HISTOLOGICAL AND BEHAVIORAL CONSEQUENCES OF REPEATED MILD TRAUMATIC BRAIN INJURY IN MICE

The majority of the estimated three million traumatic brain injuries that occur each year are classified as “mild” and do not require surgical intervention. However, debilitating symptoms such as difficulties focusing on tasks, anxiety, depression, and visual deficits can persist chronically after a mild traumatic brain injury (TBI) even if an individual appears “fine”. These symptoms have been observed to worsen or be prolonged when an individual has suffered multiple mild TBIs. To test the hypothesis that increasing the amount of time between head injuries can reduce the histopathological and behavioral consequences of repeated mild TBI, a mouse model of closed head injury (CHI) was developed. A pneumatically controlled device with a silicone tip was used to deliver a diffuse, midline impact directly onto the mouse skull. A 2.0mm intended depth of injury caused a brief period of apnea and increased righting reflex response with minimal astrogliosis and axonal injury bilaterally in the entorhinal cortex, optic tract, and cerebellum. When five CHIs were repeated at 24h inter-injury intervals, astrogliosis was exacerbated acutely in the hippocampus and entorhinal cortex compared to a single mild TBI. Additionally, in the entorhinal cortex, hemorrhagic lesions developed along with increased neurodegeneration and microgliosis. Axonal injury was observed bilaterally in the white matter tracts of the cerebellum and brainstem. When the inter-injury interval was extended to 48h, the extent of inflammation and cell death was similar to that caused by a single CHI suggesting that, in our mouse model, extending the inter-injury interval from 24h to 48h reduced the acute effects of repeated head injuries. The behavioral consequences of repeated CHI at 24h or 48h inter-injury intervals were evaluated in a ten week longitudinal study followed by histological analyses. Five CHI repeated at 24h inter-injury intervals produced motor and cognitive deficits that persisted throughout the ten week study period. Based upon histological analyses, the acute inflammation, axonal injury, and cell death observed acutely in the entorhinal cortex had resolved by ten weeks after injury. However, axonal degeneration and gliosis were present in the optic tract, optic nerve, and corticospinal tract. Extending the inter-injury interval to 48h did not significantly reduce motor and cognitive deficits, nor did it protect against chronic microgliosis and neurodegeneration in the visual pathway. Together these data suggested that some white matter areas may be more susceptible to our model of repeated mild TBI causing persistent neuropathology and behavioral deficits which were not substantially reduced with a 48h inter-injury interval. In many forms of TBI, microgliosis persists chronically and is believed to contribute to the cascade of neurodegeneration. To test the hypothesis that post-traumatic microgliosis contributes to mild TBI-related neuropathology, mice deficient in the growth factor progranulin (Grn-/-) received repeated CHI and were compared to wildtype, C57BL/6 mice. Penetrating head injury was previously reported to amplify the acute microglial response in Grn-/- mice. In our studies, repeated CHI induced an increased microglial response in Grn-/- mice compared to C57BL/6 mice at 48h, 7d, and 7mo after injury. However, no differences were observed between Grn-/- and WT mice with respect to their behavioral responses or amount of axonal injury or ongoing neurodegeneration at 7 months despite the robust differences in microgliosis. Dietary administration of ibuprofen initiated after the first injury reduced microglial activation within the optic tract of WT mice 7d after repeated mild TBI. However, a two week ibuprofen treatment regimen failed to affect the extent of behavioral dysfunction over 7mo or decrease chronic neurodegeneration, axon loss, or microgliosis in brain-injured Grn-.- mice when compared to standard diet. Together these studies underscore that mild TBIs, when repeated, can result in long lasting behavioral deficits accompanied by neurodegeneration within vulnerable brain regions. Our studies on the time interval between repeated head injuries suggest that a 48h inter-injury interval is within the window of mouse brain vulnerability to chronic motor and cognitive dysfunction and white matter injury. Data from our microglia modulation studies suggest that a chronically heightened microglial response following repeated mild TBI in progranulin deficient mice does not worsen chronic behavioral dysfunction or neurodegeneration. In addition, a two week ibuprofen treatment is not effective in reducing the microglial response, chronic behavioral dysfunction, or chronic neurodegeneration in progranulin deficient mice. Our data suggests that microglia are not a favorable target for the treatment of TBI

Repeated Closed Head Injury in Mice Results in Sustained Motor and Memory Deficits and Chronic Cellular Changes

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