This protocol provides a method for evaluating vascular damage as a result of cranial window drilling. A surgical robot offers improved consistencies across procedures while reducing the amount of training to learn the technique. During this procedure, we utilized cranial injections which can be very challenging.
We recommend extensive training for those new to the technique. To begin, set up the system as described in the manuscript and perform frame calibration following the surgical robot manual. Navigate to the surgical software and create a new project by selecting start with a clean project.
Then set the subject as mouse at the top to designate the drilling coordinate atlas to be used. Next, select start new project, and click on planning in the bottom left corner to navigate to the drilling coordinate planning screen. Create the drilling scheme for the cranial window technique to be performed by clicking anywhere on the stereotaxic atlas.
Use bregma as the reference and input the coordinates for the motor cortex as described in the manuscript. Press Enter on the keyboard to update the selected coordinates. Click store target to save these coordinates and input an appropriate name.
Then click on the move button in the bottom left to navigate back to the main drilling screen. Click on tools, then project, and save as option to reuse this template project for later projects, as it will automatically retain the drilling coordinates. First, administer subcutaneous injections of antibiotics, cefazolin, analgesic, carprofen, and buprenorphine to the anesthetized mouse.
Next, place the mouse on a heating pad and maintain anesthesia with 0.5%to 2%isoflurane via inhalation through a nose cone. Mount the animal on the surgical robot stereotaxic frame using supplied ear bars and routinely assess vitals and anesthetic depth via toe pinch. Then scrub the surgical area with chlorhexidine gluconate and 70%isopropanol for sterilization.
And place a sterile plastic wrap over the mouse and stereotactic frame to maintain sterility during surgery. Using a scalpel blade, perform a one-inch incision on the midline of the skull, starting at the back of the eyes, dry out and clean the skull using 3%hydrogen peroxide with cotton tipped applicators. To prepare the tail for easy injection, wipe down with alcohol.
Using the thumb and forefinger, bend the tail to expose the tail vein on top of the bend of the tail. Insert the syringe parallel with the vein, and slowly inject the volume of Evans blue. Once injected, wait for five minutes to allow Evans blue to circulate.
A successful injection is immediately verified as the mouse's extremities and surgical window turn blue. Once the skull is prepared for drilling, navigate back to the surgical software and click on tools. Then select project, followed by new, and select a template project from the dropdown menu to choose a template project.
Select same protocol elements. Click on planning, then select drill parameters to carry over to this new project and click start new project. Next, correct the drill and frame to account for the tilt and scaling of the mouse skull of the current animal by clicking tools, and select correct for tilt and scaling to open the corrections screen.
At the top of the screen, ensure that the drill is active by clicking on the light red drill button After correction has been performed, come out of the correction window by clicking close in the bottom middle of the screen and navigate to the drilling screen by clicking tools and then selecting drill to begin the drilling procedure. Confirm the drill site by selecting the craniotomy shape window at the top of the screen, followed by viewing the drilling target details in the dropdown below. To carry out manual pulse drilling, turn off the auto-stop feature by unchecking the checkbox next to the auto-stop option in the drill menu, which allow for controlling when the drill is off for the pulsing.
In the drill menu, select 100 micrometers as the drill depth advancement. Open up the drill site menu and navigate to the first drill site. Use the advance button or manual controls to lower the drill until it touches the skull and press set surface once the drill bit reaches the skull.
Once ready, click advance to commence drilling. And after the drill has advanced 100 micrometers, press Escape twice to stop the drill. After two seconds, repeat this cycle for the depth of the skull.
For each seed and edge point during drilling, ensure to set dura using the button in the drill menu once the dura has been reached. Measure the changes in temperature of the skull and brain using a thermocouple in combination with the three different drilling schemes connected to a data acquisition system, that allows for the measurement to be read into MATLAB. Mount a cadaver mouse onto the stereotaxic frame and robotic drill set up.
Manually drill a small hole approximately two millimeter away from where the cranial window will be made into the side of the skull, which allows for thermocouple to be slid into position under where drilling of the cranial window occurs. Begin the drilling process for each of the three schemes as demonstrated previously, resulting in spikes due to temperature change, indicating heating occurring near the brain as the drill goes through the skull. Potential for thermal damage was evaluated by measuring the change in temperature from baseline due to drilling using horizontal, point by point, and pulsed point by point methods.
Both the horizontal and point by point drilling schemes show non-significant differences in thermal changes. However, changing to a pulsed point by point method resulted in significantly less heating of the brain than both horizontal and point by point drilling. The horizontal edge drilling took 300 seconds, while point by point edge drilling took 200 seconds.
The pulse method took the longest with seed and edge drilling, taking approximately 500 seconds each. To monitor the relationship between the drilling scheme and vascular damage, Evans blue fluorescent imaging was performed. The drilling via horizontal and point by point cutting was seen to be damaging to the vasculature in the brain.
In comparison to the control groups, the pulsed point by point method had less localized damage at the seed and edge point, but still had visible Evans blue presence within the cranial window. When manually drilling it is crucial to check the craniotomy after advancing the drill to ensure the dura is not breached. In addition to cranial windows, a surgical robot can be used for standard craniotomies to improve surgical outcomes.
