The overall goal of this surgical procedure is to thin the skull without damaging the underlying tissue and implant a cranial window suitable for imaging. This method is an improvement on cranial window surgery and may allow more surgeons to perform this technique. The main advantage of this technique is that it allows for shorter surgeries with a lower average rate of complications.
Before beginning the surgery, ensure that all instruments to be used have been autoclaved to ensure sterility. Anesthetize the mouse with isoflurane. Apply veterinary eye lubricant to the eyes to prevent drying.
Then, pinch the hind toe to ensure proper depth of anesthesia. Next, remove the hair over the scalp with a three-minute application of hair removal gel. Then, place the anesthetized mouse in the stereotaxic frame on a heating pad or calibrated water recirculating blanket set to 38 degrees Celsius.
Ensure that the mask delivering anesthesia is properly placed. Administer buprenorphine subcutaneously to provide presurgical analgesia. Then, disinfect the scalp with three alternating applications of chlorhexidine or Betadine and 70%ethanol using a cotton-tipped applicator.
Lastly, inject 100 microliters of bupivacaine diluted 50%in saline under the scalp to provide local anesthesia. Fix a four-millimeter sterile circular tip to the handpiece of the Piezoelectric surgery unit. The large tip size contributes to the speed of the surgery as the entire window can be thinned simultaneously.
Set the Piezoelectric surgery unit to vibrate on the lowest setting as higher settings may break blood vessels due to intense vibrations. Fill a 10-milliliter syringe with ice-cold ACSF and hold in the hand not holding the Piezoelectric handpiece. Gently apply the vibrating surgical tip to the skull with a light circular motion and thin the bone to the desired depth to perform a thin-skull preparation.
Irrigate the skull to prevent overheating from the high-frequency vibrations by dripping the ACSF from the syringe onto the skull at a rate of one-milliliter per minute. Gently adjust the angle of the handpiece and change the angle of the tip while applying pressure to the skull to thin the skull uniformly. Be careful to not overly thin one section of the window.
Make sure to gently adjust the angle of the handpiece to thin the skull uniformly. Check the skull regularly to avoid removing too much bone and damaging the underlying tissue. Place a one-millimeter piece of hemostatic collagen foam soaked in cold ACSF on the exposed dura.
Allow it to sit on the dura for about 30 seconds to stop any microbleeds from developing. After placing a four-millimeter glass coverslip laterally over the thinned window, use the back of a small plastic or wooden cotton-tipped applicator to apply approximately one drop of cyanoacrylate glue to the corner of the window. And use the tips of the forceps to ensure that the coverslip forms a seal with the skull.
Finally, add 100 microliters of dental acrylic to the skull to hold the window in place. While the dental acrylic is still soft, use the tip of a broken wooden applicator to push back any dental acrylic that occludes the imaging window. When finished, remove the mouse from the surgical apparatus and place it in a heated cage for recovery.
Monitor continuously until the mouse has regained full consciousness as assessed by normal mobility, gait and behavior. Assess the animals twice a day for three days for manifestations of post-surgical pain including decreased activity, grooming, food and water consumption, guarding behavior or increased aggression. Administer buprenorphine subcutaneously as an analgesic eight to 12 hours after the surgery for three days as approved in our IACUC protocol.
Monitor the area of the window for dehiscence, inflammation and signs of infection. Calcium transience observed with the red calcium indicator jRGECO1a were imaged in layer four pure middle neurons in the motor cortex of a head-fixed mouse with a three-millimeter window prepared with PHASOR. This image was taken through the same window, but shows a different region of layer four motor cortex.
As seen here, time per surgery was lower for PHASOR than dental drill surgeries with statistical significance denoted as P is less than 0.05 by a two-tailed T-test. The percentage of successful surgeries defined as no bleeding or visible damage to the dura was higher for PHASOR than dental drill surgeries as assessed by a one-tailed Z-test. Once mastered, this technique can be performed in less than 10 minutes if it is performed properly.
Following this procedure, a variety of in vivo imaging methods including multiphoton imaging can be performed to visualize neurons in vivo. Thanks for watching and good luck with your experiments.
