All animal experiments were approved by the local animal welfare committee and the Austrian Federal Ministry for Science, Research and Economy.

1. Prepare the Equipment and Setup Required for Surgery

1. Position the surgical microscope, drill, heating plate and pulse oximeter to allow easy and efficient handling during surgery. Check the function of the devices based on the manufacturer's manual. Make sure that the heating plate is set at 38 °C in order to protect the animal from hypothermia during the surgery.
2. Put on a hood and a mask.
3. Perform surgical hand disinfection. Wash hands meticulously with soap. Dry hands and then use an alcoholic hand sanitizer to disinfect hands. Put on gloves after the hands are dry.
4. Prepare the sterilized surgical instruments and the equipment required for cochlear implantation. See the Table of Materials for the surgical instruments and equipment used for guinea pig CI implantation in this protocol.
5. When compound action potential (CAP) measurements are intended, prepare an approximately 3.5 cm-long piece of Teflon-insulated gold wire by carefully removing parts of the insulation from both ends using a micro forceps (approximately 3 mm from one end and 5 mm from the other end). Prepare a second piece of gold wire (approximately 2.5 cm) with uninsulated ends of approximately 5 mm. Place the prepared wires in alcohol or disinfectant.

2. Anesthesia, Medication and Animal Preparation

1. Weigh the animal.
   NOTE: Animals used are female Dunkin-Hartley albino guinea pigs. The weight of the animals used, which are approximately 4-6 weeks old, ranges from 300 to 400 g.
2. Prepare the anesthetics and medication required for surgery based on the weight of the animal. Prepare 0.72 mL of the anesthetic mixture, consisting of 0.06 mL ketamine (100 mg/mL), 0.18 mL medetomidine (1 mg/mL), 0.12 mL of midazolam (5 mg/mL) and 0.36 mL of fentanyl (50 µg/mL) for surgery on a 400 g guinea pig. See Table 1 for weight-based doses of the anesthetics.
3. Inject the mixture of ketamine, medetomidine, midazolam and fentanyl subcutaneously to the fat pad in the neck of the animal using a 27 G needle. See Table 1 for weight based doses of the anesthetics. Cover the cage and leave the animal in a quiet place for 10 min before proceeding.
4. Lubricate the eyes of the animal and keep them lubricated during the whole procedure. Shave the head of the animal, focusing on the retroauricular region to allow for a sufficient surgical access to the bulla.
5. Position the animal on the heating plate in prone position and make sure that the animal is not in direct contact with the heating plate to prevent thermal burns. Place the pulse oximeter probe on a foot of the animal. Then carefully open the mouth of the animal with a small laryngoscope and clean the entire oral cavity from food rests by using a sucker.
6. Keep the mouth of the animal opened with the laryngoscope. Carefully insert a stomach tube into the esophagus of the animal and slowly push it in the direction of the stomach until a resistance is felt.
   1. Monitor the O₂-saturation of the animal to make sure that the stomach tube is not in the trachea. Remove the stomach tube if there is a decrease in O₂ saturation and try again after the animal is completely oxygenated.
7. Inject a mixture of physiological saline, 5% glucose and enrofloxacin into the fat pad in the neck of the animal using a 23 G needle.
8. Use alcoholic hand sanitizer to disinfect hands again. Put on new clean gloves.
9. Prepare the surgical field with alternating scrubs of povidone iodine and 70% ethanol and cover the animal. Use self-adhesive drapes or towel clamps to make sure that only the surgical field stays uncovered during the procedure.
10. Subcutaneously inject 0.1 mL of 2% lidocaine solution into the area of the planned incision for sufficient local anesthesia and position the animal sideways.
11. Re-dose the animal with ¼ of the initial dose of the anesthetic mixture every 30 min after the first injection to maintain sufficient anesthesia.

3. Cochlear Implantation

1. Perform an approximately 2-3 cm skin incision 3-5 mm posterior to the pinna using a scalpel. Use bipolar cautery when necessary to minimize bleeding.
   Note: Anesthetic depth should be confirmed by the lack of pedal withdrawal prior to the first incision. This test should be repeated every 15-20 minutes to monitor anesthetic depth throughout the procedure. 
2. Carefully cut the muscles in the retroauricular area after palpating the auditory bulla using a 15 scalpel or surgical scissors.
   NOTE: Palpate the auditory bulla as a prominence under the muscles.
3. Dissect the muscles from the bulla by pushing them gently aside using a raspatory or a cotton bud. Use a retractor to expose the full length of the incision and have unhindered access to the bulla.
4. Use the tip of a 15 scalpel to drill a hole into the bulla. Carefully rotate the scalpel until the bone is perforated to allow for inspection of the middle ear structures.
5. Enlarge the bullostomy as needed to make sure that the basal turn of the cochlea and the round window niche can be adequately visualized. Position the head of the animal in an inflected position in order to access these structures. Cover the area of the bullostomy with a small piece of a compress to prevent blood and extracellular fluid from running in to the middle ear.
   NOTE: The inflected position of the head may obstruct the airway of the animal. Therefore, the oxygen saturation of the animal needs to be checked frequently.
6. Position the animal in the prone position. Expose the vertex of the animal by performing a rectangular incision and removing the skin. Dissect the periosteum and clean the bone from any other kind of tissue or blood by using a scalpel.
7. When CAP measurements are intended, finish the preparation of the 3.5 cm teflon insulated gold wire by forming a small hook with a micro forceps at the end of the wire, which has been uninsulated for 3 mm.
8. When CAP measurements are intended, guide the end of the gold wire, which has been uninsulated for 5 mm, subcutaneously to the vertex through an 18 G peripheral venous catheter using a micro forceps. Use another micro forceps with the other hand to guide the hooked end of the wire carefully in to the middle ear.
9. When CAP measurements are intended, inflect the head of the animal to visualize the area of the round window niche through the bullostomy. Use the other hand to hook the gold wire to the bony prominence of the round window niche by using a micro forceps.
10. Maintain gentle tension at the gold wire and fix it to the cranial verge of the bullostomy with 10 – 15 µL of tissue glue by using a 1 mL syringe with a 27 G needle. Avoid displacement of glue into the middle ear. See Figure 1 for an intraoperative picture of the round window area with the gold wire in situ.
11. When CAP measurements are intended, connect the uninsulated end of the gold wire to the equipment used to measure auditory potentials and perform baseline CAP measurements. See Honeder et al., 2016, for a detailed description of the CAP measurements routinely performed in our laboratory.¹²
12. Position the animal in prone position. Drill 2 holes 1 mm anterior to the lambda suture using a 1-mm burr without causing damage to the dura. Implant 2 stainless steel screws 2mm into the skull.
    NOTE: The screws serve as fixation points for the connector of the electrode. Adapt the distance between the screws with respect to the size of the connector.
13. Use an 18 G peripheral venous catheter to guide the electrode from the connector to the bulla in a tissue layer as close to the skull as possible.
14. Mix the dental cement powder with the fluid for dental cement powder using a spatula according to the manufacturer's manual.
15. Place 0.5 – 0.7 mL of semifluid dental cement between the screws by using a spatula. Position the connector of the electrode between the screws.
16. Keep the connector in position until the dental cement is hardened. Make sure that the screws are encased by the cement to allow for stable fixation of the connector.
17. Position the animal sideways. Carefully drill the cochleostomy 1 mm from the round window niche using a 0.5 mm diamond burr at a rotation rate of 5000 rounds per minute.
18. Carefully insert the electrode in to the scala tympani to a depth of 4 mm.Withdraw the electrode and repeat the insertion. See Figure 2 for the electrode used for guinea pig cochlear implantation.
19. Using a straight needle, seal the cochleostomy area with a small piece of muscle. Fix the electrode to the cranial verge of the bullostomy with 10 µL of tissue glue by using a 1 mL syringe with a 27 G needle.
20. Prepare the dental cement as mentioned in 3.14. Carefully close the bullostomy with approximately 0.3 mL of dental cement by using a spatula. Close the retroauricular incision using 5-0 absorbable sutures.
21. Turn the animal to the prone position.
22. When CAP measurements are intended, grab the posterior edge of the rectangular incision at the vertex of the animal by using a tissue forceps. Use a scissor with the other hand to make a subcutaneous tunnel of approximately 2 cm length in the neck of the animal.
23. When CAP measurements are intended, implant the 2.5 cm gold wire subcutaneously into the neck of the animal using a forceps. Solder the shorter uninsulated end to the designated pin of the connector on the vertex of the animal.
24. When CAP measurements are intended, solder the round window niche electrode (gold wire) to the appropriate pin of the connector on the vertex of the animal.
25. Apply an additional amount of dental cement on the top of the connector to fully cover the insulated pins and the electrode.
26. When CAP measurements are intended, perform postoperative measurements according to the research protocol.

4. Postoperative Care

1. Apply atipamezole and flumazenil subcutaneously after the surgery and measurements in order to antagonize the anesthesia.
2. Apply physiologic saline as fluid substitution to support the recovery of the animal from the surgery.
3. Place the animal under a heating lamp until it has fully recovered from anesthesia and starts to move within the cage.
   1. Prevent hyperthermia or burn by placing the heating lamp approximately 50 cm to the animal. Make sure that the body temperature of the animal is always between 37.5 °C and 39 °C.
4. Check the animal for symptoms of vestibular injury like nystagmus, circling or rolling over.^13,14
5. Apply buprenorphine for analgesia twice a day for two more days post-surgery.
   NOTE: Before the first application of buprenorphine post-surgery make sure that the animal has completely recovered and has stable respiration. Application of this medication while the animal is still under anesthesia may lead to respiratory depression.
6. Weigh the animal during the first 3 days post-surgery to detect possible weight loss as a surrogate marker of distress during this time.
   NOTE: Weight loss of approximately 10% during the first 3 days post-surgery can be expected and should be considered common. This weight loss is temporary and will recover within a few days.