This method can help determine the base distribution of the lobitic stem cell following intranasal delivery to the brain. The main advantage of this technique is that facilitates the non-invasive delivery and tracking of mesenchymal stem cells to the brain. Also, the multiple dosages are possible to maximize the therapeutic effects of the transplanted cells at the chronic stage after the injuries.
This technique maximizes the number of cells delivered to the site of injury and minimizes the progression of lobitic cell into other tissues. Although this technique provides insight into the use of mesenchymal stem cells in traumatic brain injury therapies, it can also be used for the investigation of non-traumatic brain injuries. For mesenchymal stem cell labeling with super paramagnetic iron oxide, add six milliliters of labeling medium to an 80%confluent mesenchymal stem cell culture in a T 75 flask for a 24-hour incubation at 37 degrees celsius and 5%carbon dioxide.
The next day, carefully aspirate the super natant and wash the cells two times with six milliliters of PBS per wash. To determine whether the cells have been successfully labeled, check the culture under a florescent microscope. To induce a CCI injury, after confirming a lack of response to pedal reflex, use electronic clippers to shave the fur from the dorsal surface of the skull, and clean the shaved area several times with a sterile cotton swab soaked in iodine.
Use a cotton swab soaked in 70%ethanol to remove the iodine after the last swab and place the animal in a stereotactic frame. Secure the mouse with the ear and nose bars and make a 2.5 centimeter midsagittal incision in the shaved skin to access the surface of the skull. Use a cotton pad to remove the tissue covering the skull and clean the skull's surface for 10 seconds with a cotton swab soaked with 3%hydrogen peroxide.
Dry the skull with a fresh cotton pad and use a pencil to draw a four millimeter circle around the coordinates of choice on the exposed bone. Using a micro drill equipped with a 0.5 millimeter diameter round bur, carefully thin the skull at the marked circle without applying pressure. Remove any bone dust with a clean and dry cotton swab and use sterile filed forceps to carefully remove the resulting bone flap.
When the dura mater has been exposed, transfer the mouse into the stereotactic frame of the CCI device, and secure the animal with the ear and nose bars so that the head is level in the rostrocaudal direction. Following the instructions on the control box, zero the impactor tip to the exposed cortical surface using the X and Y control wheels on the base of the impactor to align the impactor tip directly above the desired cortex coordinates to be impacted. Use the control box to set the experiment parameters to a velocity of five meters per second, a dwell time of 250 milliseconds, and injury depth of one millimeter to induce a mild injury.
Then, press the impact button on the control box. Swab any bleeding that occurs with a sterile cotton swab and remove the mouse from the frame. Close the incision with silk surgical sutures and apply topical antibiotics to the site before placing the mouse onto a heating pad with monitoring until full recumbency.
One day after the injury, treat the super paramagnetic iron oxide labeled mesenchymal stem cell culture with three milliliters of trypsin. After five minutes at 37 degrees Celsius, start the reaction with seven milliliters of pre-warmed DMEM medium, supplemented with 10%fetal bovine serum and collect the cell suspension into a 15 milliliter conical tube. Sediment the cells by centrifugation and re-suspend the pellet in PBS for counting.
Then, adjust the cell concentration to 1.5 times 10 to the five cells per 18 microliters of PBS. For cell delivery, after confirming the lack of response to toe pinch, scruff the mouse while immobilizing the skull. Place the tip of a pipette containing four units of hyaluronidase per microliter of PBS near the nare of the mouse at a 45 degree angle, and administer three microliters of hyaluronidase suspension into each nostril.
Place the animal face up on a clean pad for five minutes before repeating the treatment four times for a total of 100 units of hyaluronidase treatment. After the last treatment, place the mouse back onto the pad for 30 minutes, before restraining the mouse as just demonstrated. With the head immobilized, administer three microliters of the mesenchymal stem cell suspension into each nostril over a period of three seconds per solution delivery, holding the mouse in position for 30 seconds until the sample drops have completely disappeared.
After two minutes, repeat the delivery up to three times until the entire volume of mesenchymal stem cell has been delivered. Then, return the mouse to its cage with monitoring until full recumbency. To track the migration of the mesenchymal stem cells by MRI, place the anesthetized mouse onto the imaging holder of the MR imager.
Then, secure the animal in place and move the holder to the center of the MRI coil. Set the repetition time to 1500 milliseconds and the echo time to 2.8 milliseconds. Then, set the field of view to 16 by 16 millimeters, the acquisition matrix to 128 by 128, and the slice thickness to 0.75 by 0.8 millimeters with four signal averages and a 90 degree flip angle to acquire T2 star weighted scans using a spin echo sequence.
After completing the scans, retract the mouse holder from the MRI coil center, and return the mouse to its cage with monitoring until full recumbency. To track and quantify the labeled mesenchymal stem cells on the T2 star weighted images, open the data in the IT case snap software and select active label. To create segmentations of the hypointense areas and the lesion or other brain parts of interest, using different label colors for each segment.
Use the polygon tool in the main tool bar to select the hypointense areas that represent the super paramagnetic iron oxide labeled mesenchymal stem cells and click accept. The segmented areas will appear in the same color as the active label assigned to that particular segment. When all of the slices have been segmented, use the scalpel tool to develop a 3D map of the segmented areas to represent the mesenchymal stem cell distribution in the whole brain.
To perform a quantitative analysis of the volume and intensity mean of the segmented hypointense areas representing the labeled cells, click segmentation and select volume and statistics. 24 hours following intranasal delivery, super paramagnetic iron oxide labeled mesenchymal stem cells are detected as strong hypointense areas medial to the cortical injury on T2 star weighted images, indicating the targeted migration of super paramagnetic iron oxide to the injury site. This migration remains visible up to 14 days post-delivery without any noticeable reduction in signal.
Injured animals treated with PBS do not exhibit hypointense areas at any time point, indicating that the observed hypointense areas correspond to the super paramagnetic iron oxide labeled mesenchymal stem cells, and are not due to signal artifacts. The bio-distribution of the labeled mesenchymal stem cells can be visualized using 3D reconstruction, histologically by Prussian blue staining, or by florescence detection of FITC tagged super paramagnetic iron oxide within the labeled mesenchymal stem cells. Be sure to validate the MRI results with esthology or other methods.
As super magnetic iron oxide particles can remain in tissues after the cell dies, leading to false positive signals. Following this procedure, possible temperal non-invasive tracking in quantification can be applied to answer questions regarding honing the abilities and retention of mesenchymal stem cells in the brain. After this development, this technique has paved the way for researchers in field of regenerative medicine to exploring methods for improving cell honing to specific areas of the brain.
