Our protocol demonstrates a comprehensive preclinical workflow for testing the therapeutic efficacy of human-induced pluripotent stem cells after brain injury. This model of traumatic brain injury provides a high degree of versatility and reproducibility by tightly controlling the parameters of the mechanical force and therefore providing a well-defined injury. These procedures can be used to study injury and recovery processes in other brain regions and the transplantation method is amenable to use with many cell types.
Cranial drilling is challenging as the technique requires manual dexterity to master the appropriate pressure, speed, depth, and smooth movements. Document all of the events and practice extensively. To perform a unilateral craniectomy, confirm a lack of response to toe pinch in the anesthetized mouse and secure the mouse in a stereotaxic frame.
Apply antibiotic ophthalmic ointment to the eyes with a sterile cotton swab and iodine-based solution to the shaved scalp area. Remove the iodine with 70%ethanol and cover the animal with a fenestrated surgical drape. Make a 1.5 to two centimeter midline incision on the scalp and use sterile cotton swabs to clean the wound and to clear the fascia left of the midline at bregma.
Use an impactor probe to identify the craniectomy site and set the x equals zero, y equals zero stereotaxic reference point to bregma. Adjust the probe laterally to two millimeters left of the midline. Use a fine-tip surgically safe marker to landmark a five millimeter diameter circle around the presumptive impact area.
Raise and rotate the impactor out of position and complete the five millimeter circle. Briefly return the impactor into position to verify accuracy of the five millimeter circle. Then raise and rotate the impactor out of position and use a high-speed rotary micro motor kit hand tool equipped with a round tip 0.6 or 0.8 millimeter burr drill bit to make an open hole in the skull at 70 to 80%maximum speed.
Apply light pressure to the skull while drilling along the five millimeter circumferential outline within this border and use forceps to lift the resulting bone flap. Drilling through suture lines may present additional difficulty as the bone is interrupted and requires additional pressure. Blood vessels may be in close association and therefore bleeding events are likely.
To induce a mild controlled cortical impact injury, clean the impactor probe with a sterile alcohol prep pad and move the impactor probe back into position over the exposed cortex. Lower the probe until it touches the dura mater surface and mark this position as z equals zero. Withdraw the piston and move to z equals minus one millimeter and discharge the piston to impact the cortex.
Immediately raise the piston and move the arm out of position and irrigate the cortex with a generous volume of saline and use simple interrupted stitches and 5-0 silk suture to close the scalp incision. Then deliver 10 milligrams per kilogram of Cyclosporine A by subcutaneous injection into the scruff and place the mouse in a clean pre-warmed post-operative cage with monitoring until full recovery. Roughly 24 hours after the craniectomy, place the mouse back into the stereotaxic frame.
Cover the mouse with the fenestrated surgical drape. Lavage the incision site with sterile saline to clean the site and to loosen the sutures. Use a 70%ethanol soaked cotton swab to gently sterilize the incision site and use fine tweezers and ophthalmic scissors to remove the sutures.
Then irrigate the surgery and craniectomy site with abundant sterile saline. In a cell culture biosafety hood, gently swirl or tap the tube of cells to ensure a homogenous cell suspension. Use a micropipette to load approximately 7.5 microliters of cells into a Hamilton syringe through the plunger end.
Holding the syringe at a 120 degree angle with the plunger end facing down, insert the plunger taking care not to introduce an air bubble between the suspension and plunger tip. Attach the gasket assembly to the pipette needle and attach the needle to the syringe. Push the plunger to move the cell suspension into the pipette needle and attach the syringe to the stereotaxic syringe pump.
Advance the plunger to make sure the syringe pump assembly is working properly and move the needle into the coordinates for injection. Align the needle tip to bregma and set the x and y-coordinates to zero. Move the needle tip over the craniectomy to two millimeters lateral and minus one millimeter posterior to bregma and touch the needle tip to the dura mater surface.
Raise the needle slightly then make a small incision in the dura mater at the location where the needle made contact. Set the stereotaxic coordinate to z equals zero and push the plunger to confirm that the cell suspension is flowing adequately before introducing the needle into the brain. Introduce the needle into the brain to a depth of z equals minus 1.4 millimeters to place the graft at the gray matter/white matter border of the deep cortex.
Start the syringe pump to infuse the cell suspension over a 15-minute period. Use a long working distance microscope to monitor cell suspension outflow irrigating the surgery site with sterile saline during the injection to maintain tissue hydration. When all of the cells have been delivered, slowly withdraw the transplantation needle and irrigate the surgery site with additional saline before closing the incision with new sutures.
Then deliver post-operative care as demonstrated. For an adhesive tape removal test, first use a small razor knife to cut yellow and red pieces of electrical tape into three by five millimeter strips on a smooth glass surface. Bring the mice into the behavior testing room for at least 30 minutes prior to the testing and use a handling cloth to restrain the mouse by the scruff of the neck and back such that the mouse holds the forepaws away from its body and head.
Use tweezers to place an adhesive strip on the plantar surface of each forepaw and use delicate and consistent finger pressure to secure the strips to the paws. Quickly place the mouse into a plastic cylinder and start two stopwatches when the mouse has all four paws on the plastic testing box. Then use the two stopwatches to record the latencies for left paw notice, left paw removal, right paw notice and right paw removal.
In this pilot experiment, forelimb function was compared in craniectomy alone, controlled cortical impact injury and naive mice. Mice that underwent surgery exhibited transient increased latencies to notice adhesive stimuli for one to three days immediately after the surgery and transient post-operative deficits in adhesive removal from the ipsilateral forepaw. Mice that underwent controlled cortical impact, however, exhibited significant deficits in motor performance in the forepaw contralateral to the injury compared to naive mice out of post-operative day 28.
Immunostaining of mouse brain sections for human nuclear antigen reveals that human cell grafts can be clearly distinguished from host tissues in SHAM injury and controlled cortical impact brains. You must observe standard laboratory safety procedures when handling cultured human cells and when handling syringe needles that come into contact with rodents or immunosuppressive drugs. Brain tissue sections can be analyzed for the expression of neuroinflammatory markers as it is valuable to know how the graft affects the host tissue injury response and vice versa.
We were surprised to find subtle sex differences in post-injury behavior outcomes. We are now investigating whether sex plays a role in the neuroimmune response to brain injury. Steady hands, patience, and practice are crucial for developing a good craniectomy technique.
Collateral damage from an improper craniectomy can compromise the area that you wanna target for cell transplantation.
