Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Summary

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

Abstract

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

Introduction

Traumatic brain injury (TBI) is a major cause of death and disability in the United States. TBIs contribute to about 30% of all injury-related deaths^1,2. The leading causes of TBI differ among age groups and include falls, high-speed collisions during sports, intentional self-harm, motor vehicle crashes and assaults^1,2,3.

Brain diffuse axonal injury (DAI) is a specific type of TBI induced by rotational acceleration, shaking or blast injury of the brain resulting from unrestricted head movem....

Protocol

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. Pr....

Results

Table 1 illustrates the protocol timeline. The mortality rate in this model of DAI was 0%. A Mann-Whitney test indicated that neurological deficit was significantly greater for the 15 DAI rats compared to the 15 sham rats at 48 hours following intervention (Mdn = 1 vs. 0), U = 22.5, p < 0.001, r = 0.78 (see Table 2). The data are measured in counts and are presented as median and 25–75 percentile range.

Representative photomicrographs of thalamic sections o.......

Discussion

This protocol describes a rodent model of DAI. In DAI, rotational acceleration on the brain causes a shear effect that triggers axonal and biochemical changes that lead to loss of axonal function in a progressive process. Secondary axonal changes are produced by a rapid axonal stretch injury and are variable in their extent and severity^4,5,10. Within hours to days after the primary injury, biochemical changes will lead to the lo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.01 M sodium citrate	SIGMA - ALDRICH
2.5% normal horse serum	SIGMA - ALDRICH	H0146	Liquid
4 % buffered formaldehyde solution
Anti-Amyloid Precursor Protein, C - terminal antibodyproduced in rabbit	SIGMA - ALDRICH	Lot 056M4867V
biotinylated secondary antibody	Vector	BA-1000-1.5	10 mM sodium phosphate, pH 7.8, 0.15 M NaCl, 0.08% sodium azide, 3 mg/ml bovine serum albumin
bone-cutting forceps
DAB Peroxidase (HRP) Substrate Kit (with Nickel), 3,3’-diaminobenzidine	vector laboratory
embedding cassettes
ethanol 99.9 %	ROMICAL		Flammable Liquid
guillotine
Hematoxylin	SIGMA - ALDRICH	H3136-25G
Hydrogen peroxide solution	Millipore	88597-100ML-F
Isofluran, USP 100%	Piramamal Critical Care, Inc
Olympus BX 40 microscope	Olympus
paraffine	paraplast plus leica biosystem		Tissue embedding medium
phosphate-buffered saline (PBS)	SIGMA - ALDRICH	P5368-10PAK	Contents of one pouch, when dissolved in one liter of distilled or deionized water, will yield 0.01 M phosphate buffered saline (NaCl 0.138 M; KCl - 0.0027 M); pH 7.4, at 25 °C.
Streptavidin HRP	ABCAM	ab64269	Streptavidin-HRP for use with biotinylated secondary antibodies during IHC / immunohistochemistry.
xylene