The experiments were performed following the recommendations of the Declarations of Helsinki and Tokyo and to the Guidelines for the Use of Experimental Animals of the European Community. The experiments were approved by the Animal Care Committee of Ben-Gurion University of the Negev.

1. Preparing rats for the experimental procedure

NOTE: Select adult male Sprague-Dawley rats weighing 300-350 g.

1. Obtain approval for performing these experiments from the Institutional Animal Care and Use Committee.
2. Maintain rats at a room temperature of 22 ± 1 °C, with 12 hour light and 12 hour dark cycles. Provide rat chow and water ad libitum.
3. Perform all experiments between 6:00 a.m. and 12:00 p.m.
4. Use a continuous isoflurane administration system to induce anesthesia. Make sure the vaporizer system is filled with isoflurane.
   1. Anesthetize the rats with 2% isoflurane.
   2. Confirm that the rat is fully anesthetized by observing a lack of movement or pedal reflex in response to an external stimuli.

2. Induction of diffuse axonal injury

NOTE: The device consists of the following components: 1) transparent plastic cylinder, 2) iron weight (1308 g), 3) rotation mechanism consisting of a cylindrical tube, two bearings upon which the axis rotate and a head fixation (for ear pins); 4) horizontal platform on which are fixed two bearings.

1. Place the device on a heavy, stable laboratory table.
2. Attach the weight to a string that is elevated to a height of 120 cm.
3. Allow the freely falling weight to hit the bolt, activating the rotational mechanism. Using the lateral head rotation device, the rodent’s head is turned rapidly from 0 to 90°.
4. After induction of diffuse axonal brain injury, transfer the rat to a recovery room.

3. Measurement of rotational Kinematics/Biomechanical parameters.

1. Measure rotational kinematics/biomechanical parameters as follows:
   
   
   
   where F_o - force applied to animal head (kg); M – moment of force; K – kinetic energy; m – mass of the falling weight; g – gravitational acceleration; h – height (cm); D – distance between the ear pins (cm).
   NOTE: To calculate the force applied to the animal’s head (F_o), it is necessary to know the mass of the falling weight, the height at which the weight falls, and the distance between the ear pins. The other parameters remain unchanged.

4. Evaluation of Neurological Severity Score after 48 hours

NOTE: Neurological deficits were assessed and graded using a Neurological Severity Score, as previously described^17,18,19. Alterations in motor function and behavior are assessed by a point-system such that a maximum score of 24 represents severe neurological dysfunction. A score of 0 indicates intact neurological status. The following behavioral functions are assessed.

1. Assess the rat’s inability to exit from a circle (50 cm in diameter) when left in its center. Perform this for three individual sessions lasting 30 min, 60 min, and more than 60 min.
2. Test the rat for a loss of righting reflex in three sessions lasting 20 min, 40 min, and over 60 min.
3. Perform the test for hemiplegia, the inability of the rat to resist forced changes in position.
4. Raise the rat by its tail to test the flexion of the hindlimb.
5. Place the rat on the floor to test its ability to walk straight.
6. Test for three separate reflexes: the pinna reflex, the corneal reflex, and the startle reflex.
7. Rate the rat with a clinical grade based on loss of seeking behavior and prostration.
8. Test limb reflexes for placement. Perform the test on the left and right forelimbs, and then the left and right hindlimbs.
9. Perform a functional test via the beam balancing task. Beam should measure 1.5 cm wide. Run the test for sessions of 20 s, 40 s, and more than 60 s.
10. Perform beam walking test on the rat with beams of three different widths: 8.5 cm wide, 5 cm wide, and 2.5 cm wide.

5. Brain collection for histological examination after 48 hours

1. At 48 hours post injury, euthanize the rats by replacing their inspired gas mixture with 20% O₂/80% CO₂. Ensure that CO₂ is delivered at a predetermined rate in accordance with Institutional Animal Care and Use Committee guidelines.
   1. Ensure death confirmation in accordance with Institutional Animal Care and Use Committee guidelines.
2. Transcardiacally perfuse the rat with 0.9% heparinized saline at temperature 4 °C, followed by 500 mL of 4% paraformaldehyde in 0.1 M phosphate buffer saline (pH 7.4).
3. After perfusion, perform decapitation with a guillotine.
4. Perform brain collection by removing the calvarias with bone-cutting forceps to avoid damaging brain tissue.
5. Remove the brain immediately and fix in a 4% buffered formaldehyde solution for 48 h at 4 °C.
6. Block brains into 5 mm coronal sections from the olfactory bulb face to the visual cortex and bisect cerebellums and brain stems.
7. Following paraffin embedding, cut coronal and sagittal sections (5 µm) away from the thalamus by microtome sectioning.

6. Immunochemical staining and examination

1. Gently place the slices on glass slides with a soft brush, 1 slice per slide.
2. Produce immunochemical staining of βAPP.
   1. Deparaffinize slices with xylene (3 times for 5 min each) and rehydrate with gradually-reduced concentrations of ethanol at room temperature: 3 min in 100% ethanol twice, 3 min in 95% ethanol twice, 3 min in 90% ethanol, 3 min in 70% ethanol, and 3 min in DDW.
   2. Treat deparaffinized and rehydrated brain sections with 3% H₂O₂ for 15 min at room temperature to block endogenous peroxidase activity.
   3. Incubate sections with 0.01 M sodium citrate (pH 6.0) at 98 °C for 5 min for antigen retrieval.
   4. Keep the slides in the buffer for 20 min at room temperature to cool.
   5. Wash sections with phosphate-buffered saline (PBS) solution twice for 5 min.
   6. Block the sections with 2.5% normal horse serum for 1 h at room temperature and incubate overnight at 4 °C in primary rabbit anti-APP (1:4000) diluted in the blocking serum.
   7. After incubation in primary antibody, wash sections in PBS at room temperature.
   8. Incubate sections in appropriately diluted biotinylated secondary antibody for 15 min and wash with PBS for 3 min twice at room temperature.
   9. Incubate in streptavidin–peroxidase for 15 min and wash again in PBS for 3 min twice at room temperature.
   10. Incubate sections with buffered substrate solution (pH 7.5) containing hydrogen peroxide and 3,3-diaminobenzidine chromogen solution and protect from light until the color is developed.
   11. Incubate the slides with DDW at room temperature for 5 min in order to stop the reaction.
   12. Counterstain sections with Hematoxylin for 3 min at room temperature and wash for 5 min with flowing tap water.
   13. Dehydrate the slides with gradually increasing concentrations of ethanol at room temperature: 2 min in DDW, 2 min in 70% ethanol, 2 min in 90% ethanol, 2 min in ethanol 95%, 2 min in 100% ethanol, and 3 min in xylene three times.
   14. Dry and mount with mounting medium.
3. Examine the slices under microscope magnification of 200x with a 20 mm objective lens using a microscope.