Generating a Mouse Model of Traumatic Brain Injury Using an Impactor Device

プロトコル

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Induce anesthesia

1. Anesthetize the mouse with ketamine (125 mg/kg) and xylazine (10 mg/kg) injected intraperitoneally.

2. Vital signs monitoring every 15 min

1. Monitor temperature, respiratory rate, and skin color. The mouse should feel warm to the touch. The skin should appear pink and well perfused. The respiratory rate should range 50–70 breaths per minute.

3. Pre-surgical procedures

1. Weigh all the mice on the day prior to injury induction.
2. Sterilize one set of surgical instruments by autoclaving for each experimental subject. Sterilize the impacting device before use. 
3. Prepare a recovery cage by placing a clean cage over an electric heating pad set to "low" setting and positioned in a manner such that the mice can move away from the heat once ambulatory.
4. Set up the operating theater within a sterilized laminar flow hood.
   1. Position the stereotaxic operating frame.
   2. Attach the impacting device to the stereotaxic frame.
   3. Set the actuating device with the desired biomechanical parameters for velocity and dwell time.
      NOTE: In this protocol a severe brain injury is described utilizing a 3 mm diameter impact tip via a 5 mm diameter craniectomy with the velocity set at 2.5 m/s and a dwell time of 0.1 s. A wide range of biomechanical parameters may be used to induce the full spectrum of TBI.
5. Don new personal protective equipment and sterile gloves.
6. Shave the fur from the operative site using electric clippers.
7. Apply protective opthalmic ointment to the eyes of the mouse to prevent corneal injury and drying.
8. Place the mouse into the operating theater.
9. Prep the skin with an iodine based surgical scrub alternated with alcohol three times.

4. Application of controlled cortical impact

1. Incise the scalp 1 cm in the midline with a scalpel exposing the skull.
2. Position the mouse within a stereotaxic operating frame by securing the bilateral temporal bones between miniature ear bars and locking the incisors within an incisor clamp creating a stable three-point-hold on the mouse head.
3. Retract the scalp away from the operative site with a hemostat or locking forceps to ensure the scalp does not come in contact with the drill bit during craniectomy.
4. Identify the sagittal and coronal sutures on the exposed skull.
   NOTE: This protocol centers the craniectomy 2 mm left of the sagittal suture and 2 mm rostral to the coronal suture.
5. Perform a craniectomy using a drill with a trephine drill bit.
   1. To perform the craniectomy, first activate the drill at maximum speed and then apply the trephine drill bit perpendicular to the skull at the site of craniectomy.
   2. Apply gentle, even pressure to the drill once contact is made with the skull. A slight "give" will be felt once the drill penetrates through the skull. Do not penetrate the underlying dura.
      NOTE: This protocol utilizes a 5 mm trephine drill bit to perform the craniectomy.
6. Use forceps and a small gauge hypodermic needle to remove the bone flap, fully exposing the underlying dura mater.
7. Rotate the impactor tip into the operative field and lower it until it makes contact with the exposed dura mater. Once contact is made the instrument's contact sensor will make an audible tone to alert the surgeon that contact has been made. This will mark the zero point from which the deformation depth is set.
   NOTE: This protocol utilizes a 3 mm impacting tip to generate a severe injury. Tips as small as 1 mm may be used to apply more localized injury.
8. Retract the impacting tip and set the desired impact depth by lowering the impactor position on the stereotaxic frame.
   NOTE: In this protocol we describe a severe injury by setting the deformation depth to 2 mm.
9. Apply the injury by activating impactor on the actuating device.
10. Rotate the impact device out of the field and remove the animal from the stereotaxic frame.

5. Surgical site closure

1. Control bleeding from the skull and injured cortical surface with direct pressure from a sterile cotton tipped applicator.
2. Dry the skull with a sterile cotton tipped applicator.
3. Close the scalp over the craniectomy using a commercially available surgical adhesive or monofilament suture.
   NOTE: In this protocol a veterinary surgical adhesive is used to close the scalp. The bone flap is not replaced and is discarded.

6. Post-operative care and monitoring

1. Administer post-operative analgesia (e.g., sustained release buprenorphine 0.1–0.5 mg/kg administered subcutaneously providing 72 h of sustained analgesia).
2. Place the animal in the lateral decubitus recovery position in a clean pre-warmed cage.
3. Observe the animals until awake and mobile, then return each mouse to its home cage.
4. Ensure free access to food and water. Normal food and water intake typically resume within one to two hours after injury.
5. Measure body weight every three days throughout the experiment.

資料

Name	Company	Catalog Number	Comments
AnaSed Injection Xylazine Sterile Solution	LLOYD, Inc.	5939911020
Buprenorphine SR Lab 0.5mg/mL	Zoopharm-Wildlife Pharmaceuticals USA	BSRLAB0.5-182012
High Speed Rotary Micromotor KiT0	Foredom Electric Company	K.1070
Imapact one for Stereotaxix CCI	Leica Biosystems Nussloch GmbH	39463920
Ketathesia Ketamine HCl Injection USP	Henry Schein, Inc	56344
Mouse Specific Stereotaxic Base	Leica Biosystems Nussloch GmbH	39462980
Trephines for Micro Drill	Fine Science Tools, Inc	18004-50