Our protocol provides a novel methodology for creating traumatic brain injury in rats using laser irradiation to create a focus impact in the motor cortex. The main advantage of this technique are the low variability in infarct area, the low mortality rates, and the relative simplicity of the procedure which doesn't require expert handlers. Demonstrating the procedure will be Dmitry Natanel, a researcher from our laboratory.
To perform a laser-induced brain injury, first assign 20, 300 to 350 gram Sprague Dawley rats into the laser group and 20 to the control Sham-operated group. After confirming a lack of response to withdrawal reflex, place one rat on a rectal temperature regulated heating pad and use a shaver to remove enough hair from the injury site with an approximately two centimeter hair-free margin around the incision. Disinfect the exposed skin with 70%ethanol and place the rat in the prone position in a stereotaxic head holder.
Make a three centimeter incision to allow lateral reflection of the scalp to expose the area between bregma and lambda. Holding an Nd:YAG laser with peak wavelength of 1064 nanometers at a two millimeter distance from the skull, administer 50 joules times 10 points with a one-second pulse duration to the exposed area of the skull above the right hemisphere. After the laser injury has been delivered, remove the rat from the device and close the scalp with 3-0 silk surgical sutures.
Then, place the rat in its cage with monitoring until full recumbency. 24 hours after the procedure, use a 43 point scoring system to evaluate the neurological severity score. Testing the animals for neurological deficits, behavior disturbances, beam balancing task, and reflexes, and assigning higher scores for more severe disabilities.
After the assessment, harvest the brains from each experimental and control animal according to standard protocols. To assess the brain infarct volume, section the harvested brains into six two millimeter thick coronal slices and incubate each slice in 0.05%TTC for 30 minutes at 37 degrees Celsius. Following staining, scan this slices with an optical scanner with a 1600 by 1600 dots per inch resolution.
The unstained areas of the fixed brain slices are defined as infarcted. To evaluate the incidence of blood brain barrier breakage, 24 hours after the laser induced injury load a syringe with 2%Evans blue dye diluted in four milliliters per kilogram of saline solution and deliver the solution intravenously to the injured and control rats via the cannulated tail vein. Allow the solution to circulate for one hour before using surgical pincets and scissors to open the chest of the first animal.
Perfuse the exposed heart with cooled 0.9%saline via the left ventricle at 110 millimeters of mercury of pressure until a colorless profusion liquid is observed from the right atrium. Next, harvest the brain and obtain two millimeter rostral caudal slices. Separate the left brain slices from the right brain slices to allow evaluation of the injured and non-injured hemispheres separately, and weigh the samples.
Homogenize the samples using a mortar and pestle and incubate the tissue samples in 50%trichloroacetic acid for 24 hours. The next day, centrifuge the homogenized brain tissue samples for 20 minutes at 10, 000 times G and room temperature, and mix one milliliter of the supernatant with 1.5 milliliters of 96%ethanol at one to three ratio. Then, use a fluorescence detector at a 620 nanometer excitation and the 680 nanometer emission wavelength to assess the blood brain barrier breakage.
As observed by histological analysis, Evans blue staining can be used to reveal the incidence of brain injury at laser and MCAO model animals. In this representative analysis no deaths or subarachnoid hemorrhages were registered in either the control or experimental groups, and the MCAO group had a 20%rate of both mortality and subarachnoid hemorrhage. The relative body temperature changes in the rats from both groups were also similar, despite a difference in the variability of both groups.
The neurological severity scores were significantly worse in both the laser and MCAO models compared to the Sham-operated control group. Laser-induced to brain injury also caused a significant increase in infarct volume at the target hemisphere compared to the Sham-operated control group. However, the infarct volume of the laser model was smaller compared to the animals injured by the MCAO technique.
No difference in brain edema was observed between the laser-induced brain injury model and the Sham-operated control group. However, there was a significant difference in brain edema between the laser model and MCAO injured groups. Compared to the Sham-operated control group, the laser-induced brain injury and MCAO techniques both caused a significant increase in blood brain barrier breakage at the non-injured and target hemispheres.
While attempting this model, remember to precisely target the motor cortex area and to standardize the energy, the number of points being irradiated, and the total exposition time. Following this procedure, a wide plethora of behavioral tests, such as beam walking, Froedert, and other evaluations can be performed.
