Name: a novel high-resolution in vivo imaging technique to study the dynamic response of intracranial structures to tumor growth and therapeutics
Uploaded: 2013-06-16T14:00:00
Duration: 12 min 9 s
Description: Watch this Scientific Journal Video about A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics at JoVE.com

We would like to thank the Advanced Optical Microscopy Facility at the princess Margaret hospital, in particular James Jonkman for their assistance in the initial setup of channels on the 2Photon microscope. The authors would like to acknowledge the Spatio-Temporal Targeting and Amplification of Radiation Response (STTARR) program and its affiliated funding agencies. We thank Dr. Peter Tonge, Dr.Iacovos Michael and Dr.Andras Nagy for supplying the VEGFTrap plasmids and for their manuscript corrections and feedback. The continued support and discussion from staff at the BTRC has been invaluable and we would like to Commemorate Dr.Abhijit Guha for his scientific input. Work was funded by CIHR and NIH grants.

ALL ANIMAL WORK HAS BEEN CARRIED OUT UNDER AN ANIMAL CARE AND USE COMMITEE APPROVED PROTOCOL AND EXECUTED IN COMPLIANCE WITH ALL RELEVANT GUIDELINES, REGULATIONS AND REGULATORY AGENCIES.
For troubleshooting reference Table 2.
1. Bone Marrow Reconstitution (Optional) Figure 1 (30 min prep, 5 min per mouse)
All surgery should be performed using strict aseptic technique with autoclaved sterile equipment.
One donor mouse will reconstitute three host mice.
<ol>
	<li>Anaesthetize recipient NODscid mice and position, with lead to shield the head, inside the center of the Gammacell 40 'exactor' irradiator.</li>
	<li>Irradiate NODscid mice with 2.5 Gy total body irradiation (TBI).</li>
</ol>
CRITICAL STEP: Optimization of TBI may be needed due to variation in dose required for sufficient host immune cell depletion in different mouse strains.
Pause point: Host mice can be irradiated in advance but should be used within 24 hr of irradiating.
<ol start="3">
	<li>Euthanize donor mice according to the institutional animal care committee guidelines. Clean the hindlimbs and remove the tibia and femur from both, stripping all excess tissue from the bones (Figures 1ai, 1aii).</li>
</ol>
CRITICAL STEP: Fibular bones are not viable for use due to very narrow lumen.
<ol start="4">
	<li>Remove end plates from both ends of four extracted bones and flush them out using a 22 G needle and 1 ml sterile PBS until they are white in appearance (Figure 1aiii). Reference Table 2.</li>
	<li>Mix the extracted bone marrow (BM) suspension well and draw 300 &mu;l into three 27 G tuberculin needles. Extracted BM should contain approximately 2 x 107 cells and is enough for 3 host mice reconstitutions. Reference Table 2.</li>
	<li>Inject the suspension into the lateral tail vein of three previously irradiated NODscid mice from step 1.1 (Figure 1b). Reference Table 2.</li>
</ol>
CAUTION: To check sterility of the BM injected we recommend plating the remaining BM and culture for 24 hr to check for infection. Primary cause of death at this point is infection in the newly reconstituted mice.
2. Intracranial Window Generation - Figure 2 (30 min per mouse)
All surgery should be performed using strict aseptic technique with autoclaved sterile equipment and under a heat lamp to keep animals warm (Figure 2A).
<ol>
	<li>Anaesthetize recipient NODscid mice with IACUC approved anesthetic, Avertin IP injection at 0.5 mg/g and position, with lead to shield the head, inside the center of the Gammacell 40 'exactor' irradiator. Remove hair from the scalp. In addition apply tear gel to prevent corneal dehydration.</li>
	<li>Clean scalp first with Betadine solution and then with alcohol, then make an incision from the midpoint of the ears to just above the eyes. Remove scalp 3 mm either side of the first incision, exposing the skull and landmarks of the skull surface (Figure 2Bi).</li>
	<li>Elevate the periosteum by injecting 2% lidocaine:epinephrine solution and dissect away from skull surface (Figure 2Bii).</li>
	<li>Using 2.7 mm trephine drill, weaken a 2.7 mm circle of the skull over the cortex of the right hemisphere between lambda and bregma. DO NOT penetrate the bone with the drill (Figure 2Biii). Reference Table 2.</li>
</ol>
CRITICAL STEP: if drill goes through the skull the brain surface will be damaged influencing results with additional trauma. In addition excessive bleeding will occur preventing a clear window from being generated.
<ol start="5">
	<li>Remove the weakened bone flap using dissection tweezers and dental hook and deliberate but controlled force (Figure 2Biii).</li>
	<li>(Optional) If looking at tumor pathology, inject chosen tumor cell lines (with fluorescent reporter gene) into the center of the window generated in step 2.5.
	<ol>
		<li>Load 10 &mu;l 30 G Hamilton syringe with cell suspension and load needle into a stereotactic frame. 
		CRITICAL STEP: Cell number will need to be optimized for tumor cells in use and timeline of growth required. For U87 cultures we use 2 x 105 cells in 10 &mu;l per mouse.</li>
		<li>Load mouse onto the digital stereotactic frame align the needle to the center point of the window generated.</li>
		<li>Lower needle until it just touches the cortical surface and reset the digital coordinates.</li>
		<li>Lower the needle to 3.2 mm into the cortical tissue and inject at 3 mm deep.</li>
		<li>Retract needle slowly then remove mouse from the frame. 
		CRITICAL STEP: Inject solution over the duration of 1 min and leave needle in position following injection to ensure reduced back flow. 
		CRITICAL STEP: If minor hemorrhaging is encountered irrigate with sterile saline for 1-5 min to stop superficial bleeding. Proceed with subsequent steps.</li>
	</ol>
	</li>
	<li>Dampen brain surface with a drop of sterile PBS to keep the brain tissue irrigated.</li>
	<li>Float a 3 mm coverslip onto brain surface to seal fully around the 2.7 mm window generated. Reference Table 2.</li>
	<li>Dry surrounding skull bone then apply vetbond to the entire exposed skull to reseal scalp tissue to the skull bone and the coverslip in place. DO NOT apply an excess as it will leak under the glass and decrease the imaging potential.</li>
	<li>Mix fresh dental acrylic powder and solution, roughly 30% (w/v), and apply over the top of the vetbond. To ensure a tight seal overlap the acrylic slightly onto the glass coverslip edge (Figure 2Biv). Reference Table 2.</li>
</ol>
CAUTION: Dental acrylic is extremely hazardous so we recommend use of gloves and mask throughout the procedures.
CRITICAL STEP: Dental acrylic needs to remain pliable during molding to ensure a good seal and reduce excess. Build up of acrylic on the window will blur images.
<ol start="11">
	<li>Allow mice to recover in a warm cage.</li>
</ol>
3. Stereotactic Radiation (Optional) - Figure 3 (25 min per mouse)
Variations will exist in the different irradiators used and as such optimization will be required. We used a custom designed irradiator.
<ol>
	<li>Anaesthetize mice using Isoflurane at 4% for induction followed by 1.5-2% throughout the procedure with 0.5-1 liter O2 a min, and place onto custom head restrainer inside the stereotactic irradiator (Figure 3Ai).</li>
	<li>Obtain a 360&deg; cone beam CT scan with the X-ray tube running at 40 kVp and 0.05 mA through a 2 mm aluminum filter. Use the image to guide the movement of the stage, directing the radiation isocentre to the right hemisphere ensuring it is central in the Dorsal Ventral direction.</li>
	<li>Insert the hemispheric collimator, 8 mm x 11 mm block.</li>
</ol>
CRITICAL STEP: Collimators can be designed for many different setups and so can be enhanced to include and exclude different parts of the brain. 8 mm x 11 mm defines a hemispheric area of the brain.
<ol start="4">
	<li>Obtain single CT orthogonal images both from top (AP) and bottom (PA) through the collimator to further enhance the placement of the brain, anatomical bone structures can be used for reproducibility.</li>
	<li>Exchange the aluminum filter for a 0.93 mm copper treatment filter and administer half of the desired radiation dose with the X-ray tube running at 225 kVp and 13 mA from the top in an AP direction.</li>
	<li>Return gantry to the bottom position and administer the second half of the radiation dose again with the X-ray tube running at 225 kVp and 13 mA form the bottom in a PA direction.</li>
</ol>
CRITICAL STEP: It is essential to irradiate from both the top and the bottom to reduce the RT gradient through the brain tissue, it also aids with the alignment of the isocentre to the center of the brain.
4. In vivo Two Photon Laser Microscopy - Figure 3 (1-3 hr per session)
Variations will exist in the different microscopes used and as such optimization will be required. We used a Carl Zeiss LSM510 META Laser Scanning Confocal Microscope.
<ol>
	<li>Anaesthetize mice with IACUC approved anesthetic, Avertin IP injection at 0.5 mg/g and clean window using alcohol spray.</li>
	<li>
	<ol class="ualpha">
		<li>(Optional) Inject tagged vascular dye 5-10 min prior to imaging, into the tail vein prior to imaging. Alexa647-Dextran used at 0.35 &mu;g/g or APC-CD31 used at 0.2 &mu;g/g. See Table 2.</li>
		<li>(Optional) Inject Fluorescein via the tail vein at a dose of 7.7 mg/kg, 5 min prior to imaging, to delineate tumor. Reference Table 2.</li>
	</ol>
	</li>
	<li>Setup channels on the confocal microscope as determined by the fluorochrome used in the chimeric mouse generated. Table 1 demonstrates the channels described in this model.</li>
	<li>Invert mouse onto the moveable microscope stage and stabilize the head in position with moldable plasticine. (Figure 3Aii) Reference Table 2.</li>
</ol>
CRITICAL STEP: The ICW needs to be perpendicular to the laser point and as such must be positioned horizontally to ensure the imaging is optimal.
<ol start="5">
	<li>Turn on first channel laser and use to position in the center of the ICW.</li>
	<li>Use 5x lens and take an image of the entire window to use as a map for further higher resolution images.</li>
	<li>Imaging should be carried out using 10X and 20X 'long range' lenses to get the best image quality.</li>
</ol>
CRITICAL STEP: Channels and objectives on the 2PLM should be set up using engineered cells in vitro prior to the imaging of murine models to ensure they highlight the correct fluorochrome.
<table border="1">
	<tbody>
		<tr>
			<td>Fluorochrome</td>
			<td>CFP</td>
			<td>GFP / Fluorescein / FITC</td>
			<td>mCherry / RFP / DsRED</td>
			<td>APC / Alexa647</td>
			<td>SHG Autofluorescence</td>
		</tr>
		<tr>
			<td>Excitation laser</td>
			<td>458 nm</td>
			<td>488 nm</td>
			<td>543 nm</td>
			<td>633 nm</td>
			<td>Chameleon laser 820 nm</td>
		</tr>
		<tr>
			<td>Collection Filter</td>
			<td>480-520 nm</td>
			<td>500-550 nm</td>
			<td>565-615 nm</td>
			<td>650-710 nm</td>
			<td>390-465 nm</td>
		</tr>
	</tbody>
</table>
Table 1. Fluorochrome setup. Guides for the user to see the excitation laser and emission spectra collector used for each of the channels available in the model.

<ol>
	<li>Kienast, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+reveals+the+single+steps+of+brain+metastasis+formation.">Real-time imaging reveals the single steps of brain metastasis formation.</a> Nature Medicine. 16, 116-122 (2010).</li><li>Holtmaat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term,+high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nat. Protoc. 4, 1128-1144 (2009).</li><li>Hansen-Algenstaedt, N., 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=Long-term+observation+reveals+time-course-dependent+characteristics+of+tumour+vascularisation.">Long-term observation reveals time-course-dependent characteristics of tumour vascularisation.</a> Eur. J. Cancer. 41, 1073-1085 (2005).</li><li>Kherlopian, A. R., 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=A+review+of+imaging+techniques+for+systems+biology.">A review of imaging techniques for systems biology.</a> BMC Systems Biology. 2, 74 (2008).</li><li>Kedrin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+metastatic+behavior+through+a+mammary+imaging+window.">Intravital imaging of metastatic behavior through a mammary imaging window.</a> Nature Methods. 5, 1019-1021 (2008).</li><li>Perentes, J. Y., 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=In+vivo+imaging+of+extracellular+matrix+remodeling+by+tumor-associated+fibroblasts.">In vivo imaging of extracellular matrix remodeling by tumor-associated fibroblasts.</a> Nature Methods. 6, 143-145 (2009).</li><li>Brown, E., 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=Dynamic+imaging+of+collagen+and+its+modulation+in+tumors+in+vivo+using+second-harmonic+generation.">Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation.</a> Nature Medicine. 9, 796-800 (2003).</li><li>Marker, D. F., Tremblay, M. -. E., Lu, S. -. M., Majewska, A. K., Gelbard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Thin-skull+Window+Technique+for+Chronic+Two-photon+In+vivo+Imaging+of+Murine+Microglia+in+Models+of+Neuroinflammation.">A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation.</a> J. Vis. Exp. (43), e2059 (2010).</li><li>Drew, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+optical+access+through+a+polished+and+reinforced+thinned+skull.">Chronic optical access through a polished and reinforced thinned skull.</a> Nature Methods. 7, 981-984 (2010).</li><li>Yang, G., Pan, F., Parkhurst, C. N., Grutzendler, J., Gan, W. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinned-skull+cranial+window+technique+for+long-term+imaging+of+the+cortex+in+live+mice.">Thinned-skull cranial window technique for long-term imaging of the cortex in live mice.</a> Nature Protocols. 5, 201-208 (2010).</li><li>Burrell, K., Hill, R. P., Zadeh, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+in-vivo+analysis+of+normal+brain+response+to+cranial+irradiation.">High-resolution in-vivo analysis of normal brain response to cranial irradiation.</a> PLoS ONE. 7, e38366 (2012).</li><li>Tate, M. C., Aghi, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+angiogenesis+and+invasion+in+glioma.">Biology of angiogenesis and invasion in glioma.</a> NURT. 6, 447-457 (2009).</li><li>Aghi, M., Chiocca, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+bone+marrow-derived+cells+to+blood+vessels+in+ischemic+tissues+and+tumors.">Contribution of bone marrow-derived cells to blood vessels in ischemic tissues and tumors.</a> Mol. Ther. 12, 994-1005 (2005).</li><li>Nussenbaum, F., Herman, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis:+insights+and+innovations.">Tumor angiogenesis: insights and innovations.</a> J. Oncol. 2010, 132641 (2010).</li><li>Zhang, H. -. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+endothelial+progenitor+cells+into+the+neovasculature+of+malignant+glioma+xenograft.">Incorporation of endothelial progenitor cells into the neovasculature of malignant glioma xenograft.</a> J. Neuroonco. 93, 165-174 (2009).</li><li>Blouw, 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=The+hypoxic+response+of+tumors+is+dependent+on+their+microenvironment.">The hypoxic response of tumors is dependent on their microenvironment.</a> Cancer Cell. 4, 133-146 (2003).</li><li>Du, R., 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=HIF1alpha+induces+the+recruitment+of+bone+marrow-derived+vascular+modulatory+cells+to+regulate+tumor+angiogenesis+and+invasion.">HIF1alpha induces the recruitment of bone marrow-derived vascular modulatory cells to regulate tumor angiogenesis and invasion.</a> Cancer Cell. 13, 206-220 (2008).</li><li>Rajantie, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+bone+marrow-derived+cells+recruited+during+angiogenesis+comprise+precursors+for+periendothelial+vascular+mural+cells.">Adult bone marrow-derived cells recruited during angiogenesis comprise precursors for periendothelial vascular mural cells.</a> Blood. 104, 2084-2086 (2004).</li><li>Shin de Patil, V. R., 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=marrow-derived+lin(-)c-kit(+)Sca-1++stem+cells+do+not+contribute+to+vasculogenesis+in+Lewis+lung+carcinoma.">marrow-derived lin(-)c-kit(+)Sca-1+ stem cells do not contribute to vasculogenesis in Lewis lung carcinoma.</a> Neoplasia. 7, 234-240 (2005).</li><li>Purhonen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+circulating+endothelial+precursors+do+not+contribute+to+vascular+endothelium+and+are+not+needed+for+tumor+growth.">Bone marrow-derived circulating endothelial precursors do not contribute to vascular endothelium and are not needed for tumor growth.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 6620-6625 (2008).</li><li>Aghi, M., Cohen, K. S., Klein, R. J., Scadden, D. T., Chiocca, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+stromal-derived+factor-1+recruits+vascular+progenitors+to+mitotic+neovasculature,+where+microenvironment+influences+their+differentiated+phenotypes.">Tumor stromal-derived factor-1 recruits vascular progenitors to mitotic neovasculature, where microenvironment influences their differentiated phenotypes.</a> Cancer Research. 66, 9054-9064 (2006).</li><li>Burrell, K., Zadeh, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Mechanisms of Tumor Angiogenesis. , (2012).</li><li>Thorball, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FITC-dextran+tracers+in+microcirculatory+and+permeability+studies+using+combined+fluorescence+stereo+microscopy,+fluorescence+light+microscopy+and+electron+microscopy.">FITC-dextran tracers in microcirculatory and permeability studies using combined fluorescence stereo microscopy, fluorescence light microscopy and electron microscopy.</a> Histochemistry. 71, 209-233 (1981).</li><li>Tolentino, M. J., 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=Angiography+of+fluoresceinated+anti-vascular+endothelial+growth+factor+antibody+and+dextrans+in+experimental+choroidal+neovascularization.">Angiography of fluoresceinated anti-vascular endothelial growth factor antibody and dextrans in experimental choroidal neovascularization.</a> Arch. Ophthalmol. 118, 78-84 (2000).</li><li>Holash, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-Trap:+a+VEGF+blocker+with+potent+antitumor+effects.">VEGF-Trap: a VEGF blocker with potent antitumor effects.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 11393-11398 (2002).</li><li>Shinoda, J., 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=Fluorescence-guided+resection+of+glioblastoma+multiforme+by+using+high-dose+fluorescein+sodium.">Fluorescence-guided resection of glioblastoma multiforme by using high-dose fluorescein sodium.</a> , 1-7 (2003).</li></ol>

Authors have nothing to disclose.

The three channels described in all the images so far are interchangeable for three markers of interest in other models and leaves researchers with an invaluable set of tools to look an numerous cell types and interactions. Planning is required to ensure all markers and cell types have successfully integrated a reporter fluorescence molecule in a distinct channel.
In addition to the standard three channels used here we were also able integrate a fourth CFP channel (Figure 4A) and an SHG based collagen channel 7 (Figure 4B). This extends the possibility of looking at cellular interactions in vivo and in addition allows the user to undertake population mixing to study specific cell-cell interactions while retaining two channels for other markers. For example, we have looked at tumor cells (RFP) and cancer stem cells (CFP) at a ratio of 1:3 with an interest in comparing their intratumoral interactions (Figure 4A). We observed that 7 days post implantation of the mixed population the ratio was upheld and both cell populations could still be seen.
The CFP channel provides technical issues due to the overlap with the GFP channel in both excitation and emission spectra and as such requires post imaging processing to define the true CFP+ cells from those that are actually GFP+. Post imaging processing can be carried out on the 2-photon microscopes built in Zeiss LSM software directly whereby, the GFP image is subtracted from the CFP image resulting in the true CFP cells being left behind (Figure 4A). The premise for the ratio subtraction is dependent on equally bright images and is due to the CFP laser (458 nm) fluorescing both CFP and GFP cells whilst the GFP laser (488 nm) is too high to fluoresce the CFP cells. In addition to CFP we have also been able to use previously published setups to look at the SHG levels and so depict the collagen fibers which make up the basement membrane surrounding vasculature, (Figure 4B).
Another adaptation of this model has taken advantage of a custom built small animal irradiator which has the ability to use stereotactic guided radiation to irradiate sections of tissue as small as 2 mm in diameter. By targeting the window with irradiation it is possible to look at the effect radiation has on the underlying tissue. We have recently published a study looking at the effect radiation has on normal brain tissue with respect to the recruitment of BMDCs to the vasculature, looking specifically at their role in post radiation tissue changes. We found that the recruitment of the BMDCs was both time and dose dependent in normal tissue 11.
It is feasible to study the mechanisms of delivery and integration of therapeutics providing the drug is tagged with a fluorescent marker. During our research we have been able to track the production of VEGFTrap, an anti-angiogenic drug which blocks all VEGF signaling in the local area of production 25. By genetically modifying our tumor cells to express the VEGFtrap gene coupled with an IRES to EGFP (Figure 4Ci) and using RFP 'donor' BM we were able to image the EGFP:VEGFtrap production, BMDC interaction and vasculature simultaneously (Figure 4Cii). This demonstrated the versatility of the model and channels available. It is also feasible to look at kinetics of the drug due to the promise of single-cell resolution. Fluorescein (GFP+) is used to delineate tumors during surgery to ensure maximal resection is achieved 26. By injecting intravenously whilst imaging it is possible to demonstrate that the delineation occurs NOT through the active uptake of the Fluorescein into the tumor cells themselves, but instead through the collection of the drug into the stromal area with time, seen by the lack of co-localization 10 min following the injection of the Fluorescein (Figure 4D).
Overall our strategy combines novel and existing techniques to achieve a unique experimental platform, which is advantageous in the study of dynamic cellular interactions. This strategy has proven to be an invaluable approach for examining high-resolution single-cell dynamic evolution of BMDC, tumor and normal brain vascularity in response to therapy, intracranially. Intravital imaging can provide a better understanding of the molecular regulators of BMDC recruitment, migration and differentiation in intracranial brain tumor vasculature as well as many other dynamic processes adaptable to other research fields. Inhibiting putative factors that regulate BMDC in combination with other therapeutic strategies can aid the identification of the precise timing of combinatorial therapies. Furthermore, this strategy can be adapted to many future projects utilizing not only in vivo intravital staining dyes, but also small molecular inhibitors and nanoparticles that are fluorescently tagged allowing researchers to trace the distribution and migration patterns of more targeted therapeutics as well as look longitudinally at their kinetics.

A better understanding of the structural and biological changes that arise in the brain in response to various pathologies, and therapeutic interventions is critical for improving treatment strategies. However, one of the current challenges in studying these structural and biological changes, particularly regarding the intracranial pathologies, is the inaccessibility of the tissue and the inability to study the temporal evolution and dynamic progression of changes in an in vivo setting. The generation of the "window" technology has previously proved successful in examining soft tissue changes through tumor development 5-7. Development of ICW models proves to be more technically challenging, due to the necessity to remove the skull bone without damaging on instigating infection in the underlying cerebral tissue. Previous papers have attempted to thin the skull to visualize the tissue 8-10 however, to produce high resolution clear images full removal of the skull bone is required 11. Repeat long-term imaging (30 days+) has only recently become a feasible option through imaging 1,11, previously shorter time frames have been studied 5.
Over the past decade a major goal has been to elucidate the origin of neo-vasculature following pathological stimuli, in particular in response to tumor formation and progression, to provide novel therapeutic targets for the treatment of tumors. Much controversy remains around the source of new vasculature in the brain during tumor development or following radiation. Traditionally vascularization has been considered to occur through angiogenesis, a process by which new vessels form from the sprouting of pre-existing vessels 12. More recent studies however, suggest that the previously assumed embryonic process of vasculogenesis may be playing a more considerable role in the formation of pathological vasculature. Vasculogenesis involves the recruitment of the adult angioblast counterparts from the bone marrow which in turn are then directly involved in the formation of new vessel endothelium 12-14. Accumulating evidence indicates that endothelial precursor cells are mobilized from the bone marrow to initiate de novo vessel formation in response to oncogenic mediators 15-17. However, these studies provide contradictory evidence for the direct contribution of these bone marrow derived cells (BMDCs) to vessel endothelium with percentage contribution varying with the type of pathological stimulus and response to treatment 18-21.
Therefore, establishing reproducible experimental methods that allow high-resolution intravital imaging for repeated long-term study of the process of BMDC integration into tumor vasculature and in response to therapy is invaluable. Standard histological techniques fail to provide the dynamic information required to determine the long-term survival, differentiation and integration of the cells that give rise to neovasculature and therefore cannot demonstrate definitively the mechanisms of cell interaction.
We have shown using our experimental approach that there is a varying degree of BMDC recruitment following both intracranial ionizing radiation and tumor growth, a recruitment that is however, pathology site specific and not an invasion of the entire intracranial tissue 11,22. We have shown that the recruitment follows a time sensitive pattern demonstrated by the repeated imaging of a single animal 11,22. Similarly, in vivo imaging can also provide invaluable insights into tumor cell mimicry whereby fluorescently tagged tumor cells can be imaged and tracked with an intra-vital CD31 antibody for endothelial cells to highlight the possibility of tumor cell transdifferentiation to directly form its own endothelium.
The versatility of the model is enhanced by the ability to image 4 fluorescent channels, providing an exhaustive number of combinations for the study of varying cellular and biological process. We are able to intravitally image CFP (blue), GFP (green), Cherry/RFP (red), and Alexa647/APC (far-red), simultaneously in a single field repeatedly over time. Researchers can genetically modify cells to express fluorochromes for each of the channels as well as purchase dyes and antibodies to highlight structural changes of particular interest. To date dyes and antibodies commonly used in studies include CD31 and dextran which both highlight the microvasculature and its changes in tissue 1,11,23,24, although we utilized the far-red channel for this purpose both are available for use in the other channels mentioned. Similarly, other dyes including Sytox Orange, which will highlight areas of apoptosis, and markers such as those from the company Visen, have been specifically designed for use in vivo. Further to the four commonly used fluorescent channels mentioned, a second harmonic generation (SHG) channel can be added and optimized to image the endogenous collagen fibers of the model 7, visualizing the basement membrane surrounding vasculature.
To demonstrate the adaptability of our model, as well as the cell-cell interactions mentioned above, we have been able to study drug-cell interactions. We have looked at drug inhibitors like AMD3100, an SDF-1 inhibitor, and its role in signaling networks involved in the BMDC recruitment 11. Similarly we have genetically engineered out U87 glioma xenografts cells to express VEGFTrap, a VEGF inhibitor 25, in conjunction through an IRES to a GFP molecule. By using RFP+ BM we are able to study the role VEGF has on the recruitment of BMDCs to the vasculature. Most recently we have utilized the model to study drug kinetics looking at the mechanism behind the tumor-delineating drug Fluorescein 26, at a cellular level. Through the use of a custom-built in house small animal irradiator we have been able to integrate stereotactic delivered radiation to assess the response of both tumor and BMDCs to treatment.
Through the use of our novel intravital imaging method researchers will gain insight into the single-cell real-time changes that occur in various pathologies and systems, aiding the elucidation of many functional and biological features of tissue changes.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>NODSCID mice</td>
			<td>Jackson Lab</td>
			<td>001303</td>
			<td>8 week old</td>
		</tr>
		<tr>
			<td>B5/EGFP Mice</td>
			<td>Jackson Lab</td>
			<td>003516</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ACTB/DsRED mice</td>
			<td>Jackson Lab</td>
			<td>005441</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tear Gel</td>
			<td>Novartis</td>
			<td>T296/2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2% Lidocaine-Epinephrine</td>
			<td>Bimeda MTC</td>
			<td>25SP</td>
			<td>Use neat</td>
		</tr>
		<tr>
			<td>Vetbond</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Self curing acrylic kit</td>
			<td>Bosworth</td>
			<td>166260</td>
			<td>Use ~30% (w/v)</td>
		</tr>
		<tr>
			<td>10K MW Dextran-Alexa647</td>
			<td>Invitrogen</td>
			<td>D22914</td>
			<td>Use @ 0.6 mg/kg in Saline</td>
		</tr>
		<tr>
			<td>CD31-APC</td>
			<td>BDPharmingen</td>
			<td>551262</td>
			<td>Use @ 0.3 mg/kg in Saline</td>
		</tr>
		<tr>
			<td>AK-fluor (Fluorescein) 10%</td>
			<td>AKORN inc.</td>
			<td>NDC 17478-253-10</td>
			<td>Use @ 7.7 mg/kg in Saline</td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>Purdue Products</td>
			<td>NDC 67618-150</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Fine Tweezers</td>
			<td>VWR</td>
			<td>82027-402</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fine Dissection Scissors</td>
			<td>VWR</td>
			<td>25870-002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>22G Needle</td>
			<td>BD</td>
			<td>305156</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>27G 0.5 ml TB syringe</td>
			<td>BD</td>
			<td>305620</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Handheld Micro-Drill</td>
			<td>Fine Science Tools</td>
			<td>18000-17</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2.7 mm Trephine drillbit</td>
			<td>Fine Science Tools</td>
			<td>18004-27</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 &mu;l 30G Hamilton syringe</td>
			<td>Sigma Aldrich</td>
			<td>20909-U</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass Coverslip 3 mm</td>
			<td>Warner Instruments</td>
			<td>64-0720 CS-3R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescence goggles</td>
			<td>BLS</td>
			<td>FHS/T01</td>
			<td>Basic head frame</td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>stoelting</td>
			<td>51950</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse restrainer for IV injection</td>
			<td>Brain Tree Lifescience</td>
			<td>MTI</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a novel high-resolution in vivo imaging technique to study the dynamic response of intracranial structures to tumor growth and therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

The optional step of reconstituting the BM of NODscid mice should result in an 80% uptake of the fluorescent 'donor' BM in 100% of the NODscid 'host' mice, however optimization of the TBI will be required to enhance the reconstitution in other non-immunocompromsed strains. If mice are unsuccessfully reconstituted they will get sick and die rapidly following the procedure, weakened mice may require additional food and water sources.
The ICW once finished should look like that seen in Figure 2Biv. It is ideal to have a ridge of acrylic surrounding the glass coverslip as this gives strength to the join with the skull. Perfect windows are reproducible and allow for repeated imaging for up to 8 weeks following their generation (Figure 2C). Images produced through these optimal windows will look as those seen in Figure 3Bi. Imperfections in the window generation will produce images of poor quality for example, air bubbles under the window will prevent whole fields of view being imaged, areas will appear dark as the air prevents the laser imaging (Figure 3Bii). With excess glue and acrylic on the coverslip there will be a high level of background fluorescence and areas of the field being blotted out as seen in Figure 3Biii, similarly if there is dirt on the window small dots of background fluorescence will be visible in the field (Figure 3Biv).
Success of ICW imaging is predetermined by the integrity of the surgical technique, however, even optimal ICWs can encounter problems during the imaging session. As such, a range and degree of clarity exists in the images generated as seen in Figure 3. The publication quality image portrayed in Figure 3C occurs when everything is optimal. Although it is not possible for all mice, it is feasible to expect 80% of the images generated to look like this. One of the major flaws in the current microscopy setup is the use of inverted lasers. Mice have to be positioned on their backs and this results in excessive stress and discomfort that may lead to labored breathing during the imaging. This produces a 'lined' image as the breathing interferes with the averaging that occurs during the imaging, whereby, each pixel row is imaged four times and the average of the four is displayed in the final image. Any movement during the averaging, such as that caused by labored breathing, will create an artifact that appears as a line on the image, as seen in Figure 3D. Mice that are insufficiently anaesthetized during imaging may encounter this problem also. The 'lined' effect can be decreased by limiting the image averaging to on, however this will in turn reduce the image quality and may not eliminate the problem. Alternatively mice can be repositioned or retried when breathing has normalized. The use of an upright 2PLM would negate this problem as mice could be imaged 'in the prone position'. An additional problem with imaging on an inverted 2PLM includes the limited accessibility for positioning the ICW once the mouse is on the microscope, and this leads to 'segmented' images like those seen in Figure 3E. Here the laser and coverslip are not positioned perpendicular to one another and as such the imaging occurs at an angle. This results in the generation of a 'segmented' image where the sides of the field of view are not imaged. The problem is easily solved with the repositioning of the mouse to ensure the coverslip is completely horizontal once on the head mount as seen in Figure 3Aii.
The model presented here was specifically used to examine the role of BMDCs in the vascularization of tumor tissue and demonstrates the ability to use three different channels simultaneously (Cherry, GFP, Far-red - Alexa647 and APC) making the CFP and SHG redundant for this particular story. We were able to image mice longitudinally for up to 8 weeks, studying the recruitment and integration of BM cells into the vasculature at a single cell level, with no detrimental effects caused by the window. This model demonstrates the ease of collecting dynamic information on the source and formation of vasculature and the interaction of different cell types of interest, previously lost through end point histological analysis.
<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Problem</td>
			<td>Reasoning</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>1.4</td>
			<td>Bone shatter</td>
			<td>Blunt tools</td>
			<td>Collect bone marrow form a fresh mouse using sharpened scissors or fresh scalpel blade, bone fragments will inhibit TV injection</td>
		</tr>
		<tr>
			<td>1.5</td>
			<td>Low extraction (low viscosity)</td>
			<td>Bone endplates cut too distally; poor collection method</td>
			<td>Bones should be cut as proximally as possible and collected with the bone inside collection tube to prevent splash back 
			Time should be spent to extract the BM maximally and with care 
			If necessary pool more than one mouse into 1 ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>High Extraction (high Viscosity)</td>
			<td>Low collection buffer</td>
			<td>Dilute out solution with extra 0.1% BSA, split to three recipient mice up to 500 &mu;l per mouse maximum</td>
		</tr>
		<tr>
			<td>1.6</td>
			<td>Bad Intravenous injection</td>
			<td>Poor vasodilation and vessel visibility</td>
			<td>Enhance dilation with heat lamp. Place mouse in tail vein restrainer with built in light source to aid access</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Mice Sick</td>
			<td>Infection</td>
			<td>Sacrifice mice according to institution rules. Ensure tails are cleaned prior to injection and check sterility of extracted BM in culture</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Mice die</td>
			<td>Poor BM uptake</td>
			<td>Check % of fluorescent BM uptake of dead mouse. 
			Optimize TBI for strain of mice being used 
			Increase amount of BM injection (example use one donor mouse for two recipients)</td>
		</tr>
		<tr>
			<td>2.4</td>
			<td>Minor hemorrhaging</td>
			<td>Dura breached during drilling</td>
			<td>Pressure with gel pads and and continuous sterile saline wash</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Major hemorrhaging</td>
			<td>Brain damaged by drilling</td>
			<td>Sacrifice mouse according to institution guidelines</td>
		</tr>
		<tr>
			<td>2.8</td>
			<td>Air-bubbles under coverslip</td>
			<td>Poor contact with cortical surface prevents proper placement</td>
			<td>Remove coverslip and add extra PBS to float the coverslip onto the window to remove bubbles</td>
		</tr>
		<tr>
			<td>2.10</td>
			<td>Slippage of coverslip during gluing</td>
			<td>Acrylic mass is heavy, placing pressure on the coverslip and moves the coverslip out of the way</td>
			<td>Tweezers should be used to hold coverslip down while vetbond and acrylic is applied</td>
		</tr>
		<tr>
			<td>4.2</td>
			<td>Bad Intravenous injection</td>
			<td>Poor vasodilation and vessel visibility</td>
			<td>Enhance dilation with heat lamp. Place mouse in tail vein restrainer with built in light source to aid access</td>
		</tr>
		<tr>
			<td>4.4 
			Figure 3C</td>
			<td>'Lined' images</td>
			<td>Labored or irregular breathing</td>
			<td>Remove mouse from frame and allow to recover 
			Increase level of anaesthetic administered and adjust position to ensure neck is not overly flexed or extended to prevent breathing</td>
		</tr>
		<tr>
			<td>Figure 3C</td>
			<td>'Segmented' image</td>
			<td>Brain is not parallel to objectives</td>
			<td>Adjust position of coverslip to ensure it is flat</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Vessel not visualized</td>
			<td>injection not seen intravascularly</td>
			<td>Redo injection into alternative tail vein, warm tail to ensure good vasodilation</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>High background</td>
			<td>Dirty coverslip, 
			Air Bubbles, Acrylic</td>
			<td>Wipe coverslip with damp 70% ethanol cloth, do not soak as may penetrate under acrylic and damage the brain tissue</td>
		</tr>
	</tbody>
</table>
Table 2. Troubleshooting. A guideline for corrective steps required for problematic areas of the procedures.
<img alt="Schemata 1" fo:content-width="5.5in" fo:src="/files/ftp_upload/50363/50363schemata1highres.jpg" src="/files/ftp_upload/50363/50363schemata1.jpg" /> 
Schemata 1. Experimental flow diagram. This demonstrates the timeline of events through experimental steps 1-4. Mouse models are setup over a week, to ensure the BM reconstitutes properly, and are not treated with drug until day 7 of the tumor to ensure the tumor grafts. <a href="https://www.jove.com/files/ftp_upload/50363/50363schemata1large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50363/50363fig1highres.jpg" src="/files/ftp_upload/50363/50363fig1.jpg" /> 
Figure 1. BM Reconstitution.(A) BM extraction procedure i. Dissection of the hind limb bones. ii. Dissected femur and tibia from the hind limbs, cleaned and ready for extraction. iii. Bones with end plate removed and flushed through, demonstrating the white appearance of the empty bones. (B) Schemata demonstrating where lateral veins for injection are positioned in the tail.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50363/50363fig2highres.jpg" src="/files/ftp_upload/50363/50363fig2.jpg" /> 
Figure 2. ICW generation. (A) Aseptic setup recommended (B) i. Exposed skull surface reveals the landmarks required for surgery, ICW should be positioned on the right hemisphere equidistant from the bregma and lamda. ii. Periostieum lifted with lidocaine solution, ready for removal. iii. Dental hook required for the removal of the bone fragment generated with the drill. iv. Finished ICW with dental acrylic. (C) Three example windows demonstrate the reproducibility of the method.
<img alt="Figure 3" fo:content-width="4.75in" fo:src="/files/ftp_upload/50363/50363fig3highres.jpg" src="/files/ftp_upload/50363/50363fig3.jpg" /> 
Figure 3. 2PLM anticipated results. All images show green BM, red tumor, Blue (pseudo colored far red) vasculature. (A) i. Demonstrates the head frame which retains isoflurane flow inside the small animal irradiator. The 8 x 11 mm collimator can also be seen moving around through the gantry. ii. Mouse in the inverted positioned in the head frame required for imaging, moldable plastercine ensures all windows can be accommodated. (B) Demonstrative photos of the outcomes of problems with the window generation from i. an optimal window, ii. a window with air bubbles trapped underneath, iii acrylic spillage over the coverslip and iv. dirt on the window itself. (C) Highlights the problems that occur with imaging following successful generation of an ICW i. optimal imaging ii. breathing artefacts create a 'lined' image iii. coverslip not perpendicular to the laser generate a 'segmented' image. <a href="https://www.jove.com/files/ftp_upload/50363/50363fig3large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 4" fo:content-width="5.5in" fo:src="/files/ftp_upload/50363/50363fig4highres.jpg" src="/files/ftp_upload/50363/50363fig4.jpg" /> 
Figure 4. Functional uses and adaptations of the Model. (A) CFP post imaging processing whereby GFP image is subtracted from CFP image to reveal the true CFP image that can be overlayed the other three channels in white. Green BM, Red tumor, White CSCs, Blue (pseudo colored far red) vasculature (B) Demonstration of the collagen fibers that can be imaged with SHG. Green Dextran, red BM, Cyan Collagen (C) i. VEGFTrap cells in vitro demonstrate the GFP signal produced with the VEGFtrap. ii. In vivo imaging demonstrates the VEGFtrap cells clearly and in addition highlights the ease of switching channels to demonstrate the system you are looking at. Green VEGFTRap + Tumor, Red BM, Blue (pseudo colored far red) vasculature. (D) Demonstrates the collection of Fluorescein in the stroma of the tumor and not in the cells directly, shown by the lack of green signal overlay with red tumor. Green Fluorescein, Red tumor. <a href="https://www.jove.com/files/ftp_upload/50363/50363fig4large.jpg" target="_blank">Click here to view larger figure</a>.

Watch this Scientific Journal Video about A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics at JoVE.com

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

a-novel-high-resolution-vivo-imaging-technique-to-study-dynamic

Research

JoVE Journal

Medicine

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...