This protocol allows to study the stimulation consequences on neural networks helping to unravel the enigma surrounding DBS. And to determine the impact of a stimulation on brain dynamics. The main advantage of using FD repair during a stimulation is that we can visualize the in vivo consequences of the acute stimulation on brain dynamics and then refine in vivo the stimulation protocols.
This method is of particular relevance in the fields of neurology and psychiatry. As it is in these medical specialties in which DBS has its major impact as an anthroponotic strategy. To begin, lay the anesthetized animal supine on the CT bed.
For CT imaging, secure the face mask or nose cone to the rat. Locate the head in the center of the field of view of the CT scanner. Proceed to acquire the CT image using acquisition parameters according to the scanner's specifications.
For MR Imaging, lay the animal supine on the MRI bed. To avoid movements during MRI acquisition, secure the head to a stereotactic frame placed on the scanner bed. Also, secure the rest of the rat body with silk tape.
Once the position is correct, acquire the MRI image. Use an image processing software to spatially normalize CT and MRI using an automatic rigid registration algorithm based on mutual information. Localize the bregma line in the co-registered image.
And measure the distance in the AP, ML, and DV access from bregma to the medial prefrontal cortex according to the Paxinos and Watson Rat Brain Atlas. Shave the area between the ears and the eyes. Place the animal in the prone position on the stereotactic frame.
Ensure immobility of the head by using the rat ear bars. Be careful not to insert the ear bars too deep as this may damage the eardrum. Use the head holding adapter for rats to maintain the animal in the correct position during the surgery.
Apply Athymic lubricating gel to the rat eyes to prevent dryness during surgery and cover them with sterile gauze. Make a longitudinal incision in the skin overlying the skull between the ears. Extending 1.5 to two centimeters from lambda to bregma.
Expose the skull with the help of two or three clamps. Remove the periosteum with scissors and clean the blood with saline solution To expose bregma and the sagittal sutures. Remove the excess saline solution with gauze.
Before positioning the electrodes, straighten them with plastic tweezers to ensure the correct placement during the surgery. Place one electrode on the holder of the right arm of the stereotactic frame. Move the right arm holding the electrode through the stereotactic frame.
And place the tip of the electrode exactly over bregma. Try to bring the electrode tip as close as possible to the skull without touching it To avoid electrode deformation. Note the resulting coordinates for bregma provided by the stereotaxic frame.
Using a surgical pen, make a mark on the skull indicating the initial position of the electrode. Move the holder to the AP and ML coordinates obtained earlier. And make a mark on the skull with a surgical pen indicating the position of the electrode target.
Remove the right arm of the stereotactic frame holding the electrode. Be careful not to touch anything with the electrode. Using a small electric drill, make a hole through the skull in the target position until the dura is visible.
Use a cotton bud to stop the bleeding. Drill four holes along the skull to place four screws. To increase the dental cement surface area and locate the ground.
Then attach the screws. Locate the right arm of the stereotactic frame with the right electrode. Move the arm to the calculated position which should coincide with the hole.
Then lower the electrode until it touches the dura mater. This position will serve as zero level in the DV direction. Using the DV coordinates obtained earlier, insert the tip of the electrode in the DV direction.
Attach the ground to one of the screws closest to the electrode and apply dental cement around the electrode and screws. Repeat the same electrode placement procedure for the other hemisphere of the brain. Apply more dental cement to form a cap without covering the electrode and wait until it hardens.
Use iodopovidone solution to disinfect the surgical area. Remove the rat from the stereotactic frame and proceed with CT imaging as demonstrated earlier to evaluate the correct placement of the electrodes. Fast the rat for eight to 12 hours prior to each PET scan.
Fill a 27 gauge syringe with approximately 37 megabecquerel of the FDG solution in the least possible volume as measured in an activimeter. Place a heating pad under the animal's tail or use infrared light to dilate the tail veins. Inject the FDG solution through one of the lateral tail veins.
Place the animal back in the cage and allow 45 minutes for radio tracer uptake before initiating the image acquisition session. For the D2 study, delivered DBS during the FDG uptake period. Prepare the isolated stimulator and the required wires in a vast and quiet room with enough space for the animal cages and minimal influence of potentially disturbing stimuli.
Connect the stimulation wires to the swivels to allow animals to freely move within the cages and to the stimulator. Set the stimulation parameters as described in the text manuscript. Use an oscilloscope to check the current mode, frequency, and pulse width.
Confirm the biphasic wave form with a rectangular pulse shape. The CT image clearly visualized the electrode inserted into the rat brain. The imaging modality used in this study also provided good anatomical information and facilitated the registration of FDG-PET images.
A fused PET-CT image of the same animal spatially registered to the same stereotaxic space. The brain metabolic differences were observed between PET sessions as T-maps superimposed on sequential one millimeter brain slices from an MRI registered to the reference CT image. These differences consisted of increases and decreases in FDG uptake shown as warm and cold colors respectively.
A detailed summary of the statistical results obtained from the analysis indicated the modulated brain region and the brain hemisphere in which the modulation was observed. The T statistic, the cluster size, the direction of modulation, and P-values obtained at peak and cluster levels. It is crucial to maintain an adequate aesthetic level and of the head during the surgery.
Moreover, coordinate calculation and a straightness of the electrodes are essential. New operators are advised to be meticulous and follow all the steps. After completing the whole procedure animals can be subjected to behavioral test to evaluate the stimulation impact on different cognitive domains.
In addition, a number of image studies can be performed to assess the DBS consequences at the cellular level.
