Oral Supplementation of n-3 Polyunsaturated Fatty Acids (n-3-PUFA) Can Prevent TBI-Induced Visual, Motor, and Emotional Deficits in Mice

Abstract

Traumatic brain injury (TBI) causes neuroinflammation and can generate long-term pathological consequences, including motor and visual impairments, cognitive deficits, and depression. In our previous study, we found that Fat1⁺-transgenic mice with higher endogenous n-3 polyunsaturated fatty acids (n-3 PUFA) were protected from post-TBI behavioral deficits and exhibited reduced levels of TBI-induced microglial activation, inflammatory factors, and sphingolipid ceramide, a lipid mediator of inflammation and cell death. This study's objective was to evaluate if feeding n-3 PUFA (EPA and docosahexaenoic acid, DHA 2:1) could restrict the elevation of ceramide in brain tissue and prevent TBI-mediated sensory-motor and behavioral deficits. Wildtype C57/BL6 mice were gavage pre-fed with PUFA (EPA: DHA = 2:1) at 500 mg/kg body weight/week for 2 weeks before and 4 weeks after exposure to left side focal cranial air-blast (50 psi) TBI or sham-blast (0-psi). Saline-gavaged mice served as controls. Following blast injury, various motor, visual, and behavioral tests were conducted, and brain tissues were collected for histological and biochemical assays. Lipidomics analysis confirmed a significant elevation of EPA in the plasma and brain tissue of PUFA-fed mice. TBI-Blast brain tissues were found to have elevated ceramide levels in control mice but not in PUFA-fed mice. Moreover, PUFA-fed mice demonstrated protection against motor impairment, photoreceptor dysfunction, depression, oculomotor nerve degeneration, and microglia activation in the optic tract. Our results demonstrate that EPA-mediated suppression of ceramide biosynthesis and neuroinflammatory factors in PUFA-fed mice is associated with significant protection against the visual, motor, and emotional deficits caused by TBI.

Keywords: Ceramide; Emotional deficits; N-3 PUFA; Neuroinflammation; Traumatic brain injury (TBI); Visual deficits.

Fig. 1

Schematic diagram of the experimental timeline, study groups, treatments, observations and tests, and tissue harvest

Fig. 2

Analysis of different Polyunsaturated fatty acids (PUFAs) in the tissues of saline-fed (Saline) and PUFA-fed (PUFA) mice. Relative mole percentages of PUFA [20:5n3, Eicosapentaenoic acid (EPA); 22:6n3, Docosahexaenoic acid (DHA); 20:4n6 Arachidonic acid (AA)] in the Plasma (A) and brain (B) of saline and PUFA- fed mice. Data are presented as Mean ± SEM, and levels of significance are shown as P values (n = 5; *p < 0.05, t-test)

Fig. 3

Retinal function assay by Electroretinogram of saline-fed (Saline) and PUFA-fed (PUFA) mice treated with 0-psi (sham) or 50-psi (blast) TBI. Pre-blast a-wave and b-wave amplitude values served as reference for each group. Data are presented as percentages value of pre-blast in a-wave (A) and b-wave (B) (n = 4 Saline-Sham, 5 Saline-Blast, 4 PUFA-Sham, 4 PUFA-Blast (*p < 0.05 (* indicates Saline Sham vs Saline Blast); ^#p < 0.05, ^##p < 0.01 (.^#,## indicates PUFA Blast vs Saline Blast; ANOVA; t-test)

Fig. 4

Analysis of open field behavior, motor coordination, and depression in saline-fed (Saline) and PUFA-fed (PUFA) mice with (50-psi, blast) or without (0-psi, sham) mild TBI. Mice were divided into four groups, Saline-Sham, Saline-Blast, PUFA-Sham, and PUFA-Blast, each containing 6–8 mice. Open-field motor testing was conducted, and data are represented as percentages values of fold-over control (Saline-Sham) (A). The Tail suspension testing was conducted, and data are presented as line graphs of cumulative immobility per minute over the 5 min test, in which more immobility reflects more depression-like behavior (B–C). The Rotarod Motor Test was conducted, and the data are presented as a line graph representing the time to fall from the rotarod counted as seconds in the trial and test (D–F). Data are presented as Mean ± SEM, and levels of significance are shown as p (*) values [*p < 0.05; t-test, Chi-squared test]

Fig. 5

Immunohistochemical analysis of oculomotor neurons with choline acetyltransferase (ChAT) immunostaining at midbrain level in sections from saline-fed (Saline) and PUFA-fed (PUFA) mice with and without mild TBI. Representative images of ChAT-immunolabeling of the oculomotor nucleus of Saline-Sham, Saline-Blast, PUFA-Sham, and PUFA-Blast littermates (A–D). The bar diagram represents the number of perikarya at the oculomotor nucleus in the brain sections of Saline-Sham, Saline-Blast, PUFA-Sham, and PUFA-Blast mice (E). Perikarya are represented by the dark-stained nuclei, and neuronal loss was estimated by counting these nuclei. Data are presented as Mean ± SEM (n = 5–7), and levels of significance are shown as P values (*p < 0.05, t-test)

Fig. 6

Analysis of microglial activation in the optic tract by immunostaining for IBA1 from saline-fed (Saline) and PUFA-fed (PUFA) mice with and without mild TBI. The histogram shows the number of active microglia (A) and density (microglia/µm.²) (B) at the right side of the optic tract in the brain section. Data are presented as Mean ± SEM, and levels of significance are shown as P values (n = 5–6 each for Saline-Sham, Saline-Blast, PUFA-Sham, PUFA-Blast; *p < 0.05, **p < 0.01, t-test)

Fig. 7

Enzyme activity of sphingomyelinase (SMase) (fluorescence unit/ug of protein) in the brain tissues of saline-fed (SF) and PUFA-fed (PUFA) mice treated with 0-psi (sham) or 50-psi (blast) one month after TBI. No significant changes were noted in acidic SMase (aSMase) activity across groups (A). A significant increase in neutral SMase (nSMase) activity was noticed in Saline-Blast mice compared to their sham counterpart (B). Data are presented as Mean ± SEM, and levels of significance are shownas P values (n = 5 each for Saline-Sham, Saline-Blast, PUFA-Sham, PUFA-Blast; *p < 0.05, t-test)

Fig. 8

Analysis of Sphingolipid profile in the brain tissues of Saline-fed (Saline) and PUFA-fed (PUFA) mice treated with 0-psi (sham) or 50-psi (blast) 1 month after TBI. Histogram represents the level (pmol/mg of protein) of Ceramide (Cer), Hexosyl-Ceramide (HexCer), and Sphingomyelin (SM) in the brain tissues of Saline-Sham, Saline-Blast, PUFA-Sham, PUFA-Blast mice (A). An increased level of C18 and C24 Cer species was noticed in the brain tissues of Saline-Blast mice (B). An increased level of C18 HexCer species was noticed in the brain tissues of Saline-Blast mice (C). An increased level of C18 SM species was noticed in the brain tissues of Saline-Blast mice (D). Data are presented as Mean ± SEM and levels of significance are shown as P values (n = 4 Saline-Sham, 5 Saline-Blast, 4 PUFA-Sham, 4 PUFA-Blast; *p < 0.05, t-test)