This intracranial injection protocol is significant because it allows precise injections, day-to-day monitoring, and accurate tumor volume measurement for more effective study of mechanisms of brain metastasis. The main advantage of this technique is that it allows for serial monitoring of intercranial tumor growth. Demonstrating the procedure will be Jonathan Spehar, a graduate research associate from the Sizemore Laboratory, and Anna Bratasz, an imaging research scientist from the Ohio State University Small Animal Imaging Shared Resource.
Before beginning the procedure, twist the stage lock on the drill to allow a drill bit adapter and sterile one millimeter drill bit to be inserted into the drill and manually tighten the bit locks to lock the drill. Attach the drill to the stereotactic frame and set the digital injector delivery rate to 0.4 microliters per minute, with a target of two microliters. After confirming a lack of response to pedal reflex, place the anesthetized mouse onto the frame and use the blunt end of a cotton tip applicator to position the teeth in the trough of the mouth bar.
Press the left ear bar against the medial canthus of the left ear to stabilize the skull and use the screw on the stereotactic frame to lock the skull into place. Then secure the other side of the skull with the right ear bar in the same manner. To make a calvarial window, clean the scalp with three sequential alternating Betadine solution and 70%ethanol scrubs and use a sterile scalpel to make a three millimeter incision through the skin along the central median aspect of the cranium following the sagittal suture line.
Identify and orient the sterile drill bit perpendicular to the bregma, making sure to reset the digital vernier scale to zero and move the drill bit two millimeters lateral to the sagittal suture and one millimeter anterior to the coronal suture. Move the skin away from the drill and using the highest speed and paying attention to the landmarks, carefully drill a hole, roughly 0.5 millimeters deep, through the calvaria, cooling the drill site with drops of sterile saline as necessary. Once the calvarial window has been made, carefully raise the drill and remove the drill from the stereotactic frame.
For cancer cell injection, attach the automatic injector unit to the stereotactic apparatus and load the six to eight microliters of cells thoroughly resuspended in DPBS into a sterile Hamilton syringe. Load the syringe onto the injector and dispense a small volume of solution onto a disposable sterile drape to prime the needle for injection. Use a cotton tip applicator to wipe the syringe with 70%ethanol and align the needle tip to the center of the calvarial window until it nearly touches the exposed cerebrum.
Reset digital vernier scale to zero and slowly insert the needle into a three millimeter depth into the brain. Leave the needle in the brain for at least 60 seconds before selecting run on the injector screen to begin the cell delivery to the injection site. When all of the cells have been delivered, allow the needle to rest in the brain for at least three minutes to allow the brain parenchyma to acclimate to the injection before retracting the needle from the brain at a rate of 0.75 millimeters per minute.
For magnetic resonance imaging of the tumor burden, 10 to 20 minutes before imaging at the appropriate experimental time point, administer 100 microliters per 20 grams of body weight gadolinium-based contrast agents to the mouse by standard intraperitoneal injection. Anesthetize the mouse post-injection and place the anesthetized mouse on a heated holder. Place the holder into a 9.4 T magnet equipped with a mouse brain surface coil and obtain a localizer image.
Then image the mouse brain using a T2 weighted rare sequence and post gadolinium-based contrast T1 weighted rare sequence. To measure the total tumor volume, open the MRI data file of interest in ImageJ and use the free hand selections tool to draw an outline around the tumor. Then open the analyze tab and select measure to obtain the area of the selected region.
When all of the tumor containing slices for an individual mouse at that specific time point have been assessed, export the values into an appropriate data analysis program. Here, a representative overview of the tumor volume quantification for a single mouse at day 7 and day 10 post murine mammary tumor cell injection can be observed. The evaluation of 30 slices per scan revealed that for this analysis, at day seven post-injection, five slices exhibited a tumor burden.
While at day 10, nine slices exhibited a tumor burden. The tumor area in each image was then measured as demonstrated, allowing the change in tumor volume to be tracked over time. Each cell line and each recipient mouse model is unique and therefore requires optimization of the number of cells injected and the MRI schedule to ensure imaging accuracy.
Following this procedure, the brain can be harvested for essentially any downstream assay, including single cell isolation or histopathological analysis.
