The overall goal of the following experiment is to reproducibly transplant stem cells into the hippocampus. First, athymic rats are subjected to stereotactic radiotherapy, simulating what might occur during clinical management of brain cancer. In the lab, human neural stem cells are grown for transplantation, 1 million cells are typically required for the treatment of a single rat.
Two days following irradiation, the stem cells are slowly injected into the hippocampus at a precise location. Ultimately, the protocol shows the migration of transplanted stem cells throughout the hippocampus. I first had the idea for these methodologies when I started to consider the prospects of using stem cells to reverse the inhibition of neurogenesis After cranial irradiation, Generally individuals knew to this method will struggle because growth of stem cells is not trivial and it requires daily attention and care under antibiotic free growth conditions.
Visual demonstration of this procedure will demystify the clinical process used for imagery reconstruction for the precise irradiation of small, well-defined brain volumes. Demonstrating the irradiation procedure will be Dr.Dante Rowa, a clinical professor of my department. The preparation of stem cells will be demonstrated by Ms.Mary Land and Catherine Tran technicians in my lab and Dr.Muja, a postdoctoral scholar will demonstrate the procedures required for transplantation.
We utilize image guided radiotherapy software for accurate animal positioning prior to treatment and modulated radiotherapy for targeted irradiation with minimum radiation damage to other parts of the brain. 24 hours after an MRI scan, generate another cross-sectional image volume by placing the sedated animal in a radiation oncology CT scanner. Place the rat in the same position as used for the MRI scan and set the CT unit to scan the skull region to generate the CT treatment planning data.
Obtain 106 CT images with a 0.8 millimeter image thickness. Next, transfer the MRI and CT image data to the Eclipse treatment planning software. Use eclipse to fuse the MRI and CT data based on bony anatomy and soft tissue matching.
This is a time variable step and depending on image quality, this process may take from one to three hours. Once a satisfactory image fusion is obtained, contour the target regions to be irradiated as well as adjacent critical organs. Irradiation can be delivered to the whole brain or restricted to specific volumetric regions such as individual hippo campi.
The contours provide not only the location of these organs, but volume information that will be critical for tailoring the dose distribution. To tailor the dose distribution. Enter information on the target dose, target conformity and dose constraints to critical organs into the dose optimization window in eclipse.
Once a plan is generated, eclipse provides the calculated dose superimposed to the axial coronal and sagittal images, as well as in dose volume histogram format. It is at this point when the plan must be evaluated to determine if it is suitable for delivery or needs to be improved. To begin the irradiation process, place a sedated rat under a paper blanket to keep it warm.
After a few minutes, place the rat onto the treatment table of the radiotherapy linear accelerator covered with the blanket, but with the skull exposed. Be sure to place it in the same position used to generate the CT scan. This step is critical to ensure accuracy in dose delivery.
Next, acquire orthogonal x-ray images of the sedated rats using the onboard imaging system in the radiotherapy linear accelerator coregister the orthogonal x-ray images to a reference image set provided by Eclipse and proceed to apply the necessary position corrections for proper image alignment. Deliver the irradiation as a 10 grade dose with a six field IMRT plan for a single hippocampus and with a two arc rapid arc plan for both Hippo campi irradiation. After treatment delivery is complete, remove the wrap from the treatment room and allow it to recover in a heated holding cage.
To prepare for surgery, use electric clippers to remove the fur from the head of an anesthetized A TN rat. Clean the shaved area with repeated applications of povidone iodine and 70%ethanol working within a laminar flow hood. Prepare the stereotaxic instruments, micromolar drill, dry bed sterilizer and controllers.
Keep the animal warm during surgery by placing it on a heated surface. Sterile gloves are worn prior to and throughout surgery and sprayed with 70%ethanol. Surgical equipment is maintained in a dedicated laminar flow hood.
To maintain aseptic conditions, cover the animal with a small surgical drape so that only the head is exposed. Position the animal's head firmly within the stereotaxic frame by inserting ear bars into the external auditory atu. Take extreme care when sliding the tip of the air bar into the ear canal.
Place the incisor bar by hooking the rat's upper incisors and adjusting the bar height to the standard reference point. Then tighten the nose clamp. Center the position of the head between the ear bars with minimal lateral movement to achieve stereotaxic zero.
After the animal is in position, apply lubricating eye ointment to prevent drying. Apply the ointment once more during surgery. Using a sterile scalpel, make a two centimeter midline skin incision along the scalp.
Clean the area using a sterile cotton tip applicator. Next, insert a dissecting retractor to hold the periosteum open. Apply firm pressure to the area if bleeding occurs.
Using a cotton tipped applicator dipped in 1%hydrogen peroxide, clear away the soft tissues covering the skull until the sutures bgma and lambda are visible. The most difficult aspect of stereotaxic surgery is precise positioning of animal head in stereotaxic frame. To mark precise stereotaxic coordinates on animal skull, Use a wrap brain atlas to determine the precise stereotaxic coordinates using the B bgma as a reference in this study, stem cell transplantation is done at four distinct sites using the following stereotaxic coordinates to mark the locations on the skull, first, identify the bgma.
Next zero. All three coordinates on the digital display controller. Once zeroed, use the predetermined coordinates to locate the transplantation sites and to make a mark with a fine point marker attached to a probe holder on the frame.
Use a drill to make 0.35 millimeter holes through the skull at each marked location. Take care to prevent damage to the Durham membrane. If bleeding occurs during drilling, apply firm pressure using a cotton tipped applicator.
The animal is now ready for transplantation. The human neural stem cells used for transplantation should be prepared ahead of time and kept on ice until needed. When ready, fill a five microliter 30 gauge Hamilton micro syringe with the cell suspension.
Attach the filled syringe to a small probe holder on the stereotaxic frame. Once moved into the correct position, carefully insert the tip of the needle into the hole in the skull until it reaches the surface of the brain. Zero the DV Coordinate on the digital display controller and then gently insert the needle to the desired depth.
Keep the needle in place for one minute. After this time, inject cells at a rate of 0.25 microliters per minute for a total of one microliter. Keep the needle in place for eight minutes before retracting to prevent capillary reflux of the injection contents back through the needle track slowly retract the needle from the transplantation site at a rate of 0.5 millimeters per minute.
Repeat this procedure for the remaining transplantation sites. When the injections are complete, remove the skin retractor and use blunt tweezers to gently pull the skin back over the skull. Close the wound with the application of four to five sterile stainless clips after surgery.
Place the animal into a heated cage and monitor its recovery. One month following transplantation, brain samples were sectioned and stained with BRDU to detect transplanted NSCS and counter stain with hematin. The needle track represented here by the red line indicates the injection trajectory that deposited NSCS at the transplant release site designated as TR as seen.
Here, the transplanted NSCS showed extensive migration from the corpus cossum and CA one regions to the dente gyrus and ca three regions of the hippocampus. It is important that the acquire Mr.I image set is used accurately to a corresponding CT image set. Proper image fusion is critical for identification and subsequent contouring of the hippocampal regions prior to treatment procedure, acquire or orthogonal x-ray images to ensure that the animal is accurately positioned To ensure success of transplantation maintains sterile surgical en environment at all times.
Mark Otax coordinates precisely and remove micro syringe after injection gradually to prevent fluid reflux. After watching this video, you should have a good understanding of undertaking a clinically relevant radiation procedure using an animal model of your choice and how to surgically transplant stem cells to a precise region of the brain.
