Hi, I'm John Ya.And I'm Dan Alba. Today we'll demonstrate functional MRI will. Simultaneous deep brain stimulation in a rat.
We can use this technique to map functional responses throughout the brain to deep brain stimulation at a variety of targets. This procedure involves stereotactic electrode implantation FMRI acquisition, an analysis of functional responses. The first step is electrode implantation.
We will implant an electrode unilaterally into the subthalamic nucleus. We will start by anesthetizing the animal completely and monitoring physiological parameters, including oxygenation and body temperature throughout the procedure. Begin preparing the surgical site by removing hair and attach the animal to a stereotactic frame.
Use ointment to lubricate the animal's eyes during the procedure. Apply antiseptic to the site and apply drapes to the sterilized field. To complete preparation of the surgical site.
Remove the skin and fascia overlying the skull, ensuring that bgma and lambda are uncovered. Use a scalpel to roughen the skull surface to improve cement attachment later in the procedure. And use sterile cotton in electrocautery for hemostasis over the bone surface and cut tissues use.
Use a needle attached to the stereotactic arm to touch the exact location of bgma as a reference for craniotomy location and mark the location on the skull with the needle for the subthalamic nucleus. This will be 3.6 millimeters posterior torema and 2.5 millimeters lateral from the midline. Use a small bit drill to make a small hole in the skull cleaning and probing the edges of the hole.
Use a small gauge needle to thoroughly perturb the dural surface and clean any additional bleeding or leakage of cerebral spinal fluid. Now we will place an MRI compatible screw in the skull for anchoring. Drill a hole in the skull contralateral and posterior to the future electrode location.
Place the screw deep enough to be secure, but without pressing on dura or cortex, we use an MRI compatible homemade two channel tungsten micro electrode for stimulation. Inspect the electrode to ensure it is straight and the leads are not split. Place the electrode on the stereotactic arm in a vertical position and touch the tip to bgma.
Move the electrode to the position of the target brain region using the stereotactic frame for measurement and touch the cortical surface. Lower the electrode using the stereotactic frame for measurement. Ensure that there is no bending in the electrode.
During this process, place an initial layer of cement first, ensuring a dry skull service. After the first layer is dry, bend the electrode posteriorly, then apply additional cement to increase stability. Allow the cement to dry completely before animal removal.
The animal may now be resuscitated or immediately prepared for functional MRI scanning. The second step is preparing the animal for FMRI and FMRI acquisition. This may be performed immediately after surgery or several days later.
A headpiece should be used to completely immobilize the animal's head with respect to the coil. The animal should be endotracheally, intubated and ventilated. A variety of anesthetics may be used.
We use dme, toine, and combin with low dose isof. Fluorine paralysis should be performed using pantonium acquire cap geometry near the junction of the ventilation tubing with the endotracheal tube. Adjust ventilator settings to stabilize end TAL CO2.
In the normal range, generally near 3%temperatures should be monitored using a rectal probe fixed to the base of the tail. A circulating hot water bath should be adjusted to stabilize body temperature within the normal range. Pulse oximetry may also be acquired in the hind paw area, placed toothpaste over the area to be scanned to improve image quality.
Place a surface coil directly over the brain regions to be scanned. Secure the coil firmly to the headpiece and holder. Connect the stimulating system to the electrode connector and secure the leads in place.
Cover the animal for improved insulation in the chilled environment of the magnet. Now it is time to insert the animal into the center of the magnet. Use a three plain S scout image for precise positioning of the animal within the scanner globally.
Shim with reference to the cortical surface to improve homogeneity of the magnetic field. Then use FMAP to shim locally at the brain regions to be used for functional scans. Acquire a single T two weighted slice in a mid sagittal plane.
Create a single shot gradient. Echo EPI scan with matrix size of 96 by 96, field of view of 2.56 by 2.56 centimeters. Repetition time of 1000 milliseconds, echo time of 14 milliseconds and slice width of one millimeter with four dummy scans followed by 70 repetitions.
Each scan with these parameters should last one second. For a total scan time of 70 seconds align this EPI scan to the anterior commissure of the previous T two sagittal image for geometrical consistency between subjects set up stimulation, frequency, pulse width and stimulation paradigm, and synchronize the stimulations trigger to the RF outputs from the scan sequence. Here we use a frequency of 130 hertz pulse width of 90 microseconds and a stimulation paradigm of 60 seconds of rest, followed by 30 seconds of stimulation, and finally 120 seconds of rest.
Then set up the stimulation current type, including the desired current amplitude and bipolar current output run. The EPI scan ensuring that the stimulation triggers appropriately after acquisition check to ensure a robust response using the data analysis tools described in the following section. If this does not occur, check the stimulator connections.
After all scans are required, anatomical scans should be run to measure approximate electrode position and ensure electrode position within the intended target. Afterward, the rat may be resuscitated and preserved for further experiments. Finally, after all necessary experiments have been run, the animal may be euthanized in the brain perfuse with formalin.
With the electrode tract removed, hide resolution T two weighted scans may be performed to identify the electrode tract and its relationship to nearby structures. In this case, the electrode tract terminates just ventral to the internal capsule in the subthalamic nucleus confirming implantation accuracy. Next, we'll process and analyze the results of our functional scans.
For this, we'll use a custom written program which runs within matlab, though other software packages for FMRI analysis may be used in a similar fashion in matlab. Use SPM codes to apply rigid body across all stimulus trials. Then create an average segment of images, conserving the time course of the scans, manually perform skull stripping after application of a cutoff threshold.
Now we will generate correlation coefficient maps for the brain regions of interest. Noise reduction may be performed using a minimum cluster size or three by three low pass smoothing. The stimulation paradigm should be input and a short paradigm delay may be set to account for hemodynamic delay.
For each voxel, the correlation coefficient of signal intensity change over time may then be calculated with respect to the stimulation paradigm. This may be mapped out over each slice to identify neuro anatomical regions where ball response strongly correlates to the experimental protocol. Regions of interest may be manually defined or use a polygon as an approximation.
Percent signal change within this region of interest may then be determined using the pre stimulation signal as a baseline. These values may then be exported for statistical analysis. Here we have performed deep brain stimulation at the subthalamic nucleus and the responses on our correlation coefficient map are in brain regions distant from that nucleus.
Here we see positive, bold, and cortical regions anterior to the location of stimulation in the most posterior slice. The artifact from the stimulating electrode is visible when the change in bold signal intensity over time is calculated for a sample cortical region of interest. The result is displayed on screen with the stimulation paradigm displayed here by a yellow bar.
This may be used to quantify the bold response and for further analysis, we have just demonstrated deep brain stimulation with simultaneous functional MRI in er rodent model. Thank you for watching and good luck with your experiments.I.
