We would like to acknowledge Dr. Joshua B. Rubin for the generous gift of the plasmids for the lentivirus system as well as Mahil Rao for helpful suggestion on the preparation of the GL261-luc cells.
We thank Students Supporting Brain Tumor Research (SSBTR), The Barrow Neurological Foundation and The Wallace Foundation for their generous support.
Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Institutional Animal Care and Use Committee of St. Joseph's Hospital and Medical Center.

1. Cell Culture
<ol>
 <li>The GL26 cell line was obtained from the Division of Cancer Treatment and Diagnosis (DCTD) National Cancer Institute (NCI), Frederick, MD. To facilitate a quantitative measurement of tumor growth rate GL261 cells were made bioluminescent using the Lentiphos HT System (Clontech Laboratories, Inc., Mountain View, CA) with the Lenti-X HT Packaging Mix (Clontech Laboratories, Inc.) and the FUW-GL plasmid (a generous gift from the laboratory of J.B. Rubin, MD, PhD). Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 10% tetracycline-free Fetal Calf Serum (FCS; Clontech Laboratories, Inc.). GL261 cells were also stably transfected with the gene encoding luc2 using the pGL4.51[luc2/CMV/Neo] vector (Promega Corp, Madison, WI) and FuGENE 6 Transfection Reagent (Roche Applied Science, Indianapolis, IN) following conditions specified by the manufacturer. The luc2 gene is a codon optimized version of firefly luciferase that provides a significantly higher light output than the standard luc gene. Stable transfectants were selected and maintained in DMEM media containing 10%FCS and 100 &mu;g/ml Geneticin (G418, Invitrogen Corp., Carlsbad, CA).</li>
 <li>Prior to implantation the cultured cells are harvested by trypsinzation, washed once in DMEM without serum and resuspended in DMEM without serum at a concentration of 1 x 107 cells/ml.</li>
</ol>
2. Surgery Setup 2
<ol>
 <li>A sterile environment is maintained throughout the surgery, including all surgical instruments, supplies, gloves, drapes, etc..</li>
 <li>Ten week old C57BL/6-cBrd/cBrd/Cr (albino C57BL/6) mice are purchased from the National Cancer Institute at Frederick Animal Production Program and used at an average weight of 20 grams (NCI, Frederick, MD).</li>
 <li>Animals are anesthetized by intraperitoneal injection of xylazine (5 mg/kg), ketamine (50 mg/kg) and buprenorphine (0.05 mg/kg). Toe pinch is done to ensure that the animal is adequately anesthetized before the surgery is begun. Movement (even if slight) of any part of the animal is an indication of a reduction in the level of anesthesia. Animal is immediately given an additional 3.3 mg/kg xylazine and 26.6 mg/kg ketamine. Body temperature is maintained using a lamp and sterile dressings covering the body.</li>
 <li>Once the animal is properly anesthetized, they are placed on the cushioned bed of the stereotactic headframe (Model 900 Small Animal Stereotaxic, David Kopf Instruments)[Figure 1].</li>
 <li>A small amount (1/4 inch) of AKWA Tears Opthalmic Lubricant Ointment is applied over the corneas.</li>
 <li>The animal is secured in the stereotactic frame by opening the animal's mouth (by placing your index finger and thumb around the animal's jaw) and sliding the top front teeth into the indentation in the stereotactic frame. The clamp is tightened to secure the animal's head into the headset making sure that the head is aligned and the eyes are centered, making sure however not to exert undue force on the animal's head.</li>
 <li>The O2 hose is taped down near the animal's nostrils. Oxygen flow rate is 0.5 l/min.</li>
 <li>The lower back and tail is taped down to the stereotactic bed being careful not to compromise respirations.</li>
 <li>The surgical incision site is shaved. Povidine-Iodine Swab Sticks are used to wash the area between the eyes going back to the area between the ears with iodine, making sure the iodine does not drip into the animal's eyes.</li>
 <li>The cells are prepared for implantation just prior to the surgery and are periodically mixed to ensure they don't settle.</li>
 <li>A 10 &mu;l, 50 mm World Precision Instrument (WPI), Sarasota, FL, syringe with 26 gauge beveled needle is loaded with the cell inoculum. If the cells seem to be clumped together it may be necessary to reload the syringe.</li>
 <li>The 10 &mu;l syringe is then placed into the UMP3-1 UltraMicroPump micro injector (WPI, Sarasota, FL).</li>
</ol>
3. Intracranial Implantation 2
<ol>
 <li>The skin incision is made using a size 15 scalpel blade and toothed forceps. A 10-15 mm incision is made longitudinally from between the animal's eyes, moving towards the animal's ears exposing the bregma (junction of the sagittal and coronal sutures at the top of the skull). Be sure to properly identify the bregma for it can be easily confused with the sinus area which is distal to the bregma [Figure 2].</li>
 <li>After the animal is sedated it receives an intra-incisional injection of 0.25% (2.5 mg/ml) bupivacaine.</li>
 <li>A burrhole is made 0.1 mm posterior to bregma and 2.3 mm to right of the midline by slowly twisting a 16 gauge 1&frac12; inch needle by hand, applying little pressure to the needle while twisting until the skull is penetrated and the brain is exposed.</li>
 <li>The syringe needle is moved down into position using the microdrive stereotactic syringe holder until it just touches the brain's surface. From this position, the needle is advanced into the brain to a depth of 3 mm and kept in place for 3 minutes.</li>
 <li>The needle is withdrawn .4 mm to a total depth of 2.6 mm below the surface of the brain, creating a small pocket where the cells are to be infused. It is optional at this point to take an X-ray image of the needle in the animal to ensure proper placement and depth.</li>
 <li>The cell suspension is infused over 3 minutes using the micro injector set to a volume of 2000 nL (2 &mu;L) with an infusion rate of 667 nL/minute.</li>
 <li>The needle is left in place for 2 minutes to prevent leakage from the site of infusion.</li>
 <li>The needle is slowly withdrawn completely.</li>
 <li>The burrhole is filled with bone wax using a Penfield dissector.</li>
 <li>The incision is sutured using a 4-0 (1.5 Metric) vicryl suture making sure no large gaps are left in the skin. Vicryl is a suture material that dissolves, and therefore sutures do not need to be removed when the incision is healed.</li>
 <li>After surgery the animals are placed in a cage under a heating lamp that is set at the height required to warm the bottom surface of the cage to 30&deg;C. When the animals are fully awake (as judged by normal movement in the cage) they are returned to group housing. Oral ibuprofen is added to their drinking water for 5 days post-operatively. 100 mg of children's ibuprofen (100mg/5ml) is added to a standard 473 ml rodent water bottle. Animals are observed daily and imaged and weighed every 3 days. Two weeks post-implantation observation is increased to twice per day. Animals are euthanized when they show signs of declining health which includes hunched posture, reduced mobility and visible body weight loss (&ge;20%). These symptoms are a published response to the tumor and they reproducibly appear approximately 1 day prior to death due to the tumor.</li>
</ol>
4. In vivo Bioluminescence Imaging 3
<ol>
 <li>Start the [Living Image] software.</li>
 <li>The IVIS Imaging System is Initialized by clicking the [Initialize IVIS System] button on the bottom right side of the control panel.</li>
 <li>Select the [Luminescent] Imaging Mode on the top left side of the control panel.</li>
 <li>To determine the optimal time of imaging after luciferin injection a kinetic study is necessary [Figure 3]. This description is for firefly luciferase;
 <ol class="lalpha">
 <li>Inject 10&mu;l/g body weight of D-luciferin firefly (15mg/ml in PBS; Caliper Life Sciences Catalog XR-1001 or a similar product from another vendor) into the animal, as described below.</li>
 <li>Wait 3 minutes, and then anesthetize the mouse by placing it into the gas anesthesia chamber (2% Isoflurane gas in O2).</li>
 <li>Turn off the gas anesthesia to the chamber and open the anesthesia valve and the vacuum to the IVIS manifold. Immediately place the sedated animal on the temperature controlled imaging platform, making sure the mouse's nostril is properly placed in the gas anesthesia manifold. The first image should be taken approximately 5 minutes after the luciferin injection. Up to 5 animals can be imaged at one time in the IVIS Spectrum instrument. If less than 5 animals are to be imaged, it is possible to plug the unused manifold(s) to conserve on isoflurane gas.</li>
 <li>Continue to take images every 3 minutes by creating a sequence for up to an hour to generate a kinetic curve for luciferin expression.
 <ol>
 <li>Click on the [Sequence Setup] button in the control panel.</li>
 <li>The sequence editor appears.</li>
 <li>In the control panel, specify the settings for the first bioluminescent image in the sequence.
 <ol class="lroman">
 <li>We recommend starting with Medium Binning.</li>
 <li>We also recommend Auto-Exposure to determine the optimal exposure time.</li>
 <li>Select the [Delay] button in the sequence editor and specify a delay time of 3 minutes between each acquisition.</li>
 </ol>
 </li>
 <li>Click [Add] in the sequence editor. Acquisition parameters are then added to the table.</li>
 <li>Repeat step 4 for each image in the sequence.</li>
 </ol>
 </li>
 <li>Once the curve is established, the optimal imaging time can be determined by plotting the signal strength (intensity) versus time. Image animals at the time of highest in vivo photon count to get the strongest and most accurate signal.</li>
 </ol>
 </li>
 <li>Early experiments used intraperitoneal (i.p.) injection of luciferin, however, i.p. injections occasionally resulted in intermittent results showing little to no bioluminescence in the tumor. We hypothesized that the occasional random lack of signal was due to the delivery of the luciferin to the bowel or other internal organs. We therefore began to use subcutaneous (s.c.) luciferin injections and saw greater imaging reproducibility [Figure 3].</li>
 <li>Twenty minutes after s.c. injection, the animals are anesthetized by placing them in a chamber with 2% Isoflurane gas in O2 until they are unresponsive.</li>
 <li>The anesthetized animal(s) are moved to the imaging chamber. Ophthalmic ointment should be used for the kinetic study because of the length of imaging. It is not needed for the other imaging procedures because they are short in duration.</li>
 <li>The image is acquired at medium binning with a 5 minute exposure time. The auto acquisition option may also be used.</li>
 <li>If the signal is saturated and/or faint at medium binning, binning or exposure time can be adjusted.</li>
 <li>The program files and subsequent image comments are saved in the user's computer directory.</li>
</ol>
5. 3D Imaging 3
<ol>
 <li>Click on the [Imaging Wizard] tab in the [Sequence Setup] window of the control panel.</li>
 <li>Select the [Bioluminescence] imaging mode on the Imaging Wizard start screen and click [Next].</li>
 <li>In the "Bioluminescence - DLIT" window of the imaging wizard, select the [Firefly] reporter probe. The emission / excitation of the selected firefly source spectrum will appear with the six corresponding filter selections to be acquired.</li>
 <li>The last screen will appear with default choices that include Auto exposure acquisition parameters and a of Field of View C. Default settings work very well; however, these settings can be modified if necessary.</li>
 <li>Click [Next] and the sequence editor window will be populated with the sequence of six spectral regions at 20 nm wide filters (560 nm, 580 nm, 600nm, 620 nm, 640 nm, and 660nm). Press [Acquire Sequence]. The first filter (560 nm) will include a structured light pattern using a laser galvanometer to establish surface topography.</li>
 <li>Select the [Surface Topography] tab in the Tool Palette. Surface smoothing can be applied to account for any sharp angles created during the reconstruction process. The default low smoothing is recommended.</li>
 <li>Click [Create] and the tomography analysis box will appear. Draw a crop box that includes the entire animal and then click [Next].</li>
 <li>The threshold tool will appear as a purple mask over the selected region. The mask should automatically be set to match the photograph of the animal. If necessary, adjust the threshold of the mask to more appropriately fit the outline of the animal.</li>
 <li>Click [Finish] and the reconstructed mesh will appear. The reconstruction can then be saved in the results tab.</li>
 <li>Following the creation of the animal's surface topography, proceed to the [DLIT 3D Reconstruction] drop down on the Tool Palette.</li>
 <li>Under the [Analyze] tab select all six wavelengths to perform the reconstruction. Deselect images that yielded saturated pixels or counts below 600. Leave settings in the [Parameters] tab as default.</li>
 <li>Under the [Properties] tab, "Muscle" should be listed as the default choice for Tissue Properties, and "Firefly" should be listed as the source spectrum.</li>
 <li>Click [Reconstruct] under the Analyze tab, and the 3D reconstruction of the animal's surface and the corresponding reconstruction of the signal source should appear [Figure 4].</li>
 <li>To determine signal location and intensity, select the Voxels button on the 3D Tools tab of the Tool Palette.
 <ol>
 <li>Draw a square around all displayed voxels and the total flux measurements is shown in the bottom of the volume tab.</li>
 <li>Click on [Center of Mass] to identify the signal location of the selected voxels. Coronal, sagittal and transaxial slices of the animal will appear and using the [Measurement Cursor Display] the distance from the animal's surface to the voxel center can be measured.</li>
 </ol>
 </li>
 <li>Please refer to the Living Image Software User's Manual for further information on co-registration of organ atlases and other advanced features.</li>
</ol>
6. Data Analysis 3
<ol>
 <li>After the image is acquired and saved, access the program file by clicking the [Browse] button and selecting the file.</li>
 <li>The image information can be found under [View] &rarr; [Image Information].</li>
 <li>The intensity of the signal can be quantified by selecting the Region Of Interest (ROI) [ROI Tools] button. Make sure the image is analyzed under the [Photon] mode by selecting "Photon" on the drop-down list on the top left corner of the image control panel.</li>
 <li>Select the [Measurement ROI] button from the "Type" drop-down list. Select the ROI shape of interest; options include Circle, Square, and or Grid. Cover all areas of intensity on the acquired image.</li>
 <li>The ROI position is set by dragging the ROI shape selection to the region containing the bioluminescent signal.</li>
 <li>The signal intensity of the ROI is computed by clicking the [Measure] button. The ROI label displays the intensity. ROI's can be managed and saved using the Living Image software.</li>
</ol>
7. Representative Results:
Successful cell implantation is evident when implanted cells are detectable using the IVIS spectrum on the day of surgery. Both GL261-luc and GL261-luc2 cells are detectable, however, the luc2 gene will provide a higher level of bioluminescence [Figure 5]. Images taken soon after implantation may have non-specific signaling on the animal's paws and nose which should be disregarded as background. Signal located at the site of implantation is real and the signal will increase with time [Figure 6]. The decline in signal intensity on day 6 is reproducible and most likely due to loss of tumor take by some of the implanted cells. Quantitative measurements of tumor burden are reproducible in that they should rise steadily until the animal ultimately succumbs to the disease. However, growth curves will largely depend on the state of implanted cells, and or unavoidable minor variability in the implantation procedure. Our laboratory has elected to image implanted animals every three days.
<img alt="Figure 1" src="/files/ftp_upload/3403/3403fig1.jpg" /> 
Figure 1. After the mouse is properly anesthetized, it is placed in the stereotactic frame. The mouse head is secured using the mouth clamp.
<img alt="Figure 2" src="/files/ftp_upload/3403/3403fig2.jpg" /> 
Figure 2. After the skin is opened the main anatomical landmarks are identified including include the bregma, the coronal and sagittal sutures. The burrhole is made 2.3mm to the right of the bregma by slowly twisting a 16 gauge 1&frac12; inch needle with a small amount of pressure until the skull is penetrated and the brain is exposed.
<img alt="Figure 3" src="/files/ftp_upload/3403/3403fig3.jpg" /> 

Figure 3. A kinetic comparison of subcutaneous (<img src="/files/ftp_upload/3403/3403fig3_1.jpg" alt="Figure3.1" />) versus intraperitoneal (<img src="/files/ftp_upload/3403/3403fig3_2.jpg" alt="Figure3.2" />) luciferin injection was performed to demonstrate the utility of a subcutaneous luciferin injection and to identify the optimal time to image following luciferin administration. Three minutes after the luciferin was injected the mouse was sedated, placed in the IVIS Spectrum instrument and imaged every 3 minutes for up to an hour and every 6 minutes after that to generate a kinetic curve of bioluminescence. This demonstrated that a subcutaneous route of luciferin administration was superior to an intraperitoneal injection in our hands, and that the optimal time to image the animals was approximately 25 minutes following the luciferin injection when GL261-luc cells were used.
<img alt="Figure 4" src="/files/ftp_upload/3403/3403fig4.jpg" /> 
Figure 4. Multiple views of a 3-Dimensional reconstruction of the intracranial implantation of GL261-luc2 cells co-registered with the mouse skeleton and brain.
<img alt="Figure 5" src="/files/ftp_upload/3403/3403fig5.jpg" /> 
Figure 5. Photon counts obtained from tumors resulting from GL261-luc cells vs GL261-luc2 cells. Results are an average of 5 animals.
<img alt="Figure 6" src="/files/ftp_upload/3403/3403fig6.jpg" /> 
Figure 6. Graph of GL261-luc tumor cell growth in an albino C57BL/6 mouse. Bioluminescence was measured every 3 days and plotted as in vivo photon count versus days post-implantation. Photographs show bioluminescence at various time points. Coloration is an indication of bioluminescence (pixel intensity) which is relative to tumor cell number (color bar is shown to the right). After the animal succumbed to the disease the brain was dissected and luciferin was added topically to obtain the ex vivo image shown in the figure inset.

<ol>
	<li>Candolfi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+glioblastoma+models+in+preclinical+neuro-oncology:+neuropathological+characterization+and+tumor+progression.">Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression.</a> J Neurooncol. 85, 133-148 (2007).</li><li>Stafford, P., Abdelwahab, M. G., do, K. i. m., Preul, Y., Rho, M. C., M, J., Scheck, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ketogenic+diet+reverses+gene+expression+patterns+and+redudes+reactive+oxygen+species+levels+when+used+as+an+adjuvant+therapy+for+glioma.">The ketogenic diet reverses gene expression patterns and redudes reactive oxygen species levels when used as an adjuvant therapy for glioma.</a> Nutr Metab. 7, (2010).</li><li>Caliper Life Sciences, Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Living Image Software Version 4.0. VivoVision Systems. , (2010).</li><li>Paxinos, 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> The Mouse Brain in Stereotaxic Coordinates. , (2001).</li><li>Jouanneau, E., Poujol, D., Gulia, S., Le, M. I., Blay, J. Y., Belin, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+are+essential+for+priming+but+inefficient+for+boosting+antitumour+immune+response+in+an+orthotopic+murine+glioma+model.">Dendritic cells are essential for priming but inefficient for boosting antitumour immune response in an orthotopic murine glioma model.</a> Cancer Immunol Immunother. 55, 254-267 (2006).</li></ol>

Free Access to this article is sponsored by Caliper Life Sciences.

The cell inoculum is infused at a depth of 2.6 mm from the surface of the brain after creating a 0.4 mm pocket. To ensure proper placement and depth of needle an X-ray can be taken using a C-Arm or similar X-ray image intensifying device; however, this is optional. Complications of the surgery may arise if the animal is not properly sedated, at which point the animal may move during cell infusion. This can cause leakage of cell mix or bleeding from needle track. Leakage of cells causes ectopic growth of tumor cells. It is also important not to puncture the ventricle which can be done if the burrhole is made medial to the 2.3 mm described in the protocol 4. Proper placement of the needle and cell spread was tested by infusing a mouse with 2 &micro;l methylene blue dye and dissecting the brain tissue to check the location of the infused dye.
In this protocol we have used the IVIS Spectrum in vivo imaging system and the Living Image software (v 4.0) designed for use with this instrument. (Caliper Life Sciences). Any comparable in vivo imaging system and image analysis tools can be used to obtain similar results. These systems provide some advantages over traditional magnetic resonance imaging (MRI) to follow the growth of an experimental intracranial tumor. The most obvious is the relative costs of the 2 instruments &ndash; animal MRI machines are far more expensive and typically require the services of a skilled MRI technician. In vivo imaging such as what was described here can be done by the end user. The bioluminescence data is quantitative, while quantitation of MRI data is time-consuming and somewhat inexact. Furthermore, MRI images show edema and inflammation in addition to tumor cells and it can be difficult to separate tumor from treatment effect. For these reasons obtaining accurate volumetric measurements of growing tumor can be a challenge. Bioluminescence requires ATP, therefore only living tumor cells contribute to the tumor size data. Despite this, there are some advantages to MRI if a machine is available. Cells do not have to be labeled with a bioluminescent marker to be visualized by MRI. The ability to visualize peri-tumoral edema may be an advantage for some experimental protocols. A strength of both technologies is that using one does not preclude the use of the other, so data can be obtained on the growth of the tumor as well as the presence of peri-tumoral edema and inflammation from the same animal when both technologies are available to the researcher.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent or supply</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>GL261-luc2 Bioware Ultra</td>
 <td>Caliper Life Sciences</td>
 <td>GL261-luc2</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Dulbecco's Modified Eagle Medium 			(DMEM)</td>
 <td>Invitrogen</td>
 <td>10313039</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Geneticin (G418)</td>
 <td>Gibco (Invitrogen)</td>
 <td>11811-023</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Fetal Calf Serum (FCS)</td>
 <td>Invitrogen</td>
 <td>26140079</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Phospate Buffered Saline (PBS)</td>
 <td>Invitrogen</td>
 <td>70011044</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>C57BL/6-cBrd/cBrd/Cr Mice</td>
 <td>NCI-Frederick</td>
 <td>&nbsp;
 </td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>AKWA Tears Lubricant Opthalmic Ointment</td>
 <td>Akorn Inc</td>
 <td>17478-062-35</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Ketaset (ketamine hydrochloride)</td>
 <td>Wyeth</td>
 <td>11570775</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Sedazine (xylazine hydrochloride)</td>
 <td>Wyeth</td>
 <td>10031894</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Small Animal Stereotaxic Instrument</td>
 <td>Kopf Instruments</td>
 <td>900</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>UltraMicroPump with 			SYS-Micro4 Controller</td>
 <td>World Precision Instruments</td>
 <td>UMP3-1</td>
 <td>If not available, it is possible to infuse 			manually</td>
 </tr>
 <tr>
 <td>10&mu;l syringe with 26 gauge 			beveled needle</td>
 <td>World Precision Instruments</td>
 <td>SGE010RNS</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Adison Forceps</td>
 <td>World Precision Instruments</td>
 <td>500092</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Penfield 			Dissector</td>
 <td>Codman</td>
 <td>65-1015</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>16g 1&frac12; Precision Glide Needle</td>
 <td>Beckton, Dickinson and Company (BD)</td>
 <td>305198</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Surgical Blade Handle</td>
 <td>BD</td>
 <td>371030</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Size 15 Blade</td>
 <td>BD</td>
 <td>371315</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>4-0 Vicryl Suture</td>
 <td>Ethicon</td>
 <td>VCP496G</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Bone Wax</td>
 <td>Medline</td>
 <td>DYNJBW25</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Povidine-Iodine Swab Sticks</td>
 <td>Medline</td>
 <td>MD93901</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>D-Luciferin Potassium Salt</td>
 <td>Caliper Life Sciences</td>
 <td>122796</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Forane (Isoflurane)</td>
 <td>Baxter</td>
 <td>1001936060</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>OPMI Pentero Microscope</td>
 <td>Carl Zeiss, Inc.</td>
 <td>&nbsp;
 </td>
 <td>Any surgical microscope will suffice</td>
 </tr>
 <tr>
 <td>Xenogen IVIS Spectrum with optional anesthesia 			system</td>
 <td>Caliper Life Sciences</td>
 <td>&nbsp;
 </td>
 <td>&nbsp;
 </td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas at JoVE.com

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.

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 ...

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

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

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 ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

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.

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.

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

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...