Dendritic arbor complexity and spine density changes after repetitive mild traumatic brain injury and neuroprotective treatments

Highlights

• Repeated TBI decreases total dendritic length in the hippocampus.

• Repeated TBI reduces dendritic spine quantity and density in hippocampus.

• Activating Nrf2 and PPAR-γ after injury protects against some neuronal changes.

Abstract

Traumatic brain injury has been described as the signature affliction of recent military conflicts and repetitive TBIs, particularly associated with military and athletic activities, typically result in more severe clinical effects. The majority of TBIs are mild, but they can result in long term cognitive deficits for which there is no effective treatment. One of the most significant deficits observed in TBI patients is memory loss, which suggests that TBI can induce pathological changes within the hippocampus. tert-butylhydroquinone (tBHQ) and pioglitazone activate the Nrf2 and PPAR-γ transcription factors, respectively, and both have been shown to be neuroprotective in model systems. We examined the morphological changes within the hippocampus following repetitive mild TBI and simultaneous treatment with both factors. We utilized a closed head injury mouse model with five injuries over 5 weeks. Our results showed marked morphological changes among the dendrites and dendritic spines of the neurons of the dentate gyrus of the hippocampus. We observed decreases in overall dendritic length, as well as in the quantity and density of dendritic spines. Our treatment partially ameliorated these effects, suggesting that the Nrf2 and PPAR-γ transcription factors may be important targets for future drug development in the treatment of TBI in humans.

Introduction

Brain trauma represents one of the most significant causes of disability and death in the United States, accounting for approximately 30% of all injury-related deaths (Faul et al., 2010). Approximately 2.5 million TBI-related hospital visits occur each year in the United States (Peterson et al., 2015). Military personnel, in particular, are at an increased risk. In fact, among US military forces alone, there have been over 384,000 brain injuries sustained since 2000 (Defense and Veterans Brain Injury Center, 2018). Along with military personnel, athletes are at a particular risk for repeated injuries, which has gained increasing public interest in recent years (Hoge et al., 2008, Powell and Barber-Foss, 1999). Mild TBIs are most common, however, up to 10% of mild TBI patients can suffer long-term complications for which there is no effective treatment (Gardner and Yaffe, 2015, Iverson, 2005, McCrea et al., 2003, Schneiderman et al., 2008, Terrio et al., 2009).

The development of a treatment to protect the brain from secondary damage caused by the neuroinflammation and oxidative stress, which follows the initial mechanical injury, could involve targeting transcription factor signaling. Activation of Peroxisome proliferator-activated receptor-gamma (PPAR-γ) by its agonist, oral insulin drug pioglitazone, has been shown to reduce neuroinflammation and neuronal damage following brain injury (Deng et al., 2020, Liu et al., 2017, Sauerbeck et al., 2011). Even a single post-injury dose of pioglitazone can reduce cortical oxidative damage and microglial responses (Pilipovic et al., 2015). Another transcription factor of interest is Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which is activated by tertbutylhydroquinone (tBHQ), a common food additive. We have shown previously that activation of the Nrf2 pathway with tBHQ following brain injury leads to improved cognitive performance in mouse models (Saykally et al., 2012). While both of these compounds have been shown to be neuroprotective individually, simultaneous activation of both pathways may provide a synergistic effect that leads to further improved outcomes following brain injury. We have shown previously that a combination treatment of both pioglitazone and tBHQ results in improved recognition memory following repeated closed head injuries in mice (Ratliff et al., 2020b).

Among the long-term complications of mild TBI, perhaps the most pervasive is memory impairment (Nicholl and LaFrance, 2009, Rutland-Brown et al., 2006). The hippocampus plays an integral role in the formation of new memories and has been previously shown to be susceptible to mechanical injury which may contribute to the memory impairment observed in TBI patients (Hicks et al., 1993). As such, it is important to carefully investigate changes within the hippocampus following injury and in the search for potential neuroprotective treatments. Our mouse model utilizes a repeated closed head injury to recapitulate the impact forces, pathology, and cognitive deficits observed in human TBI patients (Zohar et al., 2003). This model has been extensively tested and has been shown to cause both molecular changes and changes to the morphology of individual neurons and glial cells (Saykally et al., 2012, Saykally et al., 2018, Tashlykov et al., 2007, Tweedie et al., 2007). In this study, we have investigated the morphological changes of neurons within the hippocampus following repeated injury and the impact of post-injury treatment with tBHQ and pioglitazone. Our results suggest that, in addition to the cognitive improvements reported previously (Ratliff et al., 2020b), simultaneous activation of the Nrf2 and PPAR-γ transcription factors is able to ameliorate some of the potentially harmful morphological changes observed within the hippocampus following injury.