A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Summary

Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

Abstract

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal neurological deficits and even amnesia. Athletes participating in contact sports are at high risk of exposure to large number of mTBIs. In terms of the level of injury in a sporting athlete, a mTBI is defined as a mild injury that does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Despite previous attempts to model mTBI in mice and rats, many have reported gross adverse effects including skull fractures, intracerebral bleeding, axonal injury and neuronal cell death. Herein, we describe our highly reproducible animal model of mTBI that reproduces clinically relevant symptoms. This model uses a custom made pneumatic impactor device to deliver a closed-head trauma. This impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI.

Introduction

Traumatic Brain Injury (TBI) is defined as a head injury sustained from an external physical force, resulting in the disruption of normal brain function. It represents a significant socio-economic and public health burden, with the Center for Disease Control and Prevention 2015 Report to Congress estimating that 2.5 million Americans sustain a TBI every year. This impacts not only the patient's quality of life, but also places an extremely high economic cost on the community, currently estimated at $76.5 billion annually. The amount of actual brain damage conveyed and the acute phase symptomologies is what defines mild, moderate and severe TBI.

Mild Traumatic Brain Injury (mTBI), also referred to as concussion, accounts for over 70% of TBIs reported each year¹. It is most common among athletes who participate in high-risk contact sports including boxing and football². Unlike moderate or severe forms of TBI, the immediate damage and symptoms associated with mTBI are sometimes not as pronounced³. By contrast, the long-term effects of mTBI can be just as debilitating as those seen in the moderate and severe forms. Those who suffer from repetitive mTBI have been shown to develop chronic traumatic encephalopathy (CTE), as well as other cognitive and degenerative illnesses⁴. Therefore, it is important to gain a greater understanding of the mechanisms that contribute to the short-term symptoms and overall long-term damage that arises following mTBI.

In humans, the definition of concussion as defined by the Fourth International Conference on Concussion in Sport (Zurich 2012)⁵ states that the level of injury for sports concussion is mild, does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Indeed, a recent study investigated the effect of mTBI on cognitive impairment in high school football players, using head impact telemetry systems. This study showed the amount of times players sustained helmet impacts >20 g in a single season ranged from a low of 226 (average, 4.7 per session) to a high of 1855 (average, 38.6 per session)⁶. Most of these impacts did not result in the clinical diagnosis of a concussion; but evidence of functional changes in brain function could be observed using fMRI⁶. The brain changes that cause these functional changes are unknown, and therefore there is a pressing need to have a reliable and reproducible model to facilitate research into the effects of concussive and subconcussive mTBI.

Despite previous attempts to model mTBI in mice and rats⁷, many report adverse effects. In particular, most rodent models are limited in their repetitive nature using fewer than five mTBIs impacts, as well as having adverse pathological events including intracerebral bleeding, skull fractures, severe axonal injury, neuronal cell death, and increased mortality^8,9,10,11,12. Herein, we describe a mouse model of mTBI that is closer to the true definition of concussion in humans. This model recapitulates many of the symptoms observed in human mTBI, such as the mechanical force that results in the transient loss of consciousness with no overt gross brain pathology. Furthermore, it is advantageous in the fact that it can be utilised for both single impact and repetitive impact paradigms over long time periods as reported previously¹³.

Protocol

These studies were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes Health. The protocol was approved by the Institutional Animal Care and Use Committee at Georgetown University. Mice were housed in a temperature controlled animal facility and were kept on a 12 h light/12 h dark cycle. Food and water were available ad libitum.

1. Preparation of the mTBI Apparatus

NOTE: The mTBI apparatus includes a data acquisition (DAQ) box to control impact parameters, a high-velocity pneumatic impactor to perform the impaction, and a moulded gel filled base to decelerate head after impact.

1. Turn on compressed air and initialize the high-velocity pneumatic impactor to a pressure setting of 861.85 kPa.
2. Calibrate the DAQ control system to following parameters- piston velocity of 2.35 m/s, surface dwell time of 31.5 ms.
3. Place the molded gel-filled base (compressibility 64 kPa/mm) such that the midline is perpendicular with the trajectory of impactor tip.

2. mTBI Impaction

1. Record the weights of all mice to be used.
2. Anesthetize mice with 3% isoflurane in oxygen for 120 s in an induction chamber.
3. Transfer mice to mTBI apparatus, continuing anesthesia via a pliable, non-fixed nose cone.
4. Whilst in nose cone, position mouse head on the gel pad so that the flat surface of the skull is presented perpendicular to the impactor tip.
5. Place tape over mouse head to create a flat surface and hold ears away from the impact site.
6. Lower the polytetrafluoroethylene impactor tip to align with the sagittal midline in the center of the head. The impactor tip used is 10 mm in diameter and will cover the area of the scalp from just behind the eyes to the midline of the ears. Adjust the impactor so it is just touching the surface of the mouse head.
7. Retract the impactor tip and manually dial down the required deflection depth (7.5 mm).
8. Arm the DAQ control system and depress trigger button to impact mouse head, either a singular or repetitive impacts. Repetitive impacts are delivered in succession with no delay between each impact.
9. Immediately after the final impact, remove mouse from anesthesia and the mTBI apparatus and place in supine position.
10. Using a stopwatch measure the latency of the return of righting reflex (supine to prone), to determine loss of consciousness time, as well as time to ambulation (unimpeded walking).
11. Monitor mouse and upon recovery to normal behaviour, return to its home cage.
12. Sham mice receive identical handling and anesthesia, but receive no impact.

Results

The use of this novel mTBI device allows for single and repetitive mild head injuries without risk of skull fracture or structural brain damage. The model uses a custom made pneumatic Teflon impactor device to deliver a closed head mechanical energy impact. The impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI (Figure ...

Discussion

In humans, mTBI is characterized by a functional impairment in the absence of structural injury. This can occur with, or without, a loss of consciousness¹. Exposure to repeat concussions is currently thought to underlie the development and/or progression of neurodegenerative diseases such as CTE⁴. It is well documented that CTE is commonly found in boxers and football players and although exposure to repeat concussions (including those that do not result in a loss of consci...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Powerlab 8SP data acquisition (DAQ) control box	(AD instruments)
VIP 3000 calibrated vaporizer	Matrx
Isoflurane	Henry Schein Animal Health	29405
Oxygen			Commercially available
Compressed Air			Commercially available
Masking Tape			Commercially available
Stop Watch	Fisher Scientific	02-261-840
C57 Bl/6 Mice	Jackson Laboratories
Digital Scale and weigh container	Fisher Scientific	20031
anti-Iba1 antibody	Wako	019-19741
HRP labelled secondary	Jackson Immunoresearch	111-035-003