Administration of DHA Reduces Endoplasmic Reticulum Stress-Associated Inflammation and Alters Microglial or Macrophage Activation in Traumatic Brain Injury - PubMed (original) (raw)

Administration of DHA Reduces Endoplasmic Reticulum Stress-Associated Inflammation and Alters Microglial or Macrophage Activation in Traumatic Brain Injury

Lloyd D Harvey et al. ASN Neuro. 2015.

Abstract

We investigated the effects of the administration of docosahexaenoic acid (DHA) post-traumatic brain injury (TBI) on reducing neuroinflammation. TBI was induced by cortical contusion injury in Sprague Dawley rats. Either DHA (16 mg/kg in dimethyl sulfoxide) or vehicle dimethyl sulfoxide (1 ml/kg) was administered intraperitonially at 5 min after TBI, followed by a daily dose for 3 to 21 days. TBI triggered activation of microglia or macrophages, detected by an increase of Iba1 positively stained microglia or macrophages in peri-lesion cortical tissues at 3, 7, and 21 days post-TBI. The inflammatory response was further characterized by expression of the proinflammatory marker CD16/32 and the anti-inflammatory marker CD206 in Iba1(+) microglia or macrophages. DHA-treated brains showed significantly fewer CD16/32(+) microglia or macrophages, but an increased CD206(+) phagocytic microglial or macrophage population. Additionally, DHA treatment revealed a shift in microglial or macrophage morphology from the activated, amoeboid-like state into the more permissive, surveillant state. Furthermore, activated Iba1(+) microglial or macrophages were associated with neurons expressing the endoplasmic reticulum (ER) stress marker CHOP at 3 days post-TBI, and the administration of DHA post-TBI concurrently reduced ER stress and the associated activation of Iba1(+) microglial or macrophages. There was a decrease in nuclear translocation of activated nuclear factor kappa-light-chain-enhancer of activated B cells protein at 3 days in DHA-treated tissue and reduced neuronal degeneration in DHA-treated brains at 3, 7, and 21 days after TBI. In summary, our study demonstrated that TBI mediated inflammatory responses are associated with increased neuronal ER stress and subsequent activation of microglia or macrophages. DHA administration reduced neuronal ER stress and subsequent association with microglial or macrophage polarization after TBI, demonstrating its therapeutic potential to ameliorate TBI-induced cellular pathology.

Keywords: cortical contusion injury; docosahexaenoic acid; microglial polarization; neuroinflammation; nuclear factor kappa-light-chain-enhancer of activated B cells; secondary injury.

© The Author(s) 2015.

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Figures

Figure 1.

Figure 1.

Iba1+ microglial or macrophage population was not reduced in the DHA-treated TBI brains. (a) Experimental protocol and location of data collection. (b) Confocal images of Iba1+ microglial or macrophage in the IL and CL frontal cortices at 3, 7, and 21 days post-TBI. (c) Summary data of Iba1+ cells/TO-PRO-3+ cells. Values are mean ± SE (n = 4). (d) Summary data of CD16/32+/Iba1+ immunopositive cells. Values are mean ± SE (n = 4). *p < .05, **p < .01, ***p < .001, N/D = no data, one-way ANOVA tests used.

Figure 2.

Figure 2.

DHA treatment increased microglial ramification post-TBI. (a) Confocal Z-scan intensified images show Iba1+ microglia or macrophages in the IL frontal cortices at 3 days post-TBI. Binary images clarify the boundaries of microglial or macrophage cell morphology. Skeletonized images show the endpoints and length of each microglial or macrophage cell as individual pixels. Arrow: Ramified or surveillant microglia/macrophage. Arrowhead: Activated microglia or macrophage. Double Arrowhead: Microglia or macrophage reverting from hypertrophic and retracted processes morphology into the ramified or surveillant state. (b) Summary data of summed endpoints and summed average length of skeletonized images of Iba1+ microglia or macrophages. Values are mean ± SE (n = 4). *p < .05, **p < .01, ***p < .001, one-way ANOVA test used.

Figure 3.

Figure 3.

DHA treatment increases CD206+ microglial or macrophage population in post-TBI brains. (a) Confocal images show Iba1+ and CD206+ microglia or macrophages in the IL frontal cortices at 3, 7, and 21 days post-TBI. Confocal images of CD206+ microglia or macrophages were binaried for clarity. Arrowhead: colocalization of Iba1+ and CD206+ immunostaining (red and green). Arrow: increased CD206+ expression in microglia or macrophages in the DHA-treated TBI tissues. (b) Summary data of CD206+/Iba1+ immunopositive cells. Values are mean ± SE (n = 4). *p < .05, **p < .01, ***p < .001, N/D = no data, one-way ANOVA test used.

Figure 4.

Figure 4.

DHA reduces TBI-mediated ER stress and microglial or macrophage activation. (a) Confocal images show expression of ER stress marker protein CHOP and microglial or macrophage marker Iba1 in the IL frontal cortex at 3 and 7 days post-TBI. Arrow: Increased expression of CHOP in IL frontal cortex tissue. Arrowhead: Association of CHOP+ neurons with Iba1+ microglia or macrophages. Double Arrowhead: Vacuoles in phagocytizing microglia or macrophages. Yellow Box: Iba1+ microglia or macrophage in close contact with CHOP+ neurons. Outlines of microglial or macrophage morphology demonstrate a reramification with DHA treatment at 7 days post-TBI. (b) DHA treatment significantly reduced the expression of CHOP in neurons (n = 5) and reduced the number of CHOP+/Iba1+associations (n = 5) at 3 days post-TBI. Values are mean ± SE *p < .05, paired _t_-test used. (c) Z-sectioning images demonstrating interactions between CHOP+ neurons (green) with Iba1+ microglia or macrophages (red). Arrow: colocalization.

Figure 5.

Figure 5.

DHA-treated rats exhibit reduced NF-κB translocation to the nucleus after TBI. (a) Representative immunoblots of NF-κB nuclear and cytosolic expression in the CL and IL frontal cortices of TBI and TBI + DHA animals at 3 days and 7 days post-TBI. The same blot was probed with either antibody against nuclear TATA binding protein (TBP) or against β-actin as a loading control. (b) Summary data expressed as NF-κB expression in nuclear and cytosolic fractions (IL/CL). Values are mean ± SE (sham, n = 3; 3 days post-TBI or TBI + DHA, n = 6; 7 days post-TBI or TBI + DHA, n = 3). Paired _t_-test used. (c) Analysis of TNF-α and IL-1β in the CL and IL cortical tissues with ELISA. Values are mean ± SE (n = 5–6). *p < .05, ***p < .001, paired _t_-test used.

Figure 6.

Figure 6.

Positive correlation between reduced neuronal damage and ER stress in the DHA-treated brains. (a) Microscopic fluorescent images of sham, TBI, and TBI + DHA brains stained with FJ-C. Arrow: site of surgery. (b) Representative confocal images show FJ-C stained neurons in the IL frontal cortex peri-lesion at 3 to 21 days post-TBI. Inset: CL frontal cortex. Arrowhead: FJ-C positive degenerating neurons. Inset shows CL frontal cortex tissue at the same location. (c) Summary data of FJ-C positive cells per mm2. Values are mean ± SE (n = 4). (d) Correlation analysis of CHOP+ cells versus FJ-C+ cells (R = .588, p < .05, n = 14). *p < .05, one-way ANOVA test used.

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