1. Post Injury Omega-3 Fatty Acid Emulsion Delivery as a Means to Regulate
Inflammation from bTBI, as Measured in Rat Livers
Danyas Sarathy1, James DeMar1, Daniel Thadeio1, Joseph Long1
1Blast-Induced Neurotrauma Branch, Center for Military Psychiatry and Neuroscience Research, Walter Reed Army Institute of Research,
503 Robert Grant Avenue, Silver Spring, MD 20910.
DISCLAIMER: The views of the authors do not purport or reflect the position of the Department of the Army or the Department of Defense (para 4-3, AR 360-5). All research was conducted in compliance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals
(National Research Council), and other federal statutes and regulations relating to animals and experiments involving animals. Animals were maintained in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
Abstract
Background
Methods
Results Conclusions
Acknowledgments
References
0
5
10
15
20
25
1 2 3 4 5 6 7 8
%ARA
Treatment Group
Relative Arachidonic Acid Abundance
0
0.5
1
1.5
2
2.5
3
3.5
4
1 2 3 4 5 6 7 8
%DHA
Treatment Group
Relative Docosahexaenoic Acid Abundance
0
2
4
6
8
10
12
1 2 3 4 5 6 7 8
ARA/DHA
Treatment Group
ARA to DHA ratio
0
2
4
6
8
10
12
14
16
18
20
1 2 3 4 5 6 7 8
Omega-6/Omega-3
Treatment Group
Total Omega-6 to Omega-3 ratio
Group Number Treatment
No treatment,
house chow
No treatment,
American diet
No injury,
NaCl at 120 uL/hr
Injury,
NaCl at 120 uL/hr
Injury, Intralipid at
1.25 g/kg/day
Injury, LipOmega-3
20% at 0.25
g/kg/day
Injury, LipOmega-3
20% at 1.25
g/kg/day
Injury, LipOmega-3
20% at 2.50
g/kg/day
Fig. 1: Relative percentage of Arachidonic Acid (ARA) in Rat liver
samples across all treatment groups
Fig. 2: Relative percentage of Docosahexaenoic (DHA) in Rat liver
samples across all treatment groups
Fig. 3: Ratio of ARA (main Omega-6 contributor) to DHA (main Omega-3
contributor)
Fig. 4: Ratio of all Omega-6 fatty acids to all Omega-3 fatty acids
Student’s t-test indicates significant relationships between these treatment groups:
• House chow livers (group 1) contain significantly more DHA and ARA than American diet
livers (group 2)
• Non-treated American diet livers (group 2) had significantly lower Omega-6/Omega-3 ratio
than injured rat livers, treated with Sodium Chloride (group 4)
• Blasted rat livers treated with LipOmega-3 at 1.25, and 2.5 g/kg/day (groups 7, 8) had
significantly lower ratios (both ARA/DHA and Omega-6/Omega-3) than the control (group 5)
• Non-treated American diet livers (group 2) had significantly lower ratios (ARA/DHA and
Omega-6/Omega-3) than non-injured, sodium chloride treated livers (group 3)
Fatty Acid
Trivial
Name %
C14:0 1.51
C16:0 Palmitic 22.36
C16:1w-9 3.22
C18:0 Stearic 6.13
C18:1w-9 Oleic 28.24
C18:1w-7 4.35
C18:2w-6 LA 23.13
C18:3w-6 GLA 0.48
C18:3w-3 ALA 1.11
C20:0 0.48
20:1n-9 0.69
C20:3w-6 DHGLA 0.43
C20:4w-6 ARA 6.15
C20:5w-3 EPA 0.16
C22:0 0.19
C22:4w-6 0.24
C22:5w-6 DPAn-6 0.01
C22:5w-3 DPA 0.22
C24:0 0.11
C22:6w-3 DHA 0.59
C24:1w-9 0.09
Table 1: Representative
fatty acid table
Blast induced traumatic brain injuries (bTBIs) are one of the most prevalent dangers for the US military
service member. Multiple complications—blast over pressure wave effects and shrapnel dispersion, and
more—make bTBIs range in severity from mild to life threatening events. bTBIs are of potential harm to
brain tissue, which when compromised can be difficult to treat in the theatre of war. Thus, our lab’s main
interest is to study--in animal models--the impact of blast wave exposure on inducing TBI, and to look for
therapeutic counter measures. Recent studies have shown that supplementation of long chain
polyunsaturated fatty acids (PUFAs)—in particular, Omega-3 PUFAs—support membrane structure in
neuronal tissues and produce metabolites that suppress inflammation processes, helping to heal these
injuries. It is well known that an imbalance of Omega-6 relative to Omega-3 PUFAs in tissue is a
biomarker for inflammation. As the typical “American” diet is rich in Omega-6 PUFAs (these FAs are
shown to be pro-inflammatory) and deficient in Omega-3 PUFAs, this study seeks to assess the impact of
Omega-3 supplementation—docosahexaenoic acid (DHA), in particular—in post-blast injury rats. Adult
male rats that had been maintained on a house chow or “American” diet for one month were anesthetized
with isoflurane, and exposed to a linear over pressure wave by use of the WRAIR’s compressed air driven
shock tube. After the blast injury, rats were intravenously infused for 5 days with varying levels of
“LipOmega-3,” a proprietary emulsion that is synthetically enriched in DHA (B. Braun Melsungen AG);
control rats received saline vehicle or Intralipid mock emulsion. Rats were sacrificed 2 weeks post blast,
with liver tissue harvested. Liver tissue was homogenized and a lipid extraction was performed with
chloroform. An esterification reaction was used to functionalize the lipids into fatty acid methyl esters, for
separation by GC/MS. While the results of the study are ongoing, preliminary lipid profiles with a
minimum of five rats per treatment group was assessed by virtue of paired student t-test. The results
suggest that the highest doses of LipOmega-3, which had significantly higher DHA and lower Omega-
6/Omega-3 ratios, may be able to reduce inflammation post-blast injury. Further analysis will be done with
larger sample sizes, and use of ANOVA tests for interpretation
Blast induced traumatic brain injuries (bTBI) are ever prevalent in the theatre of war. Their long-
standing effects on service personnel encompass the entire body—from the brain to the lungs bTBIs ,
if not lethal, can potentially compromise the service member for life.
Research has shown that diet may regulate inflammation in the body. A typical “American” diet,
lacking in omega-3 polyunsaturated fatty acids (PUFAs), leaves the body susceptible to inflammation.
Given the prevalence of PUFAs in brain tissue—these come in the forms of omega-3, and omega-6
FAs—PUFAs may play a role in regulating compromised tissue. Of the Omega-3 PUFAs,
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are not inflammatory like their Omega-
6 complements—arachidonic acid (ARA), for example. Thus, the ratio between Omega-6 to Omega-3
FAs provides information on the regulation of inflammation in the body; a ratio that favors Omega-6
FAs (ARA > DHA) suggest that the body is responding poorly to inflammation.
Given the prevalence of PUFAs in the body—with omega-3’s as building blocks of the gray matter in
the brain and their ability to generate inflammation suppressing metabolites—supplementation that
favors Omega-3 FAs may help restore structure to compromised tissue, and provide resolution to
inflammation.
Table 2: Rat treatment groups
• Sprague-Dawley rats, weighing ~300g ea. were housed and fed in a
standardized (AALAC approved) facility.
• Rats were subject to eight different treatment groups, listed in Table 2.
• Post injury (by use of blast overpressure waves), rats were returned to their
cages for 2 weeks, before being sacrificed under anesthetic (isoflurane). Liver
tissues were harvested post mortem.
• Approximately 100mg of liver tissue was used per rat for lipid profile analysis.
• Liver tissue was homogenized by use of a homogenizer, and suspended in
chloroform to isolate lipids. After washes with methanol and potassium
chloride, the chloroform was evaporated with the use of a nitrogen drying block
• Lipid samples were suspended in Toluene until methylation
• An esterification reaction was performed on the toluene suspension by adding a
14% wt/vol concentrated BF3 in Methanol solution, at 100o C for one hour
• After completion of the reaction, hexanes were used to extract the now fatty
acid methyl esters (FAMEs), and were washed twice with water and methanol.
• FAME samples in hexanes were used in a Gas Chromatography/Mass
Spectrometry (GC/MS) to calculate relative lipid percentages by virtue of the
integration values returned by the machine.
• Retention times were calibrated by individually running FAME standards
through the GC/MS.
Danyas Sarathy is indebted to his mentors, Dr. James DeMar and Dr. Joe Long for their kind support
and encouragement throughout a very productive and enriching summer. Dan Thadeio’s data was
also integral to production of this poster. The authors would also like to thank B. Braun Melsungen
AG. Their contribution to the study was development and supply of the LipOmega-3 emulsion, as
well as providing us with research funding and equipment through various CRADA awards. Finally,
thanks goes to the CQL program, for making state-of-the-art research available to students.
• Given that house chow fed rat livers contain more DHA than
American diet livers, we confirm that the American diet is pro-
inflammatory (lack of Omega-3 PUFAs)
• Since livers from non-blasted rats (American diet fed) had a
significantly lower Omega-6/Omega-3 ratios than blasted rats
on the same diet treated with the drug vehicle alone (saline
and/or intralipid), it suggests that the blast exposure causes
systemic inflammation.
• Non treated American diet livers had lower ratios of
ARA/DHA and Omega-6/Omega-3 than in non-injured rats
given vehicle. This suggests that the surgical procedure used
to connect the drug infusion pumps, via jugular catheters, to
the rats may be adding to the inflammatory response of
blasted animals.
• Livers from blasted rat treated with the two highest doses of
LipOmega-3 (i.e., 1.25 and 2.50 g/kg/day) had significantly
lower ARA/DHA and Omega-6/Omega-3 ratios than non-
treated blasted rats. This suggests that intravenous therapy
with Omega-3 fatty acids (LipOmega-3 emulsion) may be able
to reduce inflammation post injury.
• Lack of a significant difference between the ARA/DHA ratios
for the two highest doses of LipOmega-3 suggests that we are
near the peak effective dose to reduce inflammation
following blast-induced injury.
Conclusions based on significance from student’s t-test
(p<0.05)
Animals
Lipid
Extraction
Methylation
and GC/MS
Profile
Oliver, T., Demar, J., Riccio, C., Edwards, A., Albert, S. V., Hill, M., . . . Long, J. (n.d.).
Efficacious Treatment for TBI: Omega-3 Fatty Acid Emulsion Delivered as a Post-Injury
Intravenous Infusion (pp. 1-22, Rep.).