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

The overall goal of this procedure is to create an intracranial model of gliomas that can be quantitatively followed over time by in vivo imaging. This is accomplished by first harvesting, cultured by luminescent mirroring glioma cells and preparing a cell suspension of one to two times 10 to the seventh cells per milliliter. The second step of the procedure is the stereotactic implantation of harvested cells into the right frontal lobe of the mouse brain.

This is followed by subcutaneous injection of Luciferian into the animal and generation of a kinetic curve for Luciferian expression. Using the IVUS spectrum in vivo imaging system, the final step of the procedure is to serially image animals and track tumor growth. Ultimately, results can be obtained that show tumor cell growth and 3D localization of the tumor through a bioluminescent signal detectable using the IVUS spectrum in vivo imaging system.

The main advantage of this technique over existing methods such as MRI is that bioluminescence imaging identifies live tumor cells only and does not require a specially trained MRI technician. It also allows for animals to be imaged simultaneously in a shorter period of time. This method can help answer key questions in the field of neuro-oncology, such as what is the efficacy of novel therapies for brain tumors.

In vivo, Though this method can provide insight into brain tumors. It can also be applied to other organ systems such as lung, prostate, and hepatic cancers. To prepare for this procedure, load at least three microliters of the glioma cell inoculum into a 10 microliter, 50 millimeter syringe with a 26 gauge beveled needle.

Make sure that the cells are not clumped together or it may be necessary to reload the syringe. The anesthetized mouse is placed in the stereotactic frame and the mouse head is secured using the mouth clamp as shown in this picture. Place the loaded syringe into the micro injector.

To begin the surgical procedure, make a skin incision using a size 15 blade and tooth forceps. Make a 10 to 15 millimeter incision between the animal's eyes, moving toward the animal's ears, exposing the bgma. Be sure to properly identify the bgma for.

It can be easily confused with the sinus area that is distal to the bma. With a 16 gauge one and a half inch needle, make the bur hole 0.1 millimeters posterior to the coronal suture and 2.3 millimeters to the right of the midline by applying a little pressure to the needle while twisting until the skull is penetrated and the brain is exposed, move the injection needle using the micro injector until it just touches the surface of the brain. From this position, advance the needle to a depth of three millimeters.

Keep the needle in place for three minutes. Next, withdraw the needle 0.4 millimeters to a total depth of 2.6 millimeters below the surface of the brain. This creates a small pocket where the cells are to be infused to ensure proper placement and depth.

X-ray images of the needle depths may be taken using a C-arm, infuse the cell suspension over three minutes using the micro injector set to a volume of 2000 nanoliters with an infusion rate of 667 NANOLITERS per minute. After the infusion, lead the needle in place for two minutes to prevent leakage from the site of infusion. Then slowly withdraw the needle completely.

Once the needle has been withdrawn, cover the bur hole with bone wax using a Penfield dissector. Finally, suture the incision using a four oh Vicryl suture, making sure no large gaps are left in the skin. In vivo.

Bioluminescence imaging is performed with the IVIS Spectrum imaging system. Start the living image software and initialize the IVIS Spectrum Imaging system by clicking the initialize IVIS system button on the bottom right side of the control panel. Select the luminescent imaging mode on the top left side of the control panel to determine the optimal time of bioluminescence imaging.

After Lucifer injection of the animal, it is necessary to first conduct a kinetic study with the sedated animal in the imaging chamber. Take the first image approximately five minutes after the Lucifer injection to generate a kinetic curve for Luciferian expression. Create a sequence to take images every three minutes for up to an hour.

Click on the sequence setup button in the control panel. In the control panel. Specify the settings for the first bioluminescent image in the sequence, starting with medium benning and auto exposure.

To determine the optimal exposure, time is recommended. Select the delay button in the sequence editor and specify a delay time of three minutes between each acquisition. Click add in the sequence editor acquisition parameters are then added to the table.

Repeat this step for each image in the sequence once the curve is established. The optimal imaging time can be determined by plotting the signal strength versus time to get the strongest and most accurate signal. Animals should be imaged at the time of highest in vivo photon count, which is about 25 minutes after Luciferian injection as shown by this curve.

To acquire the image of an experimental animal, use the auto option under acquisition time. This allows the software to determine the bin and optimal length of time for acquiring the image. Save the program files and subsequent image comments in the user's computer directory.

To begin 3D imaging, use the imaging wizard to select the firefly reporter probe. Under the bioluminescence delit window, select next and six spectral selections to be acquired will appear on the screen select acquire sequence and the images will be taken using the respective settings under the surface topography tab. In the tool palette, mask the photograph to fit the outline of the animal.

To create a reconstructed mesh of the animal's surface topography, proceed to the DLI 3D reconstruction tab on the tool palette and select the appropriate image acquisitions for signal reconstruction. Be sure that muscle is listed under tissue properties and firefly under source spectrum. Click reconstruct under the analyze tab and the 3D reconstruction of the animal surface and the corresponding reconstruction of the signal source should appear.

Data analysis can begin after the image is acquired and saved access the program file by clicking the browse button and selecting the file. The image information can be found under view image information. Quantify the intensity of the signal by selecting the region of interest or ROI tools button.

Make sure the image is analyzed under the photon mode by selecting photon. On the dropdown list on the top left corner of the image control panel, select the measurement ROI button from the type dropdown list and select the ROI shape of interest. The options include circle, square, and or grid.

Set the ROI position by dragging the ROI shape selection to the region containing the bioluminescent signal. Be sure to cover all areas of intensity on the acquired image. Finally, click the measure button to compute the signal intensity of the ROI.

The ROI label displays. The intensity successful cell implantation is evident when implanted cells are detectable by bioluminescence imaging on the day of surgery, regardless of whether the cells have been transfected with the luck or luck two gene. This graph tracks the GL 2 6 1 luck tumor cell growth in, in an albino C 57 black six mouse bioluminescence was measured every three days and plotted as in vivo photon count versus days post implantation.

The photographs show bioluminescence at various time points. Coloration is an indication of bioluminescence, which is relative to tumor cell number. Animals are euthanized at the occurrence of illness or poor welfare.

Although not a standard part of these analyses, the brain can be dissected out and Lucifer can be added topically to obtain the sex vivo image. This must be done immediately after the animal is sacrificed because the bioluminescence reaction requires a TP.This graph compares photon counts obtained from tumors resulting from GL 2 6 1 lux cells versus GL 2 6 1 L two cells. The results from an average of five animals show that the L two gene provides a higher level of bioluminescence.

This next figure shows multiple views of a three dimensional reconstruction of the intracranial implantation of GL 2 6 1 L two cells, coregistered with the mouse, skeleton and brain. A kinetic comparison of subcutaneous and intraperitoneal injections was also performed three minutes after Lucifer was injected, the mouse was sedated and imaged every three minutes for up to an hour and every six minutes after that to generate a kinetic curve of bioluminescence. As shown here, a subcutaneous root of Lucifer administration shown in red is superior to an intraperitoneal injection shown in black, and the optimal time to image the animals is approximately 25 minutes following the Luciferian injection.

Once mastered, the surgical technique can be done in an hour if it is performed properly. Following this procedure, other methods such as MRI or fluorescent imaging can be performed in order to answer additional questions. For example, whether there's edema present around the tumor or what other molecular or biochemical changes are occurring in and around the tumor.

After watching this video, you should have a good understanding of how to perform an intracranial implantation and 3D in vivo imaging of mire glioma cells.