Performing Laser-Induced Brain Injury in a Rat Model

Protocol

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

1. Laser-induced brain injury experimental procedure

1. Anesthetize the rat with a mixture of 2% isoflurane in oxygen, allowing for spontaneous ventilation. Check for sufficient anesthetic depth by pinching the tail with forceps to see if the withdrawal reflex is absent.
2. Using a rectal temperature-regulated heating pad, maintain the rat's core body temperature at 37 °C throughout the experimental procedure.
3. Remove local hair with a shaver and disinfect it with 70% alcohol and 0.5% chlorhexidine gluconate. Repeat the disinfection step two more times.
   NOTE: The surgical incision should be approximately 3 cm wide. Remove hair at least 2 cm around the incision area.
4. Place the rat on a stereotaxic head holder in a prone position. Make a 3 cm incision to reflect the scalp laterally and expose the area between Bregma and Lambda.
5. Maintain anesthesia through the nose cone.
6. Use Neodymium-YAG (Nd-YAG) laser (peak wavelength 1064 nm) to administer 50J X 10 points, with 1 s pulse duration, to the exposed area of the skull above the right hemisphere.
7. Ensure that the laser-generating part of the apparatus is 2 mm away from the exposed area to produce a laser beam. 50J X 10 points were selected after careful evaluation of different energy/surface combinations. This combination is efficient and does not cause bone destruction of the skull after administration for less than a second.
   NOTE: 2 mm is the distance between the terminal of the laser beam (from the optical cable it is passed through) and the skull bone. In case a focusing lens is used, the distance should be calculated, taking into account the angle of inclination of the lens to focus the beam in the desired area of damage. Ensure proper safety when using a laser device, including appropriate training and eye protection.
8. Remove the rat from the device and close the scalp with 3-0 silk surgical sutures.
9. Discontinue anesthesia and return the rat to its cage for recovery. Administer 0.1 mL of 0.25% bupivacaine locally to reduce the postoperative pain immediately after surgery.
   NOTE: The entire procedure should last less than 5 min if performed correctly.

Materials

Name	Company	Catalog Number	Comments
Brain & Tissue Matrices	SIGMA - ALDRICH	15013
Digital Weighing Scale	SIGMA - ALDRICH	RS 4000
Ethanol 96 %	ROMICAL		Flammable Liquid
Heater with thermometer	Heatingpad-1	Model: HEATINGPAD-1/2
Infusion Cuff	ABN	IC-500
Isofluran, USP 100%	Piramamal Critical Care, Inc	NDC 66794-017
Scalpel blades 11	SIGMA - ALDRICH	S2771
Sharplan 3000 Nd:YAG (neodymium-doped yttrium aluminum garnet) laser machine	Laser Industries Ltd
Stereotaxic head holder	KOPF	900LS