A Pediatric Concussion Model in Mice: Closed Head Injury with Long-Term Disorders (CHILD)

Summary

Pediatric concussions can result in long-term sequelae with physiological and psychological perturbations. This pediatric concussion mouse model results in long-term neuroinflammation, white matter alterations, and neuronal modifications with behavioral dysfunction. The model can be adapted to differential impact locations or repeated concussions to assess lifetime changes.

Abstract

Concussions and mild traumatic brain injuries (mTBI) during childhood represent a significant health endangerment, with many patients later in life exhibiting debilitating physiological, neurological, and psychosocial outcomes. The cellular and molecular pathophysiological mechanisms are relatively unknown, and this significant gap effectively precludes investigations into specific therapeutic strategies to mitigate chronic sequelae. No single animal concussion model recapitulates all the features reported in human subjects, and current models are designed to answer specific research questions. We set out to develop a juvenile concussion that recapitulates clinical early, and long-term symptomology encountered, termed Closed Head Injury with Long-term Disorder (CHILD). In this model, a concussive force via an electromagnetic impactor is delivered to the head of a lightly anesthetized unrestrained post-natal day 17 (P17) mouse, allowing free rotation of the head. Mice are placed on a tightly stretched tin foil across a stereotactic device, and the impactor is carefully aligned with the targeted cortical region. The electromagnetic impactor facilitates the selection of impact depth, dwell, and duration to determine injury severity. A strength of this model is that it allows head rotation, an important clinical feature. After impact, mice are immediately monitored for righting time and time to explore their environment, followed by their return to their dam. Increasing the severity of concussion results in intracranial bleeds in a subset of mice. Mice can be routinely monitored for behavior and neuroimaging over their lifespan, as desired. Various injury severities mimic the heterogeneous nature of juvenile concussions. The current standard CHILD model exhibits no skull fracture, no observable conventional neuroimaging change (similar to clinical), but leads to progressive and persistent neuronal death, altered diffusion MRI, modified neuronal activity and plasticity, increased gliosis, and progressive behavioral perturbations with age. In summary, this CHILD model mimics the early and long-term features observed in many clinical concussion patients.

Introduction

Pediatric Traumatic Brain Injury (TBI) is a major health concern as it represents the leading cause of pediatric emergency medical care visits and is associated with debilitating health outcomes later in life. Even mild TBI (mTBI), which makes up 75% of all TBI cases¹, leads to long-term abnormalities, including physiological, psychological, neurological, and cognitive dysfunctions². Despite the high occurrence of pediatric mTBI and its significant associated health risks, the pathological, cellular, and molecular mechanisms that precede the chronic deficits are still underexplored. This gap in knowledge impedes the deve....

Protocol

Experiments were reproduced on 2 sites, the University of Bordeaux (France) and the University of California Riverside (CA, USA).

In Bordeaux, all experiments were performed on male and female P17 C57Bl6J mice bred in the University animal facility. Initial breeders were obtained from a commercial source and raised under standard conditions with a 12/12-h dark/light cycle, a temperature of 21 °C ± 1 °C, and a humidity of 55% ± 10%, and food and water provided ad libitum. Experimental procedures are in accordance with the University of Bordeaux Animal Care Committee regulations, French laws governing laboratory a....

Discussion

Critical steps in the protocol
The CHILD protocol described here requires the use of an electromagnetic impactor (Figure 2A) that (1) standardizes the impact parameters (speed, depth, duration), (2) ensures reproducibility within and across laboratories, and (3) is readily available commercially. Another critical component is the impactor tip that strikes the rodent's head. A variety of possibilities can be used, ranging from silicone to metal tips, but this protoc.......

Materials

Name	Company	Catalog Number	Comments
BSA	Sigma Aldrich Chimie S.a.r.l 80 Rue de Luzais L'lsle-d'Abeau Chesnes - BP701 38297 Saint-Quentin-Fallavier Cedex France	A7030
C57Bl6J mice	Charles River Laboratory France CRLF 9 All. Moulin Berger, 69130 Écully, France		Bordeaux
Electromagnetic impactor	Leica Biosystems, Richmond, IL USA	Impact One stereotaxic impactor
Epifluorescence microscope	Olympus, BX41, Center Valley, PA
Ethylen glycol	Sigma Aldrich Chimie S.a.r.l 80 Rue de Luzais L'lsle-d'Abeau Chesnes - BP701 38297 Saint-Quentin-Fallavier Cedex France	102466
GFAP primary antibody	Millipore, Billerica, MA	AB5541	chicken anti-GFAP
GFAP secondary antibody	Molecular Probes, Invitrogen	A11034	goat anti-rabbit AlexaFluor  488
Glycerol	Sigma Aldrich Chimie S.a.r.l 80 Rue de Luzais L'lsle-d'Abeau Chesnes - BP701 38297 Saint-Quentin-Fallavier Cedex France	G5516
Heavy duty aluminium foil	Kirkland	RK611	obtained at Cosco
Micromanager software	(NIH, USA
NeuN primary antibody	AbCam, Discovery Drive, Cambridge Biomedical Campus, Cambridge, CB2 0AX, UK	AB128886	rabbit anti-NeuN
NeuN secondary antibody	Molecular Probes, Invitrogen	A11041	goat anti-chicken AlexaFluor 568
PBS	Sigma Aldrich Chimie S.a.r.l 80 Rue de Luzais L'lsle-d'Abeau Chesnes - BP701 38297 Saint-Quentin-Fallavier Cedex France	P5493
PFA	Sigma Aldrich Chimie S.a.r.l 80 Rue de Luzais L'lsle-d'Abeau Chesnes - BP701 38297 Saint-Quentin-Fallavier Cedex France	1.04003
Sodium azide	Sigma Aldrich Chimie S.a.r.l 80 Rue de Luzais L'lsle-d'Abeau Chesnes - BP701 38297 Saint-Quentin-Fallavier Cedex France	26628-22-8
Stereotaxic frame - base	KOPF instruments 7324 Elmo St, Tujunga, CA 91042, États-Unis	900-C
Stereotaxic frame - electrode manipulator	KOPF instruments 7324 Elmo St, Tujunga, CA 91042, États-Unis	960
Stereotaxic frame - u-frame	KOPF instruments 7324 Elmo St, Tujunga, CA 91042, États-Unis	900-U
Triton x-100	Sigma Aldrich Chimie S.a.r.l 80 Rue de Luzais L'lsle-d'Abeau Chesnes - BP701 38297 Saint-Quentin-Fallavier Cedex France	X100
Vectashield	Vector laboratories	H-1000-10	Mounting medium
Vibratome	Leica Biosystems, Richmond, IL USA	VTS1000
	Jackson laboratory The Jackson Laboratory 600 Main Street Bar Harbor, ME USA 04609		UCR