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. 2015 Jan 15;32(2):127-38.
doi: 10.1089/neu.2014.3514. Epub 2014 Nov 13.

Methylene blue attenuates traumatic brain injury-associated neuroinflammation and acute depressive-like behavior in mice

Ashley M Fenn  1 , John P Skendelas, Daniel N Moussa, Megan M Muccigrosso, Phillip G Popovich, Jonathan Lifshitz, Daniel S Eiferman, Jonathan P Godbout
Affiliations

Methylene blue attenuates traumatic brain injury-associated neuroinflammation and acute depressive-like behavior in mice

Ashley M Fenn et al. J Neurotrauma. .

Abstract

Traumatic brain injury (TBI) is associated with cerebral edema, blood brain barrier breakdown, and neuroinflammation that contribute to the degree of injury severity and functional recovery. Unfortunately, there are no effective proactive treatments for limiting immediate or long-term consequences of TBI. Therefore, the objective of this study was to determine the efficacy of methylene blue (MB), an antioxidant agent, in reducing inflammation and behavioral complications associated with a diffuse brain injury. Here we show that immediate MB infusion (intravenous; 15-30 minutes after TBI) reduced cerebral edema, attenuated microglial activation and reduced neuroinflammation, and improved behavioral recovery after midline fluid percussion injury in mice. Specifically, TBI-associated edema and inflammatory gene expression in the hippocampus were significantly reduced by MB at 1 d post injury. Moreover, MB intervention attenuated TBI-induced inflammatory gene expression (interleukin [IL]-1β, tumor necrosis factor α) in enriched microglia/macrophages 1 d post injury. Cell culture experiments with lipopolysaccharide-activated BV2 microglia confirmed that MB treatment directly reduced IL-1β and increased IL-10 messenger ribonucleic acid in microglia. Last, functional recovery and depressive-like behavior were assessed up to one week after TBI. MB intervention did not prevent TBI-induced reductions in body weight or motor coordination 1-7 d post injury. Nonetheless, MB attenuated the development of acute depressive-like behavior at 7 d post injury. Taken together, immediate intervention with MB was effective in reducing neuroinflammation and improving behavioral recovery after diffuse brain injury. Thus, MB intervention may reduce life-threatening complications of TBI, including edema and neuroinflammation, and protect against the development of neuropsychiatric complications.

Keywords: cytokines; fluid percussion injury; intervention; microglia; recovery.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Injury severity and righting reflex prior to control or methylene blue (MB) intervention. (A) Diagram of the experimental study design showing that mice were subjected to a sham injury (Sham) or traumatic brain injury (TBI) at day 0 and immediately (15–30 min later) provided with control of MB interventions. Rotarod acclimation and training were performed prior to injury. Several biochemical and behavioral analyses were performed 1 h – 7 d post-injury. (B) Injury severity was determined by the electromotive force induced by the fluid pulse onto the brain (mV; n=28–29). (C) Time for mice to self-right after sham injury or TBI. Bars represent the mean±standard error of the mean. Means with (*) are significantly different (p<0.05) from Sham-vehicle control (Con).
<b>FIG. 2.</b>
FIG. 2.
Methylene blue (MB) intervention attenuated traumatic brain injury (TBI)-induced edema and neuroinflammation. Adult male (3 months old) BALB/c mice were subjected to sham injury (Sham) or TBI and within 15–30 min received an intravenous injection of vehicle control (Con) or MB (2 mg/kg). The cortex and hippocampus were collected from the brain 1 d post-injury. (A) Relative brain edema was determined in the cortex (n=8–12). In the hippocampus, messenger ribonucleic acid (mRNA) expression of (B) CD14, (C) interleukin (IL)-1β, (D) tumor necrosis factor (TNF)-α, (E) CCL2, and (F) arginase (Arg) were determined (n=4–7). Bars represent the mean±standard error of the mean. Means with (*) are significantly different (p<0.05) from Sham-Con. Means with (#) are significantly different (p<0.05) from TBI-Con.
<b>FIG. 3.</b>
FIG. 3.
Methylene blue (MB) intervention reduced the percentage of myeloid (CD11b+/GR1+) cells in circulation after traumatic brain injury (TBI) but had no effect on the percent of brain-associated macrophages. Adult male (3 months old) BALB/c mice were subjected to sham injury (Sham) or TBI and within 15–30 min received an intravenous injection of vehicle control (Con) or MB (2mg/kg). At 1 d post-injury, brain and blood were collected for flow cytometry analysis. (A) Representative bivariate dot plots of CD11b/GR1 labeling of blood leukocytes. (B) Percent CD11b+/GR1+ myeloid cells in circulation (n=8–12). (C) Representative bivariate dot plots of CD11b/CD45 labeling of brain-associated macrophages. (D) Percent CD11b+/CD45hi macrophages associated with the brain (n=8–12). (E) Representative bivariate dot plots from Sham and TBI of CD11b/Ly6C labeling of brain-associated macrophages. (F) Total relative number of Ly6Chi macrophages associated with the brain (out of 10,000 live cells; n=8–12). Bars represent the mean±standard error of the mean. Means with (*) are significantly different (p<0.05) than Sham-Con.
<b>FIG. 4.</b>
FIG. 4.
Methylene blue (MB) reduced IL-1β and enhanced IL-10 expression in microglia/macrophages after traumatic brain injury (TBI). Adult male (3 months old) BALB/c mice were subjected to sham injury (Sham) or TBI and within 15–30 min received an intravenous injection of vehicle control (Con) or MB (2 mg/kg). At 1 d post-injury, enriched brain CD11b+ cells (microglia/macrophages) were isolated from whole brain homogenates. The levels of messenger ribonucleic acid (mRNA) expression for (A) CD14, (B) interleukin (IL)-1β, (C) inducible nitric oxide synthase (iNOS), (D) arginase (Arg), and (E) IL-10 were determined (n=7–10). Bars represent the mean±standard error of the means. Means with (*) are significantly different (p<0.05) from Sham-Con. Means with (+) tend to be different (p=0.1) from Sham-Con.
<b>FIG. 5.</b>
FIG. 5.
Methylene blue (MB) intervention attenuated acute depressive-like behavior 7 d post-injury. Adult male (3 months old) BALB/c mice were subjected to sham injury (Sham) or traumatic brain injury (TBI) and within 15–30 min received an intravenous injection of vehicle control (Con) or MB (2 mg/kg). (A) Body weight was assessed every day for 7 d post-injury (n=9–14). (B) Motor coordination was determined 3 d before injury and again at 1 h, and 1, 4, and 7 d post-injury (n=4–8). (C) At 7 d post-injury, depressive-like behavior was determined by tail suspension test (TST). Total average time spent immobile is shown for all groups (n=9–14). (D) Total time spent immobile was plotted versus the percentage of mice that were immobile. The vertical line at 270 sec denotes that less than 10% of sham mice were immobile for longer than 270 sec. (E) Percentage of mice from each group that were immobile for longer than 270 sec (4.5 min) in the TST. Points and bars represent the mean±standard error of the mean. Means with (*) are significantly different (p<0.05) than Sham-Con. Means with (#) are significantly different (p<0.05) from TBI-Con. Means with (+) tend to be different (p=0.1) from Sham-Con.
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