotoxic, anti-metastatic effect8. As such, investigation of hematopoietic cells in the bone marrow, particularly neutrophils, is crucial to elucidating their role in immune and tumor regulation.
Histopathology and a complete peripheral blood count are routinely used to evaluate cellular and structural alterations in the bone marrow9. However, these methods only provide static information of different cell populations or tissue microstructures. Longitudinal in vivo imaging can be used in combination with the standard methods to assess the dynamics of multiple cellular, vascular and stromal components as well as cell-to-cell interactions in a longitudinal manner. Intravital microscopy (IVM), defined as imaging of living animals at microscopic resolution10, is particularly useful for assessing dynamic cellular processes over time in the same sample, reducing the number of experimental animal required. IVM is often combined with a chronically transplanted window chamber (WC) to access the organ of interest for imaging over a duration of weeks to months. Cranial and dorsal-skinfold WC models have the longest history of use dating back to the mid 1990s. More recently, other organ-specific WC models such as those of the mammary fat pad and various abdominal organs have been developed11.
The typical approach for imaging bone marrow in vivo has mainly involved exposure of the calvaria of mice, where the thinned bone enables direct visualization of single cells with minimal surgical intervention12-14. However, the calvarial bone marrow may be distinct from that of other bones, such as the long bone, as demonstrated by a lower number of HSPCs and hypoxic cells in calvaria, which indicates reduced maintenance and development of HSPCs15. Therefore, alternative approaches for assessing cellular components in the long bone have been investigated. These include direct exposure of the femoral bone marrow16 and ectopic transplantation of split femur in the dorsal skinfold WC17. However, the former is a terminal procedure that does not permit tracking of cellular, structural and functional alterations over longer time periods, and the latter likely disturbs normal bone marrow function due to the transplantation of femur to an ectopic site inside the dorsal skinfold WC. Another method that enables orthotopic serial imaging of femoral bone marrow over time is the use of a WC in the femoral bone. One previous report demonstrated long-term imaging of microcirculation in the femoral bone marrow using a femur WC in mice18. Additionally, the authors demonstrated visualization of tumor cells in the femur, indicating its utility in monitoring bone marrow metastasis. However, this WC design was limited by its large size (1.2 cm diameter) and relatively small imaging area (4 mm diameter), which was only suitable for large mice (26-34 g, 3-6 months of age) thereby making the approach impractical for routine use.
Therefore, a new WC with a smaller overall size and larger inner imaging area was designed for the purpose of this study. The goal of this study was to provide a method for imaging various cell types in the femoral bone marrow. The femur WC model was developed in-house and was used to visualize and track neutrophils within the 3D vascular network. Using this model, IVM of the bone marrow can be performed serially over 40 days. This approach can be applied to a variety of fields for elucidating the processes of hematopoiesis, immune regulation and tumor development.
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
2. Image Acquisition Using Combined Multi-photon and Confocal Microscopy
3. Image Analysis and Quantification
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-mutilation, cellulitis and dermatitis, as well as damage to the WC for 5 days post-surgery, with no report of distress. Notably, the window remains optically clear for IVM for up to 40 days, which may enable long-term assessment of cellular and structural alterations following an intervention.
Vasculature within the bone marrow can be visualized by intravascular dyes, such as FITC-Dextran (Figure 2). Since the contrast is dependent on the injection of the dye, the acquired image represents functional vasculature where there is sufficient blood flow. In addition to the vasculature, neutrophils can be stained in vivo using anti-Ly6G antibody. Figure 3A and 3B represent the same area of the bone marrow observed at different time points, where neutrophils are seen both in the intravascular and extravascular space. These areas were characterized by the vascular landmarks. Rhodamine 6G may be used as an alternative to anti-Ly6G to stain neutrophils in vivo; however, it diffuses out of the vasculature and stains monocytes in the extravascular space in a non-specific manner. Therefore, anti-Ly6G is preferred because it enables specific imaging of neutrophils. Figure 3C represents imaging of the cortical bone, which is mainly composed of collagen fibers. The maximal depth for imaging achieved in this experiment is 180 μm (Figure 3C).
Time-lapse fluorescence imaging facilitates tracking and quantification of cellular movement in vivo. Figure 4A shows tracking of neutrophils in the bone marrow vascular niche. Imaging with no additional time interval between each scan allows for accurate tracking of cells; however, some user input is required to further improve the accuracy of tracks generated by the software. Tracking of spots (i.e., cells of interest) enables quantification of various parameters, such as mean track speed (Figure 4B). Other parameters that can be measured include, but are not limited to, track length, change in spot size, shape and intensity. This assay provides quantification of various parameters that improve the characterization of cellular movements and interactions.
Figure 1: Femur window chamber (WC), imaging stage and the surgical procedure. (A) Custom-made titanium femur WC with a U-shaped bar that serves to fix the femur in place. The WC holds an 8 mm diameter cover glass, secured by a snap ring. (B) Custom-made imaging stage. A heating element is placed on the bottom of the stage to maintain the physiological temperature of the mouse during imaging. The two alligator clips are used to hold the WC. (C–H) Surgical procedure for installing the femur WC. (C) The femur is exposed between the flexor and extensor muscles. Muscles are not removed during the procedure. (D) The U-shaped bar is placed under the femur, and (E) the WC is installed and secured using two nuts. (F) Dental cement is applied between the bone and the WC to fill the space. (G) The dermis is sutured and (H) the cover glass is fixed in place with the retaining ring. Please click here to view a larger version of this figure.
Figure 2: 3D fluorescence imaging of vascular network in the bone marrow in vivo. Representative image of vasculature in the bone marrow, imaged through the femur WC. Vasculature (green) is visualized by tail vein injection of 2 MDa FITC-Dextran. Scale bar = 100 μm. Please click here to view a larger version of this figure.
Figure 3: 3D imaging of vascular network, neutrophils and bone matrix in vivo. Images of vasculature and neutrophils in the bone marrow (A) 3 days and (B) 10 days after the femur WC surgery. Neutrophils, stained by tail vein injection of APC-labeled Ly6G antibody, are seen both in the intravascular and extravascular spaces. The arrows point to the vascular structure seen on both days, indicating the ability to track the same location over time. Scale bar = 50 μm. (C) Bone matrix can be visualized using SHG (white), in addition to vasculature (green) and neutrophils (red). The image was acquired 40 days after the WC surgery. Scale bar = 70 μm. Please click here to view a larger version of this figure.
Figure 4: Tracking neutrophil movement within the vascular network of the bone marrow in vivo. (A) Track paths of neutrophils within the vascular network. Tracks are generated automatically using the software's built-in Autoregressive Motion algorithm. Each white dot represents a center point of a particle being tracked (neutrophils). Scale bar = 50 μm. (B) Mean track speed measured for intravascular and extravascular neutrophils, indicating difference in the movement of neutrophils (Error bar = mean + SD, * p = 0.0176, two-tailed t-test). Please click here to view a larger version of this figure.
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 use in adult mice. The femur WC model can be used in a variety of mouse strains including immunocompetent and/or transgenic reporter mice.
There are critical steps during the surgery that determine the overall duration and quality of imaging. When exposing the femur, only a blunt dissection of muscles between flexors and extensors is performed; removal of these muscles may result in reduced mobility of the mouse and must be avoided. Furthermore, grinding of the cortical bone must be performed carefully. Grinding approximately 50 μm is adequate for optical imaging while maintaining the structural integrity of the femoral vascular network. Application of dental cement around the bone is crucial for sealing the surgically exposed area, preventing fluid accumulation, and maintaining a clear optical imaging window for over a month while reducing the risk of infection. The dental cement also acts to fix the WC in place, preventing the WC from rotating around the femoral bone. Lastly, the snap ring used to hold the coverglass is suitable for use with water-immersion lens, providing water-seal. The snap ring is recommended over adhesives to attach the coverglass to the WC because addition of adhesives may increase the gap between the bone and the coverglass, or place the coverglass at a slight angle relative to the imaging plane, thereby reducing the achievable penetration depth for imaging. Furthermore, the snap ring is useful when the coverglass needs to be cleaned or changed.
In addition to the surgery, there are several critical steps to achieve successful optical imaging of the bone marrow. To image inside the femur WC, the WC is placed horizontally to the imaging stage for proper immersion of the objective lens. The custom-made imaging stage is suitable for this purpose as it allows for adjustments of the angle of the WC with respect to the lens. Moreover, the design of the WC, particularly the size of the U-shaped bar, maintains the bone in a horizontal position as close to the coverglass as possible to achieve optimal penetration depth. During imaging, breathing artifacts may be observed as horizontal lines in the fluorescence images. In addition to securing the WC to the imaging stage, the temperature of the heating element under the imaging stage may be adjusted to adequately warm the animal to minimize excessive breathing. A thin layer of gauze may be placed between the mouse and the heated stage to further help reduce breathing artifacts during imaging. Depending on the biological imaging target, acquisition parameters such as frame rate, imaging volume and imaging duration may also be adjusted to collect sufficient images to address experimental questions. For example, velocity of neutrophil migration and crawling may differ under various experimental conditions, from 2.8 μm/min for resident neutrophils under basal conditions to anywhere ranging from 5-10 μm/min for neutrophils under inflammatory conditions19-21. Thus, the appropriate imaging speed and duration for neutrophil tracking in vivo would also differ based on experimental conditions. In addition, as seen from the results, tracking of cells within the vasculature would require a fast imaging camera with a high frame rate (30 frames/sec). However, if the target cells are residing in extravascular space, or if the cell-to-cell interaction of interest is expected to occur more slowly, image quality should be prioritized over imaging speed.
The influence of injected antibodies for in vivo cell staining on cellular function must also be considered. In our study, we used anti-Ly6G antibody (1A8 clone), which is typically used at high doses (150-250 μg) to deplete neutrophils from circulation and study their role in various disease models. Yipp and Kubes demonstrated that fluorochrome-labeled anti-Ly6G antibody does not disrupt leukocyte recruitment at low intravascular injection doses (1-40 μg)22. However, a more recent study suggests that fluorochromes, having great differences in molecular mass and chemical structure, can differentially influence the function of anti-Ly6G 23. The study demonstrated that FITC-labeled anti-Ly6G, but not PE- and APC-labeled antibodies, effectively depletes neutrophils in vivo at a low concentration (30 μg). In our study, we did not observe neutrophil depletion overtime using 4 μg of APC-labeled anti-Ly6G. Nevertheless, further characterization of the function of fluorochrome-labeled anti-Ly6G antibody in vivo is required to validate its use in intravital imaging. This also applies to other antibodies that have functional ability to alter cellular interactions. As an alternative to in vivo cell staining with antibodies, transgenic reporter mice24 and/or labeled fluorescent cells25 can be used to visualize neutrophils or other cell population of interest in vivo.
The femur WC model described in this protocol enables imaging for up to 40 days after the surgery. Some of the challenges for long-term imaging using this WC model include the delayed bone healing process and maintenance of a clean glass window. Notably, we did not observe signs of obvious bone healing following the WC implantation, which would include thickening of periosteum; this is not unexpected given that bone healing process typically occurs after 1 ½-3 months26,27. Maintenance of a clean and optically-clear window can be achieved by the application of the dental cement, and the use of snap ring and coverglass which provide a firm water-seal with the tissue. The coverglass can be occasionally cleaned with cotton tips from the outside to remove dust and debris. The WC has a wide imaging field, almost covering the entire length of the diaphysis region (approximately 8.4-8.6 mm28), which makes it superior to the previously published model18. However, access to the distal regions and the deeper marrow cavity is limited. In particular, the trabecular-rich metaphysis, located at the distal ends of the diaphysis, is known to be rich in HPSCs29. These areas may be accessed histologically, or by IVM as a terminal procedure. Alternatively, different imaging modalities such as micro-computed tomography can be used to image the distal regions of the bone marrow in vivo30. Such methods are complementary to IVM, providing an integrated approach for assessing anatomy and the various cellular and structural alterations and interactions within the bone marrow.
The protocol described here offers a novel approach for long-term spatio-temporal assessment of the femoral bone marrow in mice at cellular resolution. The methods presented enable in vivo serial imaging to track movements of a single cell or cellular populations over biologically relevant time scales, while simultaneously obtaining structural and functional information of the bone marrow. In addition, quantitative image analysis can be performed to further characterize cellular and functional alterations, such as tracking of cellular movements. Application of this femur WC model in various strains of mice, particularly in transgenic reporter mice31, may further enable investigation of multiple individually-labeled cell populations such as those involved in hematopoiesis. Therefore, the described protocol is widely applicable to a wide range of preclinical studies involving various biological processes within the living murine bone marrow, including investigations in hematopoiesis, leukemia, HPSC transplantation, immune regulation, the bone marrow microenvironment (e.g., hypoxic niche) and the contribution of immune cells to tumor progression and metastasis.
The authors have nothing to disclose.
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.
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 |