A Repetitive Concussive Head Injury Model in Mice

Summary

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

Abstract

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Introduction

Concussion, also called mild traumatic brain injury (mTBI), is the most frequent occurrence of traumatic brain injury (TBI) and affects millions of people in United States. Concussions can be tricky to diagnose and there is no specific cure for concussion. There is a growing recognition and some evidence that mild mechanical trauma resulting from sports injuries, military combat, and other physically engaging pursuits may have cumulative and chronic neurological consequences^1,2. However, there is still a lack of knowledge regarding concussions and their effects. Current methodology restricts the studies of pathology and treatment in humans since only neurologic assessment and imaging evaluation are available for clinical diagnosis. Animal models provide a means to study concussions in an efficient, rigorous, and controlled manner with the hope of further diagnosis and treatment of mTBI.

Studies have adapted traditional TBI models such as controlled cortical impact (CCI), fluid-percussion impact (FPI), weight drop injury, and blast injury to perform mTBI and stimulate low injury severities by changing the injury parameters. These models are beneficial to use due to their ability to replicate brain trauma morphologically similar to the clinical condition; however, they also have their own limitations. The severity of injury induced by an acceleration injury (weight drop) is often highly variable. The two results of the mild CCI — subarachnoid hemorrhage and focal contusion — are not comparable with typical human concussions. CCI and FPI require a craniotomy, which is not clinically relevant, while blast injury is a more controversial model in regards to the different exposure position and peak pressure measurements as well as variable secondary injury during the exposure^3-6_. An updated concussive animal model that can translate pre-clinical research into the clinical setting is necessary in research.

The key issue in modeling mild TBI is to define the experimental injury severity, which most closely replicates the injury in a clinical setting. Recently, different research groups developed the closed head injury or concussive head injury (CHI) model^7-10. CHI is a modification of CCI without a craniotomy, but it still uses a traditional electronic magnetic impact system to generate a head impact. A CHI can induce a concussion ranging from mild to moderate by adjusting the impact parameters. Loss of consciousness (LOC) can be observed immediately after an impact by detecting a decrease in the breathing rate or the transient termination of breathing. The period of LOC is used to determine the severity of injury. This paper includes a slightly improved and updated version of a repetitive CHI (rCHI) model in mice, along with a detailed step-by-step protocol and representative results. The rCHI model research strategies are beneficial in determining mTBI effects and potential treatments, especially since there is no individual animal model capable of imitating all of the concussion-induced pathological changes.

Protocol

All procedures were performed under protocols #201207692 approved by the Institutional Animal Care and Use Committee of University of Florida and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

1. Animal Care

1. Use 3–4-month-old male C57BL/6J mice. Provide bedding, nesting material, food, and water ad libitum. Keep the mice in ambient temperatures controlled at 20 - 22 °C with constant 12-hr light/12-hr dark cycles.

2. Pre-impaction Preparation

1. Attach a custom-made silicone rubber-coated metal tip to an electromagnetic stereotaxic impact device. Make sure the flat bottom of the tip is parallel to the surface of the probe tip (Figure 1A).
2. Anesthetize the mouse with 4% isoflurane followed by maintenance anesthesia of 2.5% isoflurane. Check the anesthesia via the flow meter. Monitor the anesthesia level until the animal reaches a surgical level of anesthesia by showing loss of pedal withdrawal reflex.
3. Put the mouse in a prone position on a heating pad. Use a funnel-shaped nose cone to keep the mouse under anesthesia. Completely shave the head using a trimmer. Use petrolatum ophthalmic ointment on the mouse's eyes to prevent dryness while under anesthesia.

3. Impact Parameters Setting

NOTE: The impact system includes a control box to set impact parameters, an actuator to perform the impaction, and a digital stereotactic frame with 3-movement axes.

1. Pre-set the velocity of the impact device to 4 m/sec and dwell time to 240 msec on the control box.

4. Positioning the Impact Center

1. Put a soft heating pad under the animal's body to keep the body temperature near 39 °C. Mount the mouse in a stereotactic frame in a prone position with the blunt-end ear bars.
2. Lower the impact tip close to the mouse's head by moving the Z-driver. Adjust the flat impact tip (9 mm diameter) by moving the X- and Y-drivers midway to the target coordinates above the sagittal suture.
3. Make sure one edge of the impact tip is vertically parallel to an imaginary horizontal line drawn between the two ears (Figure 1C). The center of impact corresponds to the central sagittal suture midway between interfrontal and lambdoid sutures (interaural 9 mm to interaural 0 mm, lateral 4.5 mm).

5. Impact Depth Setting

1. To correctly set the impact depth, use additional probe tip to replace the insulated silicone rubber-coated impact tip.
2. To make sure there is no shift of the impact center after switching tips, set the X and Y channel on the digital stereotaxic control panel to zero before switching the tips.
3. Move the probe tip to the center of the impact area by manually moving the X-and Y-drives.
4. Clip contact sensor to the mouse's tail.
5. Move the impactor (Z drive) down until the probe tip touches the surface of the impact site.
6. Set the Z channel on the stereotaxic control panel to zero.
7. Move the impact tip back to the impact area by manually adjusting X- and Y-drivers (NOT the zero buttons on the digital stereotaxic control panel) until X- and Y-drivers are zero (where the impact tip was previously positioned).
8. Retract the actuator by moving the retract switch on the control box. Manually move the impactor down (Z driver) by 4 mm.

6. Impact

1. Trigger the impact by clicking the impact switch on the control box and achieve a deformation depth of 4 mm.

7. Post-impaction

1. Measure the time from the impact until the mouse's first breath using a timer.
2. Remove the animal from the instrument and place them on a warm pad to maintain their body temperature. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.
3. Allow for recovery before returning the animal back into a clean cage. Do not return an animal to the company of other animals until fully recovered.
4. Observe and weigh the mice daily. If the mice show signs of pain, intraperitoneally inject them with Meloxicam at 1 - 2 mg/kg every 12 - 24 hr.

8. Repetitive Impaction

1. Give the mice additional injuries on days 4, 7, and 10 after the initial injury (72 hr interval between impacts).

9. Immunohistochemistry (IHC)

1. Transcardial perfusion
   1. Anesthetize the mice via intraperitoneal injection with 200 m/kg pentobarbital.
   2. Assess and assure surgical-plane anesthesia by a toe pinch. Secure the mouse in the supine position by gently taping the forepaws and hind paws to a Styrofoam work surface inside a chemical fume hood.
   3. Make an incision through the skin along the thoracic midline from just beneath the xiphoid process to the clavicle. Make two additional skin incisions at the xiphoid process and proceed along the base of the ventral ribcage laterally.
   4. Open the thoracic cavity and expose the heart by cutting through the thoracic musculature and ribcage.
   5. Secure the beating heart with blunt forceps and make a 1 - 2 mm incision in the left ventricle.
   6. Immediately insert a butterfly needle into the right atrium. Begin the infusion of 20ml saline by pushing the syringe slowly.
   7. Switch from saline to 4% paraformaldehyde. Continue perfusion with 20ml of paraformaldehyde.
   8. Decapitate the mouse and remove the skin with scissors. Isolate the brain from the skull using a bone cutter.
2. Cryostat sectioning
   1. Embed brain tissues in optimum cutting temperature (O.C.T.) formulation and freeze at -80 °C. Place the brain in the cryostat in a sagittal orientation. Cut brain sections 5 µm thick.
3. Staining
   1. Dry the frozen sections at room temperature for 1 hr.
   2. Incubate the slides with 100 μl of 2% goat serum and 0.1% Triton X-100 in phosphate buffered saline (PBS) for 1 hr at RT.
   3. Wash the slides 3 times with 300 μl of PBS. Then incubate the slides with anti-GFAP (1:200) or anti-ferritin antibody (1:200) separately over night at 4 °C.
   4. Wash the slides 3 times with 300 μl of PBS. Then incubate the slides for 2 hr at room temperature with biotin-conjugated secondary antibody.
   5. Wash the slides 3 times with 300 μl of PBS. Then incubate the slides with avidin-biotin complex (ABC) solution (1:50) at room temperature for 30 min.
   6. Wash the slides 3 times with 300 μl of PBS. Then incubate in 3,3′-diaminobenzidine (DAB) substrate solution (50 ml PBS, 10 µl H₂O₂, 10 mg DAB pill, filter before using) for 5 - 8 min. Observe the slides under the microscope until the positive cells appear.
   7. Rinse the slides in slow running tap water for 5 min. Clean slides with a lab-wipe. Then mount the sections with mounting medium and coverslip.

Results

In this model (Figure 1 A-C), there were brief periods of gasping and shallow respirations. A loss of consciousness (unconscious) is defined as a decrease in the breathing rate or transient termination of breathing before resuming a normal respiration. An impact on the center of the head caused short-term unconsciousness (7.5 ± 4.7, 7.8 ± 5.5, 10.2 ± 8.8, 9.5 ± 8.0 sec at each impact separately, Figure 1D). Mouse brains showed normal morphology by H&E...

Discussion

To mimic brain injuries morphologically similar to the clinical condition, post-concussion symptoms are expected. Post-concussion symptoms generally include headaches, dizziness, vertigo, fatigue, memory and sleeping problems, trouble concentrating as well as anxiety, and depressed mood. Since somatic symptoms may not yet be measurable in animal models, the changes of motor and cognitive function and emotional behavior are used as criteria for rationally evaluating concussion in animal models. In a previous reported stud...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anesthesia machine	Eagle Eye Anesthesia, Inc	Model 150	anesthesia
Electromagnetic Impactor	LeicaBiosystems	Impact One Stereotaxic Impactor	perform impaction
Digital Stereotaxic instrument	LeicaBiosystems	39462501	mount mouse and positioning tips
Sicilone rubber-coated metal tip	Precision Tool & Engineering, Gainesvill FL	custom-made	impact tip
Lithium Ion All-in-One Trimmer	WAHL Home Products	9854-600	shave mouse hair
paper clips	custom-made		probe tip
Cotton tipped applicators	MEDLINE	MDS202055	scrub head with saline
Tissue Tek O.C.T.	ASKURA FINETEK USA INC	4583	tissue embedding
anti-GFAP	Dako	CA93013	antibody for IHC
anti Ferritin	Sigma	F6136	antibody for IHC
VECTASTAIN Elite ABC  kit	Vector laboratories	PK-6100	IHC detection system
Permount Mounting Medium	Fisher Scientific	SP15-100
Aperio XT ScanScope scanner	Leica Microsystems Inc,		slides scanning
Leica AutoStainer XL	Leica the pathology Company	ST2010	H&E staining
DAB	sigma	D3939	IHC detection system