The overall goal of this procedure is to perform deep brain stimulation of the anterior thalamic nucleus and study gene expression changes in the rat hippocampus. This is accomplished by first anesthetizing the animal. Next, the animal is prepared for surgery.
An incision is made in the scalp and the skull is exposed. Then in the right position relative to the bgma, the bur holes are made. The DBS electrodes are inserted and stimulation is carried out.
Finally, the animal is euthanized and the brain is dissected. To remove the hippocampus, the tissue is processed for RNA extraction and CDNA preparation and QPCR are carried out. Ultimately, the results demonstrate that deep brain stimulation influences gene expression in the rat hippocampus.
This method can help answer key questions in deep brain stimulation surgery, such as what are the molecular mechanisms underlying the therapeutic effect of deep brain stimulation surgery? The implications of this technique extend towards therapy of not only epilepsy, but also other neuronal conditions such as Parkinson's disease, Alzheimer's disease, and dystonia. Because DPS is emerging as a surgical approach to treat many neuron disorders To prepare for surgery, use surgical pads to cover the workbench and ensure that there is a biohazard waste disposal available.
Weigh the rat and calculate the anesthesia dose according to the text protocol mount and secure two electrodes on the electrode holder of a stereotactic surgical frame and with a microscope, inspect the tips of the electrode for proper alignment. Secure the electrodes three millimeters apart, taking care not to touch the electrode tip on hard surfaces. Inject the ketamine xylazine.
Mix intraperitoneal to the animal and confirm that the animal has reached a surgical plane of anesthesia by checking for the toe pinch reflex, respiratory rate, and depth and regularity of breathing. Secure and position the animal on the stereotactic frame. Apply eye lubricant to the animal's eyes to protect from over drying and disinfect the scalp with three alternating scrubs, each of Betadine and either alcohol or sterile saline.
Place the animal on a circulating warm water pad or use heating lamps to maintain the animal's body temperature at an optimal level. Based on the Paxos and Watson rat brain atlas, use the following stereotactic coordinates for targeting the anterior thalamic nucleus or a NT anterior posterior negative 1.6 millimeters. Media lateral 1.5 millimeters and dorso ventral 5.2 millimeters.
Next, make an incision in the scalp sagittal to reveal the skull Using a pair of retractors, secure the incised scalp to expose the skull. Then with sterile swabs dipped in ethanol, clean the incised region to expose the sutures clearly. Locate the bgma and use a black marker to market to guide the position of the bur holes.
Make two more marks at approximately 1.5 millimeters. Media laterally on both sides from the sagittal suture and 1.6 millimeters posterior to the coronal suture. Now to make the two bur holes, use ethanol to disinfect the tip of the drill.
Using a drill held at a 45 degree angle, drill the bur holes frequently switching between the two holes to avoid excessive heat at the center of either bur hole. Continue drilling until the dura is exposed. Using a needle with its tip bent in the shape of an L, remove any broken pieces of bone that would obstruct the insertion of the electrode.
Take care to avoid damaging the underlying dura and or brain tissue while removing bone fragments. Fix the dual electrode assembly to the rotating handle of the stereotactic frame and fix the handle at a 90 degree angle. Using the adjustments in the stereotactic frame position the left electrode exactly above the bgma.
Using the stereotactic adjustments for media. Lateral positioning precisely moved the left electrode 1.5 millimeters to the left side of bgma, such that now there are two electrodes perfectly aligned along the coronal suture, but spaced apart. 1.5 millimeters media laterally from the bgma.
Then with the anterior posterior stereotactic adjustments, move the electrodes 1.6 millimeters posterior to the coronal suture. Then with the dorsal ventral adjustments, lower the electrodes to first check if the bur holes have been made in the right location, such that the electrodes can be inserted with ease without touching the rough edges of the bur holes. If so, insert the electrodes to a depth for 5.2 millimeters from the surface of the skull.
Connect the electrodes via leads to a stimulator set at 130 hertz, 2.5 volts and 90 microsecond pulse width. Deliver high frequency stimulation for a desired period of time. Performing unilateral or bilateral stimulation.
After stimulation is done carefully remove the electrodes and with three zero sutures or sterile surgical staples, suture the incision, administer buprenorphine subcutaneously, and monitor the animal until it returns to normal activity, and then return it to the housing facility. Place the brain on a pre-cool acrylic brain matrix on ice using a razor blade. Cut the brain corly at approximately seven to eight millimeters from the anterior most edge of the brain.
Make a second cut corly and posteriorly to the first cut so that an approximate five millimeter thick brain slice can be removed. Transfer the brain slice to a Petri dish with ice cold PBS using a razor blade, severed the two hemispherical sections and take care to note which section corresponds to the left and right sides respectively. Using fine forceps and scissors, carefully remove the hippocampus flash, freeze the tissue on dry ice and store at negative 20 degrees Celsius until ready for the subsequent RNA steps carried out according to the text protocol shown here is the relative expression levels of BDNF compared to the control gene beta actin across the indicated time points.
Post DBS stimulation. BDNF upregulation is observed immediately after DBS surgery, along with the peak expression at three hours post stimulation. This observation suggests that an enhanced BDNF expression and the resulting neuroprotection could contribute to the therapeutic benefit of DBS for epileptic patients.
This panel illustrates the expression of the GABA receptor, G-A-B-R-D, which shows enhanced expression in the stimulated animals compared to unstimulated controls at three hours post DBS. Considering that GABA agonists are used as effective seizure suppressors, it is interesting to observe enhanced G-A-B-R-D expression post DBS implicating a possible role for GABA and the anti-epileptic effect of DBS. Following this procedure.
Other methods like western block, chromatin immunoprecipitation, and many other standard molecular biological assays can be performed. This helps to answer questions relating to changes in protein expression, post-translational modifications, transcription factor occupancy on specific gene promoter regions, et cetera. After watching this video, you should have a good understanding of how to perform deep brain stimulation surgery in rats.
Isolate the hippocampal tissue and analyze it for gene expression changes.
