Surgical Labyrinthectomy of the Rat to Study the Vestibular System

Summary

This protocol describes the surgical labyrinthectomy of a rat, which is a useful method for studying the vestibular system.

Abstract

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including surgical or chemical labyrinthectomy and vestibular neurectomy. Surgical labyrinthectomy is a relatively simple, reliable, and rapid method. Here, we describe the surgical technique for rat labyrinthectomy. A postauricular incision is made under general anesthesia to expose the external auditory canal and the tympanic membrane, after which the tympanic membrane and the ossicles are removed without the stapes. The stapes artery, which is located between the stapes and the oval window, is a vulnerable structure and must be preserved to obtain a clear surgical field. A hole to fenestrate the vestibule is made with a 2.1-mm drill bur superior to the stapes. Then, 100% ethanol is injected through this hole and aspirated several times. Meticulous dissection under a microscope and careful bleeding control are essential to obtain reliable results. Symptoms of vestibular loss, such as nystagmus, head tilting, and a rolling motion, are seen immediately after surgery. The rotarod or rotation chair test can be used to objectively and quantitatively evaluate the vestibular function.

Introduction

The vestibular organ is essential for balance and eye control. A normal vestibular function depends upon symmetrical afferent signals from the vestibular organs in the two inner ears. Vestibular hypofunction or loss induces dizziness, nystagmus, and postural imbalance. After acute damage, the vestibular function recovers spontaneously within several days, a process known as vestibular compensation^1,2. The vestibular compensation of static deficits is a process of recovery related to the imbalance of spontaneous resting activity between the ipsilateral and contralateral vestibular nuclei. The vestibular compensation of dynamic deficits is achieved principally via sensory and behavioral substitutions (using visual or somatosensory inputs)³.These processes are attractive for neuronal plasticity studies^4,5.

A number of methods have been developed to study the vestibular system and the mechanisms underpinning neuronal plasticity during vestibular compensation, such as surgical and chemical labyrinthectomy and vestibular neurectomy^5,6,7,8. Vestibular neurectomy is a certain way to induce complete vestibular loss, but it is a more difficult and invasive procedure and may induce brain damage^8,9. This method requires greater surgical skill and takes more time than labyrinthectomy. Chemical labyrinthectomy including gentamycin, arsanilate, and tetracaine, is easier and can yield reliable results^10,11,12. However, the cochlea may also be damaged and vestibular loss may develop over time¹¹. Additionally, the effects of the chemicals on the brain, which should be preserved for accurate evaluation, are unclear. Surgical labyrinthectomy was first introduced in animal studies in 1842¹⁵ and was first reported in the rat in 1936¹⁶. This technique has since been used in many animal studies^5,17,18,19. Surgical labyrinthectomy is a specific, reliable, and relatively simple method.^13,14 Moreover, the symptoms of vestibular damage are seen immediately after surgery. Here, we describe our surgical technique for rat labyrinthectomy.

Protocol

This study was performed in accordance with the Institutional Animal Care and Use Committee of Seoul National University Hospital (14-0148-C1A1), which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.

NOTE: The experiments were performed on Sprague-Dawley (SD) male rats of 7 - 8 weeks old (200/250 g). Each animal was acclimatized to the laboratory conditions for 1 week prior to the start of the experiment. The animals were housed in a temperature- and humidity-controlled room with a constant 12-h:12-h light:dark cycle with free access to food and water.

1. Labyrinthectomy

1. Cover the autoclaved surgical instruments with a sterile pad. Disinfect the operating area with 70% ethanol. Use a sterile drape and maintain sterile conditions during the surgery.
2. Wear a gown, mask (to cover nose and mouth), cap (to cover head), and a pair of sterile gloves. The surgical instruments must only come in contact with sterile surfaces.
3. Inject tiletamine-zolazepam anesthetic (40 mg/kg body weight) intramuscularly into the medial thigh or intraperitoneally, and xylazine (10 mg/kg body weight) intramuscularly or intraperitoneally.
   NOTE: Other modes of general anesthesia, including isoflurane inhalation, can also be used.
4. Place the rat on a warming pad (initially set at 42 °C). Apply lubricant eye ointment to both eyes of the rat to prevent eye dryness while under anesthesia.
5. Place the rat on its right side. Shave the fur in the surgical area with hair clippers. Disinfect the site with 70% ethanol.
6. Inject 1% lidocaine hydrochloride subcutaneously into the left retroauricular area.
7. Using a scalpel blade #15, make a ~5.0 cm retroauricular incision. Separate the muscle and fascia to expose the external auditory canal with Iris scissors (Figure 1A). Slightly open the external auditory canal using the blade #15 or the Iris scissors (Figure 1B). Widely expose the tympanic membrane (Figure 2A).
8. Under a microscope (7.5X or 12.5X), remove the tympanic membrane and the ossicles with the exception of the stapes, using the forceps. Detach the muscles on the lambdoidal ridge with the blade #15 or the Iris scissors (Figure 2B).
9. Using a drill at low speed (<3000 rpm) with a diamond bur (2.1 or 1.4 mm), drill the tympanic bulla bone around the point of exit of the facial nerve (Figure 3A). Identify the stapedial artery. Take care not to damage the stapedial artery during the drilling process.
10. Continue drilling the vestibule superior to the stapedial artery and expose the round window and the bony lateral semicircular canal. Open the lateral semicircular canal near its ampulla. Continue drilling in the plane of the lateral semicircular canal and drill the ampulla of the superior semicircular canal. 
11. Aspirate the contents of the vestibule using a 18-G or 22-G needle. Inject and aspirate 1 cm³ of 100% ethanol 3x.
12. Close the muscles and the skin in two layers with simple interrupted sutures.
13. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Keep the rat warm until it wakens using a heating pad or a heat lamp. After awakening, move the rat to an individual cage in a room featuring the usual environmental conditions.
14. Administer antibiotics (e.g., Cefazoline 20 mg/kg, trimethoprim-sulfonamide 100 µL subcutaneously, or penicillin 300,000 IU) as needed on day 1; an analgesic (not an opioid; e.g., Metacam 2 mg/kg, Carprofen 5 mg/kg subcutaneously) can also be given.

2. Sham Surgery

1. For the sham surgery as a control, perform the same surgical and post-surgical procedures (as per section 1), including the exposure of the external auditory canal and the tympanic membrane (step 1.7), the removal of the tympanic membrane and the ossicles, and the detachment of the muscles on the lambdoidal ridge (step 1.8), but do not open the semicircular canal or inject ethanol.

3. Check the Loss of Vestibular Function

NOTE: The loss of vestibular function can be evaluated using either behavioral or vestibular function tests.^13,17,18 Behavioral tests include the evaluation of postural asymmetry and nystagmus.

1. Identify postural asymmetry which includes spontaneous or evoked barrel-rolling, falling to the left side, or moving around while leaning toward the lesioned side¹⁸. See Video 1.
2. Identify nystagmus by a visual inspection of voluntary eye movement. When spontaneous nystagmus is absent in the resting state, puff air gently over the head of the animal¹⁸. See Video 2.

Results

The success of the surgery was validated by behavioral tests. All animals exhibited the typical behavior of a unilateral loss of vestibular function. Spontaneous barrel-rolling was evident immediately after surgery, being evoked by an air puff over the head or a light touch to the body in the early recovery phase (Video 1). 3 d after surgery, the animals moved around leaning toward the lesioned side with occasional falls to the left side. Spontaneous nystagmus was observe...

Discussion

This technique is a useful method for creating sudden, permanent, and complete vestibular function loss. This could be used to study vestibular pathologies, such as vestibular neuritis, an acoustic tumor, and Meniere's disease. Many studies have used this technique to study the neuronal plasticity of vestibular nuclei or the related central process^5,17,18,19.

The...

Materials

Name	Company	Catalog Number	Comments
ASPIRATOR KB-012	KOH BONG & CO., LTD.	KB-012	Medical aspirator
Blade: #15	Fine Science Tools	#10015-00	Blades for #7 Scalpel Handles, #15
Carbon Steel Burrs	Fine Science Tools	#19007-05	shaft diameter: 2.3 mm, length: 44 mm, package of 10 burrs
Carl Zeiss Surgical GmbH	Carl Zeiss	#6627100863	Surgical microscope
Dumont #3c	Fine Science Tools	#11231-20	Standard tip 0.17 x 0.10 mm, 11 cm
Dumont #5SF	Fine Science Tools	#11252-00
Dumont #7B	Fine Science Tools	#11270-20	Serrated 0.17 x 0.10 mm, 11 cm
Extra Fine Bonn: straight	Fine Science Tools	#14084-08	Iris scissors, best suited for microdissection under high magnification
Fine Iris Scissors: straight	Fine Science Tools	#14094-11	Made from martensitic stainless steel, combined with molybdenum and vanadium
Finger Loop Ear Punch	Fine Science Tools	#24212-01	1 mm. Provides stability and control for researchers using the numbering system
Hartman	Fine Science Tools	#13002-10	Tip width: 1 cm, serrated, 10 cm
Short Scalpel Handle #7 Solid	Fine Science Tools	#10003-12	#7 short, 12 cm
Small Vessel Cauterizer	Fine Science Tools	#18000-03	Replacement tip, straight knife, keeps bleeding to a minimum and therefore provides a surgical field clear of clamps and hemostats
Strong 207S	SAESHIN	207S	Powerful torque at low speed, available with speed or on/off foot controller
Suction Tubes	JEUNGDO B&P CO., LTD.	H-1927-8	Frazier, 18 cm
VICRYL	ETHICON	W9570T	Synthetic absorbable sterile surgical suture
Weitlaner-Locktite	Fine Science Tools	#17012-13	Maximum spread: 4.5 cm, 2 x 3 blunt teeth, 11 cm
Zoletil	Virbac, France		Tiletamine-zolazepam
Rompun	Bayer		Xylazine
Rimadyl	Pfizer		Carprofen
Septra	Pfizer		Trimethoprim-sulfonamide