This method creates a paradigm for future studies by providing researchers with a reliable and translational model of concussion that allows longitudinal characterization of the molecular effects of concussion. This rat model combines microdialysis allowing continuous analyte quantification with a wedge wrap technique exerting rapid acceleration and deceleration of the head and torso that mimics an essential feature of human craniocerebral trauma. The implication of this technique extend towards therapeutical studies of concussion as it offers a valuable opportunity to study the mechanism and efficacy of pharmacologic agents in vivo and uninterrupted.
It is highly recommended to work in teams of two during the concussion and sham induction steps of this method to restrict manipulation errors and maximize efficiency during the experiment. Demonstrating the procedure will be Chloe Provost, a technician in our laboratory. To begin the procedure, shave the animal's head using an electric clipper.
Clean the shaved area using a solution of 2%isopropyl alcohol and 2%chlorhexidine gluconate three times. Then apply lubricating eye ointment to prevent dryness during anesthesia. Next, place the rat in a stereotaxic apparatus.
Insert the ear bars into the ear canals with great care and tighten the nose clamp. Afterward, fix a 26 gauge stainless steel guide cannula to the holder arm on the stereotaxic apparatus. Make a midline incision of three centimeters on the scalp.
Leave the skull clear by installing four clamps around the incision. Scrape the periosteum firmly from the skull with the surgical blade until the bregma and lambda sutures are visible. If there is bleeding, maintain firm pressure on the skull with a gauze pad or cotton tipped applicator.
Confirm if the skull is correctly aligned on the stereotaxic apparatus by comparing the dorsoventral coordinates of the bregma and lambda sutures. Identify the anteroposterior, mediolateral, and dorsoventral coordinates of the bregma suture as the reference points for the coordinates of the guide cannula. Then taking the bregma suture coordinates as references, calculate the coordinates of the guide cannula implantation site in the hippocampus.
Mark the precise implantation site using a marker. Subsequently, drill a hole with a diameter of 0.5 millimeters through the cranium at the target site of the guide cannula. Drill three other holes approximately five millimeters around this point to thread three anchor screws into the skull that will solidify the cannula after acrylic dental cement is applied.
Next, insert the cannula into the hippocampus and fix it with dental cement. Be careful not to spill excess dental cement around the site where the weight will be dropped. Leave the cement to dry for two minutes then remove the holder arm from the cannula.
Insert a stainless steel removable obturator into the cannula to avoid cerebrospinal fluid seepage and risks of infection. Afterward, remove the four clamps, pull back the retracted skin and stitch it with a surgical suture thread. Next, remove the rat from the apparatus and inject Buprenorphine subcutaneously to treat pain.
Place the rodent back in its cage with a heating pad underneath until it becomes conscious. Then return it to the animal care facility for a recovery period of seven days under close monitoring. In this procedure, remove the obturator from the cannula and insert a microdialysis probe slowly.
Next, fix the probe assembly to a stainless steel spring tethered to a liquid swivel and counterbalance lever arm with a ring stand and clamps so that the animal can move freely within its cage. The tethered rats have ad libitum access to food and water during the entire microdialysis procedure. Use a microinfusion pump to deliver perfusate to the probe and collect the dialysate from the fused silica outlet line.
At least one hour and 30 minutes before the procedure begins, turn up the probe to its working flow rate at one microliter per minute. Verify that the flow rate of the probe is consistent by measuring the volume over time with a pipette. Afterward, collect each dialysate sample in a fraction vial preloaded with one microliter of 0.25 mole per liter perchloric acid to prevent analyte degradation.
Store the sample at four degrees Celsius for subsequent analysis. Once the last sample is collected, remove the microdialysis probe from the cannula and reinsert the obturator before returning the rat to the animal care facility. To install the concussion apparatus, tape an aluminum sheet tightly to a U-shaped Plexiglass frame that contains a foam cushion.
Use a sharp razor blade to make slits in the aluminum sheet. Tape the slotted aluminum sheet. Then position the Plexiglass frame under a PVC guide tube.
Hold the PVC guide tube in place with a clamp stand at 3-1/2 centimeters above the slotted aluminum. Attach a nylon fly fishing line through the metal loop so that the bottom of the weight is at 2-1/2 centimeters over the slotted aluminum to prevent multiple hits when the rat is falling on the foam cushion following impact. Then attach the nylon fly fishing line to the clamp stand.
Pull up the weight through the PVC tube with the nylon fly fishing line. Keep it in place by inserting a hex key through the preliminary drilled holes at one meter above the slotted aluminum foil. To induce concussion, place the animal on its chest on the slotted aluminum sheet so that its head is positioned directly in the path of the brass weight.
Remove the nose cone and pull the hex key. The weight will fall vertically through the PVC tube and impact the head of the rat. As a result, the rat will undergo a rapid 180 degree rotation and land on its back.
Remove the rat from the foam cushion and place it on its back in the cage. Use a digital timer to measure the righting reflex time as a sign of recovery and injury severity. The righting reflex time is the total time from the impact until the rat wakes up and spontaneously turns back to the prone position or starts walking.
Note any signs of death, fracture or bleeding. For sham induction, place the animal on its chest on the slotted aluminum sheet so that its head lays directly in the path of the brass weight. Remove the nose cone and then remove the animal from the aluminum sheet without pulling the hex key.
Afterward, place the rat on its back in the cage. Use a digital timer to measure the righting time as an indicator of neurologic restoration. Animals from the injured group had a significantly increased righting time on average versus sham cases and appeared stunned upon regaining consciousness.
Of the 10 cases from the concussion group, only one animal showed minor signs of bleeding under the impact site following the weight drop. Significant increases in extracellular glutamate concentrations were observed in the hippocampal CA1 region during the first 10 minutes following trauma induction compared to sham injury. Please remember during the concussion induction avoid impacting the cannula with the weight as this would generate injuries to the rat's core that are significantly more severe than a concussion.
This method can be applied to the study of repeated concussions since the procedure is a close head injury which can be repeated on the same animals at different time points.
