Stereotactically-geleide ablatie van de auditieve Cortex van de Rat en de lokalisatie van de laesie in de hersenen

The overall goal of this procedure is to describe the stereotactically guided ablation of the auditory cortex and the use of a coordinate map to localize the lesion over a picture of the brain's surface. This method can help out the auditory neuroscience investigators to address the role of corticals in the pathway in the perception of sound. As well as the mechanisms and the line positional plasticity.

The main advantage of this technique is that it allows the surgical exposure and ablation of the auditory cortex by using basic methods that can be adapted by any investigator. So this method can be useful in the auditory neuroscience field, it also can be applied in other systems such as the visual and somatosensory. This method can also be used to study defects that unilateral auditory cortex ablation exert in other cortical areas.

Visual demonstration of this method is critical, because the proper identification and translation of the stereotaxic coordinates into the temporal bone is essential to accurately locate the auditory cortex. To begin this procedure, place the anaesthetized rat on a heating pad to maintain its body temperature. Then stabilize the animal's head in a stereotaxic frame using two ear bars and a bite bar.

Shave the scalp and disinfect the surgical area with povidone-iodine. Using a scalpel, make an incision along the midline to expose the skull and retract the periosteum covering the surface of the cranium. After that, use a sterile cotton tip to gently remove any blood covering the surface of the skull.

In order to visualize Bregma, Lambda, and the interaural line, make an incision in the temporal muscle near its dorsal insertion on the skull with a scalpel. After that, pull the muscle out using a needle and suture material and fix the suture material to the stereotactic frame in order to expose the temporal bone. Slowly lower a sterile straight needle until it is right above the surface of the skull, so that the tip of the needle is set at interaural zero.

Set this point as zero. Following that, target the AC using four points. Then lower the needle to right above the temporal bone to visualize each of these four points.

Mark the points with a marker on the temporal bone and connect them in order to draw a rectangle. The rectangle will serve as a guide to open a window in the bone. In this procedure, open the window using an electric drill and a small drill bit.

Drill the perimeter of the rectangle at 8, 000 rpm until the bone gives away. Cool the drilling surface by rinsing it with cold sterile saline to prevent damage to subcortical structures. When the bone gives way, be careful not to drill into the brain.

When the borders are lose, pull up the covering bone with fine forceps and store it in cold sterile saline. Using a surgical microscope, gently cut the meninges with with a microsurgical knife and remove them using two fine-pointed forceps. If bleeding occurs, rinse the surgical site with cold sterile saline.

Next, gently aspirate the AC using a surgical suction device coupled to a sterile 20 gauge blunt-tip needle. Aspirate only the six cortical layers, and not the underlying white matter. This point is critical and needs to be performed very carefully.

When the aspiration is finished, cover the surgical site with the extracted bone and apply an absorbable hemostatic gauze. Let the temporal muscle recover its original position and then suture the skin using wound clips. After that, apply antibiotic ointment to the wound.

Subsequently, inject Buprenorphine subcutaneously in the back of the rat as an analgesic, one hour and eight hours after the end of the surgery. Keep the animal on the heating pad until it wakes up and return it to its housing cage for recovery. When the research performed with the AC-ablated rat is completed, terminally anesthetize it by intraperitoneal injection of 0.1 milliliters of sodium pentobarbital.

After performing an intracardiac profusion of Ringer solution and formaldehyde, remove the skin and muscle from the head to expose the skull. Using Spencer scissors, make a transversal cut into the orbital bone. After that, remove the back of the skull and use rongeurs to cut along the top edges of the skull to expose the brain.

Be careful not to damage the brain. Once the brain is exposed, carefully remove the dura mater using fine-pointed forceps. Use a finger to gently scoop under and elevate the brain.

Then raise the brain and cut the nerves until it is free. Subsequently, immerse the brain in formaldehyde solution and store it at four degrees celsius for 24 hours. After post-fixation, carefully place the brain into a sagittal rat brain matrix, exposing the lateral surface of the brain.

Place a camera 21 centimeters above the cortex surface using a camera holder. Then, select the super-macro shooting mode, and take a picture of the brain's surface. Following that, using an image editor program, open the images and scale them down 50%Identify the Bregma, Lambda, and interaural zero references and mark their position in the pictures.

Draw the contour of the ablation over the lateral picture of the brain. Import the coordinate map where the primary and secondary regions of the auditory cortex are located to the file of the editor program. Then click on the map and drag it to superimpose to the lateral photograph of the ablated brain.

Make the Bregma and Lambda references of the coordinate map coincide with the Bregma and IA references identified in the picture of the lateral brain. Subsequently, use the Rhinal fissure as a reference to adjust the picture of the brain to the map and make them coincide. It is important to confirm that white matter is intact.

This can be done by initially immunostaining of serial section of the brain. To demonstrate the efficacy of our protocol, we kept the three AC-ablated rats survived for one week and then collected the cochleae to study the changes in the expression of the most relevant AMPA subunits present in the adult cochlea, GluA2 and GluA3 by qPCR. The comparison between the transcripts from AC-ablated rats and sham control animals showed a downregulation for GluA2 and an upregulation for GluA3 in both cochleae, which is in agreement with our previous study.

While attempting the surgery, it is important to remember three things:to drill the temporal bone at lowest speed with minimum pressure, to carefully remove the meninges to avoid breaking blood vessels, and to restrict the aspiration to the gray matter. Once mastered, the surgical exposure and ablation of the auditory cortex can be done is 45 minutes if it is performed properly. Following this procedure, physiological, behavioral, and/or immunocytochemical methods can be performed in order to answer additional questions, like how the lack of the auditory cortex affect the function of further sensory systems.