Assessing Blood-Brain Barrier Integrity in a Laser-Induced Brain Injury 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. Assign 20 rats to a group marked as laser group and 20 rats to another control group (sham-operated).
2. Subject the laser group rats to laser irradiation at 50J X 10 points in the following manner:
   1. Anesthetize 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 the absence of the withdrawal reflex.
   2. Maintain the core body temperature of the rat at 37 ˚C throughout the experimental procedure using a rectal temperature regulated heating pad.
   3. Remove local hair with a shaver and disinfect with 70% alcohol and 0.5% chlorhexidine gluconate. Repeat the disinfection step two more times.
      NOTE: The size of the surgical incision should be approximately 3 cm. Remove hair at least 2 cm around the incision area.
   4. Place the rat on a stereotaxic head holder in a prone position and make a 3 cm incision to reflect the scalp laterally and to expose the area between Bregma and Lambda.
   5. Maintain anesthesia through the nose cone.
   6. Use neodymium-doped yttrium aluminum garnet (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 at a 2 mm distance 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 considering 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.
3. Observe the rat for any signs of distress during post- anesthesia recovery. Prior to emerging from anesthesia, give 0.01mg/kg intramuscular buprenorphine for postoperative analgesia and continue with repeated doses every 12 h for at least 48 h.
4. Subject control rats to the same conditions without subjecting them to the laser.

2. Neurological severity score (NSS)

1. Evaluate the neurological severity score 24 h after the laser-induced brain injury using a 43-point score. Test the animals for neurological deficits, behavior disturbances, beam-balancing task, and reflexes, assigning higher scores for more severe disabilities.

3. Evaluation of brain injury

1. Determining the extent of blood-brain barrier (BBB) breakage
   NOTE: Assess BBB breakage 24 h after the laser-induced brain injury as follows:
   1. Administer 2% Evans Blue mixed with 4 mL/kg saline solution intravenously to rats via the cannulated tail vein and allow the solution to circulate for 1 h.
   2. Euthanize rats by exposing them to 20% oxygen and 80% CO₂ (via inspiration) 24 h after the last NSS.
   3. Harvest the intravascularly localized dye as follows:
      1. Open the chests of the rats with surgical pincettes and surgical scissors.
      2. Perfuse the animals with cooled 0.9% saline via the left ventricle using 110 mmHg until obtaining a colorless perfusion liquid from the right atrium.
   4. Harvest the brains and slice them rostro-caudally into 2 mm slices.
   5. Separate the left brain slices from the right portions to evaluate injured and non-injured hemispheres separately.
   6. Weigh, homogenize using mortar and pestle, and then incubate the brain tissues in 50% trichloroacetic acid for 24 h.
   7. Centrifuge the homogenized brain slices at 10,000 × g for 20 min.
   8. Mix 1 mL of the supernatant from the centrifuged brain with 1.5 mL of 96% ethanol at 1:3 and assess blood-brain barrier breakage using a fluorescence detector at 620 nm excitation wavelength (10 nm bandwidth) and 680 nm emission wavelength (10 nm bandwidth).
      NOTE: Both groups of rats undergo the same protocol for determining BBB breakdown.

Access restricted. Please log in or start a trial to view this content.

Materials