Omega-3 fatty acid supplementation and reduction of traumatic axonal injury in a rodent head injury model

Laboratory investigation

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Traumatic brain injury remains the most common cause of death in persons under 45 years of age in the Western world. Recent evidence from animal studies suggests that supplementation with omega-3 fatty acid (O3FA) (particularly eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) improves functional outcomes following focal neural injury. The purpose of this study is to determine the benefits of O3FA supplementation following diffuse axonal injury in rats.


Forty adult male Sprague-Dawley rats were used. Three groups of 10 rats were subjected to an impact acceleration injury and the remaining group underwent a sham-injury procedure (surgery, but no impact injury). Two of the groups subjected to the injury were supplemented with 10 or 40 mg/kg/day of O3FA; the third injured group served as an unsupplemented control group. The sham-injured rats likewise received no O3FA supplementation. Serum fatty acid levels were determined from the isolated plasma phospholipids prior to the injury and at the end of the 30 days of supplementation. After the animals had been killed, immunohistochemical analysis of brainstem white matter tracts was performed to assess the presence of β-amyloid precursor protein (APP), a marker of axonal injury. Immunohistochemical analyses of axonal injury mechanisms—including analysis for caspase-3, a marker of apoptosis; RMO-14, a marker of neurofilament compaction; and cytochrome c, a marker of mitochondrial injury—were performed.


Dietary supplementation with a fish oil concentrate rich in EPA and DHA for 30 days resulted in significant increases in O3FA serum levels: 11.6% ± 4.9% over initial levels in the 10 mg/kg/day group and 30.7% ± 3.6% in the 40 mg/kg/day group. Immunohistochemical analysis revealed significantly (p < 0.05) decreased numbers of APP-positive axons in animals receiving O3FA supplementation: 7.7 ± 14.4 axons per mm2 in the 10 mg/kg/day group and 6.2 ± 11.4 axons per mm2 in the 40 mg/kg/day group, versus 182.2 ± 44.6 axons per mm2 in unsupplemented animals. Sham-injured animals had 4.1 ± 1.3 APP-positive axons per mm2. Similarly, immunohistochemical analysis of caspase-3 expression demonstrated significant (p < 0.05) reduction in animals receiving O3FA supplementation, 18.5 ± 28.3 axons per mm2 in the 10 mg/kg/day group and 13.8 ± 18.9 axons per mm2 in the 40 mg/kg/day group, versus 129.3 ± 49.1 axons per mm2 in unsupplemented animals.


Dietary supplementation with a fish oil concentrate rich in the O3FAs EPA and DHA increases serum levels of these same fatty acids in a dose-response effect. Omega-3 fatty acid supplementation significantly reduces the number of APP-positive axons at 30 days postinjury to levels similar to those in uninjured animals. Omega-3 fatty acids are safe, affordable, and readily available worldwide to potentially reduce the burden of traumatic brain injury.

Abbreviations used in this paper: AA = arachidonic acid; APP = amyloid precursor protein; COX = cyclooxygenase; DHA = docosahexaenoic acid; EPA = eicosapentaenoic acid; LOX = lipoxygenase; NGS = normal goat serum; O3FA = omega-3 fatty acid; PBS = phosphate-buffered saline; Sir2 = silent information 2; TBI = traumatic brain injury; TRAAK = TWIK-related arachidonic acid–stimulated potassium channel; TREK = TWIK-related potassium channel; TWIK = 2-pore weakly inward rectifying potassium channel.

Article Information

Address correspondence to: James D. Mills, M.D., P.O. Box 9183, West Virginia University Health Sciences Center, Department of Neurosurgery, Morgantown, West Virginia 26506. email:

Please include this information when citing this paper: published online July 16, 2010; DOI: 10.3171/2010.5.JNS08914.

© AANS, except where prohibited by US copyright law.



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    Graphs showing that oral supplementation with either 10 mg/kg/day or 40 mg/kg/day of concentrated fish oil for 30 days increased serum total O3FA levels (A) and combined EPA and DHA levels (B). The AA/EPA ratio, a marker of systemic inflammation, was significantly lower in animals receiving fish oil supplementation than in unsupplemented animals (C). The y axis values represent the percentage increase (or decrease) compared to preinjury values.

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    Fluorescent immunohistochemical images of brainstem sagittal sections labeled with APP antibody showing multiple swollen, disconnected axons (arrows) in corticospinal tracts and medial lemnisci in unsupplemented animals subjected to impact acceleration injury 30 days previously (A). Animals receiving either 10 mg/kg/day (B) or 40 mg/kg/day (C) of pharmaceutical-grade fish oil for 30 days postinjury demonstrated very few injured axons, similar to sham-injured animals (D). Bar = 20 μm.

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    Graph demonstrating the density of APP-positive axons in corticospinal tracts and medial lemnisci in sham-injured, unsupplemented, and O3FA-supplemented rats. * p < 0.05, significantly different from the 3 other groups.

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    Graphs demonstrating the density of markers of axonal injury in sham-injured, unsupplemented, and O3FA-supplemented rats 30 days after injury (or sham injury). The density of axons positive for active caspase-3 (A) is significantly reduced in O3FA-supplemented rats as compared with unsupplemented rats, and approaches the level seen in sham-injured rats. The levels of expression of RMO-14 (B), a marker of neurofilament compaction, and cytochrome c (C), a marker of mitochondrial dysfunction, were low levels of expression. * p < 0.05, significantly different from the 3 other groups.

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    Artist's illustration showing the mechanisms by which O3FAs may play a neuroprotective role after TBI.



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