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.

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
<ol>
	<li>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.</li>
	<li>Anesthetize the athymic...

<ol>
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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-17-producing+[ggr][dgr]+T+cells+and+neutrophils+conspire+to+promote+breast+cancer+metastasis.">IL-17-producing [ggr][dgr] T cells and neutrophils conspire to promote breast cancer metastasis.</a> Nature. 522 (7556), 345-348 (2015).</li><li>Kitamura, T., Qian, B. Z., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+promotion+of+metastasis.">Immune cell promotion of metastasis.</a> Nat Rev Immunol. 15 (2), 73-86 (2015).</li><li>Granot, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+entrained+neutrophils+inhibit+seeding+in+the+premetastatic+lung.">Tumor entrained neutrophils inhibit seeding in the premetastatic lung.</a> Cancer Cell. 20 (3), 300-314 (2011).</li><li>Travlos, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+structure,+function,+and+histology+of+the+bone+marrow.">Normal structure, function, and histology of the bone marrow.</a> Toxicol Pathol. 34 (5), 548-565 (2006).</li><li>Pittet, M. 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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...

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-&#946;) 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>NRCNU-F athymic nude mice</td>
			<td>Taconic</td>
			<td>Ncr nude</td>
			<td>8-10 weeks old, female</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Baxter</td>
			<td>JB1302P</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Bioniche Animal Health Canada, Inc.&#160;</td>
			<td>DIN 01989529</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer HealthCare, Bayer Inc.</td>
			<td>DIN 02169592</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical drape</td>
			<td>Proxima</td>
			<td>DYNJP2405</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric heating pad</td>
			<td>Life Brand</td>
			<td>57800827375</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>Leica M60</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment (tear gel)</td>
			<td>Novartis&#160;</td>
			<td>T296/2</td>
			<td></td>
		</tr>
		<tr>
			<td>7.5% betadine</td>
			<td>Purdue Frederick Co</td>
			<td>67618-151-16</td>
			<td></td>
		</tr>
		<tr>
			<td>70% isopropyl alcohol</td>
			<td>GreenField</td>
			<td>P010IP7P</td>
			<td></td>
		</tr>
		<tr>
			<td>10% betadine</td>
			<td>Purdue Frederick Co</td>
			<td>67618-150-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle (#3)</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade (#15)</td>
			<td>VWR</td>
			<td>89176-368</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors curved</td>
			<td>Fine science Tools</td>
			<td>15023-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Baby-Mixter Hemostat</td>
			<td>Fine science Tools</td>
			<td>13013-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine science Tools</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra Fine Graefe Forceps</td>
			<td>Fine science Tools</td>
			<td>11151-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsted-Mosquito Hemostats</td>
			<td>Fine science Tools</td>
			<td>13008-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-drill</td>
			<td>Harvard Apparaus</td>
			<td>72-6065</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-drill burrs</td>
			<td>Fine Science Tools</td>
			<td>19007-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Femur window chamber</td>
			<td>PMCC machine shop</td>
			<td>custom design</td>
			<td>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)</td>
		</tr>
		<tr>
			<td>U-shaped bar</td>
			<td>PMCC machine shop</td>
			<td>custom design</td>
			<td>13.8mm (length), 1.6 mm (width), 3.7mm (height)</td>
		</tr>
		<tr>
			<td>Coverglass (8mm)</td>
			<td>Warner Instruments&#160;</td>
			<td>HBIO 64-0701 CS-8R</td>
			<td></td>
		</tr>
		<tr>
			<td>Retaining ring (8mm)</td>
			<td>ACKLANDS GRAINGER</td>
			<td>UNSPSC # 31163202</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuts (hexagon stainless steel)</td>
			<td>Fastenal</td>
			<td>70701</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>3M</td>
			<td>RelyX U200</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture (5-0 Monosof black)</td>
			<td>Covioien</td>
			<td>SN-5698</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey needle holder</td>
			<td>Fine Science Tools</td>
			<td>12501-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine (Temgesic)</td>
			<td>Reckitt Benckiser</td>
			<td>DIN 0281251</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam (Metacam)</td>
			<td>Boehringer Ingelheim</td>
			<td>DIN 02240463</td>
			<td></td>
		</tr>
		<tr>
			<td>Amoxicillin (Clamavox)</td>
			<td>Pfizer</td>
			<td>DIN 02027879</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD2000S</td>
			<td></td>
		</tr>
		<tr>
			<td>APC- Anti-Mouse Ly-6G (Gr-1)&#160;</td>
			<td>eBioscience</td>
			<td>17-9668</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-photon microscope LSM 710</td>
			<td>Carl Zeiss</td>
			<td>Zeiss LSM 710 NLO</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging stage</td>
			<td>PMCC machine shop</td>
			<td>custom design</td>
			<td>15.9cm (length), 11cm (width), 0,9cm (height)</td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td>Imaris 8.0</td>
			<td>Image analysis software described in Section 3 of the Protocol&#160;</td>
		</tr>
		<tr>
			<td>Zen 2012</td>
			<td>Zeiss</td>
			<td>Zen 2012</td>
			<td>Image acqusition software described in Section 2 of the Protocol&#160;</td>
		</tr>
	</tbody>
</table>

femur window chamber model for in vivo cell tracking in the murine bone marrow

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.

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...

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Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

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.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

The overall goal of this procedure is to perform long-term microscopic imaging and tracking of neutrophils in vascular structures within the murine bone marrow through an implanted chronic window chamber over the femoral bone. This method can help answer key questions in immunology and oncology about the role of monocytes in the regulation of an infection, tumorigenesis, and tumor metastasis. The main advantages of this technique are that it enables long-term microscopic access to the femoral bone marrow, a central site of hematopoiesis, and provides dynamic information otherwise difficult to obtain by conventional methods.Though this method is specific to neutrophil imaging within a vascular network, it may also be used to visualize other immune cell populations of interest. On the day of the surgery, confirm the appropriate level of sedation by lack of response to a toe pinch of an athymic nude mouse. Then, place the mouse on an electric heating pad, covered with a surgical drape, under a stereo microscope.Next, apply ointment to the animal's eyes, and sterilize the surgical area with two sequential wipes with 7.5%povidone-iodine, 70%isopropyl alcohol, and 10%povidone-iodine. When the animal has been prepped, use a scalpel with a number 15 blade to make a 10 millimeter longitudinal skin incision approaching the femur from the lateral side. Then, blunt dissect with forceps and hemostats to expose the femur between the flexor and the extensor muscles.When the femur can be observed, insert a U-shaped bar under the bone, and install the window chamber. Using two nuts, secure the bar to the window. Then, tighten the nuts with forceps.Use the forceps to readjust the musculature of the flexors and extensors back to their original locations. Fill the space between the bone and the window chamber with dental cement to fix the window chamber in place. Then, use a micro drill to grind a six by two millimeter area into the cortical bone to gain optical access to the marrow cavity.When an almost transparent periosteal layer of the bone has been obtained, place an eight millimeter cover glass onto the window, and use a snap ring to secure the cover glass in place. Now, close the dermis with a 5-0 suture and a needle holder. Then, place the mouse in a new cage with clean bedding, with monitoring until it is fully recovered.To obtain combined multiphoton and confocal microscopy images through the window chamber, first, inject the appropriate fluorescent vasculature neutrophil labeling solutions into the mouse by tail vein. Clamp the ends of the window chamber with alligator clips to affix the mouse to custom-made stage. Then, place the imaging stage onto the combined multiphoton and confocal microscope.When the mouse is secure, turn on the two-photon laser and the confocal system, and click Start System to launch the image acquisition software. To set up the imaging channels, under the Acquisition tab, click Smart Setup, and select the lasers for imaging. Choose Best signal, and click Apply.Manually add the second harmonic generation image acquisition channel by turning on the two-photon laser in the Laser window, and setting the appropriate acquisition wavelengths for second harmonic generation. Under the Locate tab, click Oculars online, and locate the region of interest through the microscope by visual inspection. Click Oculars offline, and open the Acquisition tab.In the Channels window, select the FITC Track, and click on Live to preview the FITC-Dextran image with a 5x objective. Adjust the focus as necessary. Then, click Stop under the Acquisition tab to stop the preview.Now, manually select the 20x water objective and view the FITC-Dextran image again, adjusting the master Gain and Pinhole under the Channels window to achieve the best signal and contrast. When an optimal image has been obtained for all three channels, select all of the channels in the Channels window, and click Snap under the Acquisition tab to acquire a 2D snapshot. Next, to acquire a 2D time-lapse image, select the Time Series to open the Time Series window, and enter zero for the intervals and 40 for the total number of scans to obtain 40 images continuously without a time interval.To analyze the images, open the appropriate image analysis software, and open an image file. To generate object tracks using the time-lapse images, in the Surpass view, open the image to be analyzed, and click on the icon with an image of orange dots to open the new Spots window. After setting Tracking parameters, click the Fill gaps with all detected objects box to start the tracking.And click the blue arrow to go to the next step. Under the Filter Type, select the Track Duration filter, and click on the green arrow to finish the tracking. Then, check the generated tracks, going back to the previous step as necessary.To measure the cellular movement, defined by the Object tracks in the time-lapse images, under the Edit Tracks window, select the Tracks box, and select a track that corresponds to the movement of the cell of interest. The selected track will be highlighted in yellow in the time series image. Finally, under the Statistics tab, click on the icon with an image of a graph to open the Statistics window.Then, under Selection, click on the down arrow to generate the Track Speed Mean. Vasculature within the bone marrow can be visualized by intravascular dyes, such as FITC-Dextran. Since the contrast is dependent on the injection of the dye, the acquired image represents the functional vasculature where there is sufficient blood flow.These images of the same area of the bone marrow at different time points demonstrate the presence of neutrophils in both the intravascular and extravascular spaces over a period of several days. The imaging of this cortical bone, mainly composed of collagen fibers, was performed by second harmonic generation, further illustrating the infiltration of the vascular tissue by neutrophils. These neutrophils were tracked in the bone marrow vascular niche with no additional time interval between each scan, allowing for the accurate tracking of their movement.The speed of the tracks generated by the image analysis software can then be measured to analyze the velocity of each cell. Following this procedure, other methods, such as using ex vivo label cells or transgenic reporter mice, can be used to evaluate other biological processes in situ, such as the influence of immune cells on tumor progression and metastasis. After watching this video, you should have a good understanding of how to install window chamber on the murine femoral bone for tracking neutrophils through the bone marrow vascular niche and collagen structures.

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Research

JoVE Journal

Immunology and Infection

8.1K Views.  Princess Margaret Cancer Centre. The overall goal of this procedure is to perform long-term microscopic imaging and tracking of neutrophils in vascular structures within the murine bone marrow through an implanted chronic window chamber over the femoral bone. This method can help answer key questions in immunology and oncology about the role of monocytes in the regulation of an infection, tumorigenesis, and tumor metastasis. The main advantages of this technique are that it enables long-term microscopic access to the femoral...