Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain

This study was carried out in strict accordance with both Spanish regulations (Royal Decree 53/2013 – Law 32/2007) and European Union guidelines (Directive 2010/63/EU) on the care and use of animals in biomedical research.

1 . Preparation of the Rat

NOTE: We performed the experiments in male rats to avoid any hormonal changes.

1. Anesthetize the animal using a mix of ketamine hydrochloride (30 mg/kg) and xylazine hydrochloride (5 mg/kg) injected intramuscularly; with this dose, the rat should be deeply anesthetized for around 1 h.
2. Pinch the rat's toe; absence of a withdrawal reflex indicates that the animal is fully unconscious. If the rat responds to the pinch, give supplementary anesthesia at one third of the initial dose.
3. Shave the scalp and disinfect the surgical area with povidone-iodine.
4. Place the animal on a heating pad to maintain a temperature of 38 °C and stabilize the animal's head in a stereotaxic frame using two ear bars and a bite bar. Use caution to avoid piercing the tympanic membrane with the ear bars.
5. Protect the eyes by applying a drop of ophthalmic gel or serum saline to each eye.

2. Location of the AC in the Temporal Bone of the Rat

1. Using a scalpel, make an incision along the midline to expose the skull and retract the periosteum covering the surface of the cranium.
2. Use a sterile cotton tip to gently remove any blood covering the surface of the skull to visualize bregma, lambda, and interaural 0 according to the Paxinos and Watson atlas of the rat brain²².
3. Make an incision in the temporal muscle near its dorsal insertion on the skull with a scalpel. Pull the muscle out using a needle and suture material, and fix the suture material to the stereotactic frame; this will expose the temporal bone. If bleeding occurs, rinse with cold sterile saline.
4. Place a sterile straight needle in the stereotactic micromanipulator, making sure that it is fully secured.
5. Slowly lower the needle until it is right above the surface of the skull, so that the tip of the needle is set to interaural 0. Set this point as zero, and determine coordinates from this point.
6. Depending on the brain area of interest, vary the stereotactic coordinates. Determine these coordinates by utilizing the Paxinos and Watson atlas of the rat brain²². Once the coordinates are determined, move the needle to match those coordinates.
7. Target the AC using the coordinates of the following four points: A: A/P = -5.8 mm, M/L = +/- 6.4 mm; B: A/P= -2.7 mm, M/L= +/- 6.4 mm; C: A/P= -2.7 mm, M/L= +/- 8.67 mm; D: A/P= -5.8 mm, M/L= +/- 8.67 mm. Lower the needle to right above the temporal bone to visualize each of these four points. Using a marker pen, mark the points on the temporal bone and connect them in order to draw a rectangle; the rectangle will serve as a guide to open of a window in the bone (Figure 1).

3. Surgical Exposure of the AC

1. Open the window using an electric drill and a small drill bit (0.6 mm Ø). Drill the perimeter of the rectangle at 8,000 rpm until the bone gives away. Cool the drilling surface by rinsing with cold sterile saline to prevent damage to subcortical structures. When the bone gives way, a drop in resistance can be detected. Be careful to not drill the brain.
2. When the borders are loose, pull up the covering bone with fine forceps and store it in cold sterile saline.

4. Ablation of the AC

1. Using a surgical microscope (10X), gently cut the meninges with a microsurgical knife and remove them using two fine-pointed forceps. If bleeding occurs, rinse with cold sterile saline.
2. Gently aspirate the AC using a surgical suction device (pressure -0.24 bar) coupled to a sterile 20 G blunt tip needle. This point is critical and needs to be performed very carefully: aspirate only the six cortical layers and not the underlying white matter.
3. Aspirate until the perforating arteries stop bleeding.
4. When the aspiration is finished, cover the injured area with the extracted bone and apply an absorbable hemostatic gauze.
5. Let the temporal muscle recover its original position, and then suture the skin using wound clips (9 mm). Apply antibiotic ointment (see the Table of Materials). Continue to apply ointment to the wound twice daily for three days.
   NOTE: Each application consists of a thin layer applied on the wound.
6. Inject buprenorphine subcutaneously in the back of the rat (0.05 mg/kg) as an analgesic 1 h after the surgery, and then every 8 h during 72 h.
7. Keep the animal on the heating pad until it wakes up, and return it to its housing cage to recover.
8. House animals individually to prevent cage mates from touching the sutured area, and provide some enrichment items. Change the sawdust daily to prevent infection, and carefully check that the animal recovers properly and does not show any signs of discomfort.

5. Tissue Collection

Caution: When handling paraformaldehyde (PFA), both solid and aqueous, wear personal protective equipment (PPE) and use a safety cabinet.

NOTE: Prepare 750 mL formaldehyde solution by dissolving 4% (w/v) PFA into 1x phosphate buffered solution (PBS) using heat (55 °C). Filter the formaldehyde solution with filter paper. Prepare Ringer's Solution by dissolving 8.5 g of NaCl, 0.25 g of KCl, and 0.2 g NaHCO₃in 1,000 mL of water (solution pH = 6.9).

1. Let the animal survive for as long as it is needed for the study. When the research performed with the AC ablated rat is completed, terminally anaesthetize it by intra-peritoneal injection of 0.1 mL of sodium pentobarbital (60 mg/kg). Assess the depth of anesthesia by toe pinch and the absence of withdrawal reflex.
2. When the animal is deeply anesthetized, perform an intracardiac perfusion²³ of 125 mL of Ringer's Solution followed by 750 mL of formaldehyde solution by using a needle gauge of 1.8 mm of inside diameter.
3. When the perfusion is finished, decapitate the rat at the first cervical vertebra.
4. Use scissors to remove the skin and muscle from the head and expose the skull. Use scissors to cut and open the foramen magnum and remove the back of the skull.
5. Make a transversal cut into the orbital bone using Spencer scissors, and use rongeurs to cut along the top edges of the skull to expose the brain. Be careful not to damage the brain.
6. 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. Raise the brain and cut the nerves until it is free. Immerse the brain in formaldehyde solution and store it at 4 °C for 24 h.

6. Localization of the AC Lesions

1. After post-fixation, carefully place the brain into a sagittal rat brain matrix exposing the lateral surface of the brain.
2. Place a camera 21 cm above the cortex surface using a camera holder, select the "super macro shooting" mode, and take a picture of the brain surface.
3. Place the brain into a coronal rat brain matrix exposing the dorsal surface of the brain, and take another picture.
4. Using an image editor program, open the images and scale them down 50% to make it easier to work with them. Identify the bregma, lambda, and interaural 0 references on the picture according to Paxinos and Watson coordinates¹⁹, and mark their position in the pictures (Figure 2). Draw the contour of the ablation over the lateral picture of the brain. Calculate the perimeter.
5. Import the coordinate map where the primary (A1) and secondary regions (Dorsal and Ventral Cortices) of AC are located¹⁸ to the file of the editor program where you are working with the pictures. Click on the map and drag it to superimpose it to the lateral photograph of the ablated brain.
   1. Make the bregma and lambda references of the coordinate map coincide with the bregma and lambda references identified in the picture of the lateral brain.
   2. Use the rhinal fissure as a reference to adjust the picture of the brain to the map, and make them coincide (Figure 2B).
6. Calculate the percentage of the lesion relative to the area occupied by the AC.