Methylene blue is neuroprotective against mild traumatic brain injury

Abstract

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Methylene blue (MB) has known energy-enhancing and antioxidant properties. This study tested the hypothesis that MB treatment reduces lesion volume and behavioral deficits in a rat model of mild TBI. In a randomized double-blinded design, animals received either MB (n=5) or vehicle (n=6) after TBI. Studies were performed on 0, 1, 2, 7, and 14 days following an impact to the primary forelimb somatosensory cortex. MRI lesion was not apparent 1 h after TBI, became apparent 3 h after TBI, and peaked at 2 days for both groups. The MB-treated animals showed significantly smaller MRI lesion volume than the vehicle-treated animals at all time points studied. The MB-treated animals exhibited significantly improved scores on forelimb placement asymmetry and foot fault tests than did the vehicle-treated animals at all time points studied. Smaller numbers of dark-stained Nissl cells and Fluoro-Jade(®) positive cells were observed in the MB-treated group than in vehicle-treated animals 14 days post-TBI. In conclusion, MB treatment minimized lesion volume, behavioral deficits, and neuronal degeneration following mild TBI. MB is already approved by the United States Food and Drug Administration (FDA) to treat a number of indications, likely expediting future clinical trials in TBI.

Keywords: MRI; antioxidant; mitochondria; oxidative stress; vasogenic edema.

Figures

FIG. 1.

T2 MRI. (A) Representative T2 maps are shown for vehicle- and methylene blue (MB)-treated animals at 3 h, and 1, 2, 7, and 14 days post-traumatic brain injury (TBI). The arrows indicate hyperintense lesions in the ipsilesional cortex. The white boxes in the second panel indicate the enlarged image of the lesion area demonstrated to the right of each panel to further demonstrate the evolution of the lesion with time. (B) Bar graph shows the progression of lesion volumes in MB-treated animals compared with vehicle-treated animals (mean±SEM, n=6 per group,*p<0.05).

FIG. 2.

Line graphs of the (A) cylinder and (B) foot fault tests for the vehicle- and methylene blue (MB)-treated animals at pre-traumatic brain injury (TBI), and at 1, 2, 7, and 14 days post-TBI (mean±SEM, n=6 each group, *p<0.05, **p<0.01 between vehicle- and MB-treated groups).

FIG. 3.

Multiple regression correlation plots are demonstrated for T2 MRI lesion volume versus (A) asymmetry and (B) foot fault, with different symbols indicating data from different days post TBI.

FIG. 4.

Nissl staining 14 days post-traumatic brain injury (TBI). (A) Mosaic images of the vehicle and methylene blue (MB)-treated animals. The red box indicates the location of the zoom-in images. (B) Magnified images of a vehicle- and an MB-treated TBI animal obtained using a 60×objective. The red arrows indicate typical dark-stained neurons. (C) Bar graph of the number of dark-stained Nissl bodies for vehicle- and MB-treated animals (mean±SEM, n=6 each group, *p<0.01, ##p<0.01). Color image is available online at www.liebertpub.com/neu

FIG. 5.

Fluoro-Jade® B staining 14 days post-traumatic brain injury (TBI). (A) Representative images of vehicle- and methylene blue (MB)-treated animals at 60×magnification. (B) Bar graph of the number of Fluoro-Jade B positive cells per field for vehicle- and MB-treated animals (mean±SEM, n=6 each group, *p<0.01, ##p<0.05). Color image is available online at www.liebertpub.com/neu

FIG. 6.

Multiple regression correlation plots are demonstrated for T2 MRI lesion volume versus (A) Nissl staining and (B) Fluoro-Jade® B staining analysis at day 14 post-traumatic brain injury (TBI).