Ls and were approved by the University of California at Los Angeles (UCLA) Chancellor’s Animal Research Committee (ARC).Experimental DesignTwo-day pregnant female Sprague-Dawley rats weighing between 280 and 300 g were obtained from Charles River Laboratories, Inc. (Portage, MI, USA). The animals were housed with 12-h light/dark cycles and a maintained temperature of 22?24uC. Animals were randomly divided into two MedChemExpress HA15 dietary groups:Effects of Diet and Brain Trauma in Spinal CordTable 1. Fat composition of the experimental diets (g/100 g diet).Fat sources Hydrogenated coconut oil Safflower oil Flaxseed oil DHA EPA Other n-3s doi:10.1371/journal.pone.0052998.tn-3 adq 7.45 1.77 0.48 1.2 0.24 0.n-3 def 8.1 1.9 0 0 0omega 3 fatty acids adequate (n-3 adq) diet vs omega 3 fatty acids deficient (n-3 def) diet. The dietary treatment started with the mothers and the male offspring were weaned to the same diet as their dam. After 14 weeks, the animals were subjected to moderate FPI, and continued their diet until sacrificed one week later.Diet CompositionThe two custom diets (n-3 def, n-3 adq) were prepared commercially (Dyets, Bethlehem, PA, USA) and contained the same basal macronutrients, vitamins, minerals, and basal fats (hydrogenated coconut and safflower oils). Vitamin-free casein Alacid 710 (NZMP North America Inc., CA, USA) was included. The n-3 adq diet contained an extra 0.5 flaxseed oil (linoleic acid), 1.2 DHA and 0.24 EPA (Nordic Naturals, Inc. Watsonville, CA, USA), relative to the n-3 def diet. (Table 1). The individual’s daily intake of DHA was about 480 mg per kilogram of animal weight.western blots as previously described [10]. Tissue was first homogenized in a lysis buffer and the total protein was separated by electrophoresis on a polyacrylamide gel and electrotransferred onto a PVDF (nitrocellulose for BDNF) membrane (Millipore, Bedford,MA). After blocking, the membranes were rinsed with TBS-T and incubated with the primary antibody for actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), BDNF (1:300, Santa Cruz Biotechonology, Santa Cruz, CA, USA), pTrkB (1:200, BD Biosciences, Sparks, MD, USA), TrkB (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), pCREB (1:1,000, Millipore, Bedford, MA, USA), CREB (1:200, Millipore, 24195657 Bedford, MA, USA), syntaxin-3 (1:300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), iPLA-2 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), or 4-HNE (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA ). Immunocomplexes were visualized by chemiluminescence using the commercial kit ECL plus (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). Respective protein sizes were compared to the Benchmark pre-stained protein ladder (Invitrogen Technology, Carlsbad, CA, USA). Protein bands were digitally scanned and quantified using the ImageJ software. Actin was used as an internal control. The phosphorylated proteins were normalized to their respective non-phosphorylated partners.Fatty Acids Analysis by Gas ChromatographyThe lipids content of the MedChemExpress ICG-001 cervical SC were extracted and analyzed by gas chromatography. Lipids were first extracted by homogenizing tissue on ice in a 2:1 (vol:vol) chloroform:methanol solution with 50 ug/mL of butylated hydroxytoluene added to prevent lipid oxidation. Tricosanoic acid methylester (C23:0) was added to each sample to function as an internal standard. After extraction, lipids were methylated by heating at 90uC for 1 hour in14 (w/v) boron trifluoride-methanol reage.Ls and were approved by the University of California at Los Angeles (UCLA) Chancellor’s Animal Research Committee (ARC).Experimental DesignTwo-day pregnant female Sprague-Dawley rats weighing between 280 and 300 g were obtained from Charles River Laboratories, Inc. (Portage, MI, USA). The animals were housed with 12-h light/dark cycles and a maintained temperature of 22?24uC. Animals were randomly divided into two dietary groups:Effects of Diet and Brain Trauma in Spinal CordTable 1. Fat composition of the experimental diets (g/100 g diet).Fat sources Hydrogenated coconut oil Safflower oil Flaxseed oil DHA EPA Other n-3s doi:10.1371/journal.pone.0052998.tn-3 adq 7.45 1.77 0.48 1.2 0.24 0.n-3 def 8.1 1.9 0 0 0omega 3 fatty acids adequate (n-3 adq) diet vs omega 3 fatty acids deficient (n-3 def) diet. The dietary treatment started with the mothers and the male offspring were weaned to the same diet as their dam. After 14 weeks, the animals were subjected to moderate FPI, and continued their diet until sacrificed one week later.Diet CompositionThe two custom diets (n-3 def, n-3 adq) were prepared commercially (Dyets, Bethlehem, PA, USA) and contained the same basal macronutrients, vitamins, minerals, and basal fats (hydrogenated coconut and safflower oils). Vitamin-free casein Alacid 710 (NZMP North America Inc., CA, USA) was included. The n-3 adq diet contained an extra 0.5 flaxseed oil (linoleic acid), 1.2 DHA and 0.24 EPA (Nordic Naturals, Inc. Watsonville, CA, USA), relative to the n-3 def diet. (Table 1). The individual’s daily intake of DHA was about 480 mg per kilogram of animal weight.western blots as previously described [10]. Tissue was first homogenized in a lysis buffer and the total protein was separated by electrophoresis on a polyacrylamide gel and electrotransferred onto a PVDF (nitrocellulose for BDNF) membrane (Millipore, Bedford,MA). After blocking, the membranes were rinsed with TBS-T and incubated with the primary antibody for actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), BDNF (1:300, Santa Cruz Biotechonology, Santa Cruz, CA, USA), pTrkB (1:200, BD Biosciences, Sparks, MD, USA), TrkB (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), pCREB (1:1,000, Millipore, Bedford, MA, USA), CREB (1:200, Millipore, 24195657 Bedford, MA, USA), syntaxin-3 (1:300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), iPLA-2 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), or 4-HNE (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA ). Immunocomplexes were visualized by chemiluminescence using the commercial kit ECL plus (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). Respective protein sizes were compared to the Benchmark pre-stained protein ladder (Invitrogen Technology, Carlsbad, CA, USA). Protein bands were digitally scanned and quantified using the ImageJ software. Actin was used as an internal control. The phosphorylated proteins were normalized to their respective non-phosphorylated partners.Fatty Acids Analysis by Gas ChromatographyThe lipids content of the cervical SC were extracted and analyzed by gas chromatography. Lipids were first extracted by homogenizing tissue on ice in a 2:1 (vol:vol) chloroform:methanol solution with 50 ug/mL of butylated hydroxytoluene added to prevent lipid oxidation. Tricosanoic acid methylester (C23:0) was added to each sample to function as an internal standard. After extraction, lipids were methylated by heating at 90uC for 1 hour in14 (w/v) boron trifluoride-methanol reage.
