The overall goal of this procedure is to achieve a standardized follow-up in the in vivo behavior of stem cells transplanted into the mouse brain. This is accomplished by first labeling the bone marrow derived stromal cells, which co-expressed EGFP and luciferase with fluorescent micron sized iron oxide particles. The second step is to appropriately inject the MPIO labeled BMCs into the mouse brain tissue.
Next, the survival and localization of cell grafts are followed non-invasively using bioluminescence imaging and magnetic resonance imaging. The final step is to validate the in vivo imaging results using postmortem histological analysis. This methods can provide insight into the behavior of stem cells after transplantation into the mouse brain, but it can also be applied to other systems such as model organisms and studies of a disease.
Visual demonstration of this method is critical as the animal handling steps are difficult to learn because intravenous and intracerebral injections are difficult to perform and contain certain risks for animal survival. To begin this procedure, add 75 times 10 to the six glacial blue micron sized iron oxide particles to a sub confluent growing cell culture. Incubate them for another 16 hours to allow particle uptake via endocytosis, then wash away the remaining particles.
Allow the cells to grow for an additional 24 hours to obtain cell co fluency and homogenous distribution of the G-B-M-P-I-O. After that, use fluorescence microscopy to visually validate the particle uptake by calculating the ratio of EGFP positive and G-B-M-P-I-O positive cells to the total amount of EGFP positive cells. Next, wash the G-B-M-P-I-O, labeled BMSC luciferase EGFP cells twice with 10 milliliters.
PBS treat the cells with tripsin EDTA and harvest them afterward. Reward then wash the cells and resuspend them at a final concentration of 133 times 10 to the sixth cells per milliliter in PBS. Finally, keep the cells on ice until transplantation.
In this procedure, anesthetize the mouse by intraperitoneal injection of the mixture of ketamine at 80 milligrams per kilogram and xylazine at 16 milligrams per kilogram. Wait for five minutes for the mouse to fall asleep, then shave its head to allow for sterile manipulations. Next place the mouse in a stereotactic frame, disinfect the skin with alcohol.
After that, apply ointment to its eyes To prevent dehydration. Make a midline scalp incision to expose the skull in order to target the hippocampus. Drill a hole in the skull at two millimeters posterior and two millimeters lateral using a dental drill burr.
At this point, vortex the cell suspension briefly, then aspirate the cell suspension in the syringe. Subsequently lower the needle syringe to a depth of 2.5 millimeters under the dura and allow pressure equilibration for one minute. After a minute, retract the needle to a position of two millimeters under the dura.
Then inject three microliters of cell suspension using an automated micro injection pump at the speed of 0.70 microliters per minute. After the injection, wait for an additional five minutes to prevent backflow of the injected cell suspension. Next, slowly retract the needle.
Afterward, disinfect the skin borders, then suture the skin. Inject 300 microliters of 0.9%sodium chloride solution subcutaneously in order to prevent dehydration. At the end, place the mouse under a heating lamp to allow recovery from anesthesia to perform bioluminescence imaging.
Anesthetize the mouse with a mixture of 3%iso, fluorine, and oxygen. Then place the mouse in the photon imager. Reduce the anesthesia level by adjusting the isof fluorine to 1.5%Next, inject d Lucifer intravenously at 150 milligrams per kilogram of body weight.
Acquire image for five minutes using the photo vision software. After that, perform image processing using the M three vision software and quantify the observed signal using fixed regions of interest. To perform magnetic resonance imaging, anesthetize the mouse with 3%ISO fluorine in a mixture of oxygen and nitric oxide.
Then place the mouse in the restrainer of a horizontal 9.4 Tesla MR system. Reduce anesthesia level by adjusting the isof fluorine to 1%Next, apply ointment to the eyes of the mouse to prevent dehydration. After that, use a rectal probe to monitor the body temperature.
Then place a sensor underneath the mouse belly to monitor the breathing rate. Maintain the breathing rate at 110 plus or minus 10 times per minute, and keep the body temperature constant within a narrow range of 37 plus or minus 0.5 degrees Celsius. Then place the surface RF coil on top of the mouse head.
Position the mouse in the middle of the magnet. Next open ParaVision 5.1 Set sequence parameters according to the accompanying manuscript. Then acquire a set of 10 coronal T two weighted spin echo images in order to obtain specific anatomical information.
NT two star weighted gradient echo images in order to study stem cell migration with an in plain resolution of 70 micrometers squared. In this step, sacrifice the mouse and remove the brain. Fix the brain tissue at room temperature in 4%paraldehyde in PBS for two hours.
After two hours. Dehydrate the brain tissue by placing it in different gradients of sucrose at four degrees Celsius. On the next day, freeze the brain tissue using fluid nitrogen and store the tissue at minus 80 degrees Celsius until sectioning when ready.
Section the brain tissue in 10 micrometer thick sections using a cryostat. Afterward under a fluorescence microscope screen the unstained cryo sections for blue fluorescence from the G-B-M-P-I-O particles and green fluorescence from the EGFP expressing cells. Then screen the unstained cryo sections for green red background fluorescence from the inflammatory cells.
The described glacial blue MPIO labeling procedure results in highly efficient labeling of BMSC, luciferase EGFP cells, which can easily be validated by fluorescence microscopy. Next, upon grafting of G-B-M-P-I-O, labeled BMSC, luciferase EGFP cells in the CNS of mice, the survival of the cellular graft can be monitored by in vivo bioluminescence imaging based on luciferase activity. Additionally, the exact localization of grafted cells can be monitored by in vivo magnetic resonance imaging based on the iron content of the glacial blue MPIO.
Finally, histological analysis shows the validation of the results obtained by bioluminescence imaging and magnetic resonance imaging. Stable survival was confirmed by a stable EGFP expression in time After its development. This technique paved the way for researchers in the field of molecular imaging to explore cellular and molecular events after stem cell transplantation in different animal models for neurodegenerative diseases.
After watching this video, you should have a good understanding of how to perform molecular imaging, applying bioluminescence and magnetic resonance imaging, and adequately validate in vivo results using postmortem histological evaluation.
