Millions of mild traumatic brain injuries (TBIs) occur every year in the United States, with many people subject to multiple head injuries that can lead to chronic behavioral dysfunction. We previously reported that mild TBI induced using closed head injuries (CHI) repeated at 24h intervals produced more acute neuron death and glial reactivity than a single CHI, and increasing the length of time between injuries to 48h reduced the cumulative acute effects of repeated CHI. To determine whether repeated CHI is associated with behavioral dysfunction or persistent cellular damage, mice receiving either five CHI at 24h intervals, five CHI at 48h intervals, or five sham injuries at 24h intervals were evaluated across a 10 week period after injury. Animals with repeated CHI exhibited motor coordination and memory deficits, but not gait abnormalities when compared to sham animals. At 10wks post-injury, no notable neuron loss or glial reactivity was observed in the cortex, hippocampus, or corpus callosum. Argyrophilic axons were found in the pyramidal tract of some injured animals, but neither silver stain accumulation nor inflammatory responses in the injury groups were statistically different from the sham group in this region. However, argyrophilic axons, microgliosis and astrogliosis were significantly increased within the optic tract of injured animals. Repeated mild CHI also resulted in microgliosis and a loss of neurofilament protein 200 in the optic nerve. Lengthening the inter-injury interval from 24h to 48h did not effectively reduce these behavioral or cellular responses. These results suggest that repeated mild CHI results in persistent behavioral dysfunction and chronic pathological changes within the visual system, neither of which was significantly attenuated by lengthening the inter-injury interval from 24h to 48h.

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Published in PLOS ONE, v. 11, no. 7, e0159442, p. 1-23.

© 2016 Bolton Hall et al.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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This study was supported by the National Institute of Neurological Disorders and Stroke T32 NS077889 and National Institute of Neurological Disorders and Stroke F31 NS087878-02 (ANBH); National Institute of Neurological Disorders and Stroke P30 NS051220, Kentucky Spinal Cord and Head Injury Trust 14-13A, Kentucky Spinal Cord and Head Injury Trust 9-13 (KES).

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S1 Fig. Hemotoxylin and Eosin stain following repeated closed head injury (rCHI) or repeated sham injury (rSHAM).

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S2 Fig. Acute microgliosis in the optic tract after single and repeated closed head injuries (CHI).

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S3 Fig. PHF-1 after repeated closed head injury (CHI) compared to positive control tissue.