The nuclear factor erythroid 2-like 2 activator, tert-butylhydroquinone, improves cognitive performance in mice after mild traumatic brain injury
Highlights
► Transcription factor Nrf2 activator, tBHQ, reduces visual memory loss after mild TBI. ► HSP70 expression is elevated by the injury and treatment in the mouse brain. ► HSP70 inhibition blocks tBHQ neuroprotection of cultured model neurons from mild TBI.
Introduction
There are reported 1.7 million people who suffer traumatic brain injuries each year in the United States (CDC, 2011). Injuries range from mild to severe with approximately 80% presenting with mild injuries (Kraus and Nourjah, 1988). Traumatic brain injuries have become the signature affliction of deployed forces with most of these categorized as mild injuries (Hoge et al., 2008). While these traumas are not life threatening they still have a substantial impact on the individual initiating cognitive and other health problems (Rimel et al., 1981, Dacey et al., 1986, McAllister et al., 1999, Vakil, 2005, McAllister, 2011). Magnetic resonance imaging and single-photon emission scans have shown that mild injury causes brain lesions with atrophy apparent even 6 months post trauma (Hofman et al., 2001). This is recapitulated in animal studies where extended loss is noted after trauma (Smith et al., 1995). One of the most common symptoms that results from mild traumatic brain injury (mTBI) is memory loss (Binder, 1986, Warden, 2006, Halbauer et al., 2009). This may result from damage to the hippocampus (Rempel-Clower et al., 1996), which is sensitive to mechanical injury (Kotapka et al., 1991, Hicks et al., 1993, Rempel-Clower et al., 1996, Umile et al., 2002). Among the different aspects of memory, working memory or short-term memory is often impaired following mTBI (McAllister et al., 1999, Halbauer et al., 2009). Visual memory is a form of working memory that is specifically affected in patients. It has been observed that mild TBI patients have visual memory deficits, as measured by the Shum visual learning test, but they do not show any difference in spatial memory measured by an electronic maze test (Shum et al., 2000). Memory deficits following traumatic injury have been recapitulated in animal models. TBI induced in mice (Tashlykov et al., 2009) has shown neuronal loss in the hippocampus and associated cognitive deficits and similar results have been found in the rat (Hicks et al., 1993, Smith et al., 1994). Importantly, animal models have shown that closed head injury results in cognitive deficits (Zohar et al., 2003) when evaluated for visual memory (Biegon et al., 2004, Edut et al., 2011), spatial memory (Rubovitch et al., 2010, Baratz et al., 2011), non-spatial memory (Zohar et al., 2011), or anxiety (Edut et al., 2011). A chronic loss of memory, even slight, complicates several other cognitive functions.
Traumatic brain injury is a complex disease with heterogeneous actions and dysregulation throughout the brain resulting in neuronal loss. The primary injury results in a focal injury at the site of impact with local neuronal loss. It is not just these immediate events that result in neuronal deficit, but the pathobiology that occurs following the primary injury. These processes include disruption of cellular homeostasis by calcium release, oxidative stress, and apoptosis signaling cascades that persist for days or weeks following injury. It is the multifactorial dysregulations that occur following the primary injury, often called the secondary injury that results in extended neuronal loss as well as diffuse axonal injury. Diffuse axonal injury is a serious condition where the axon is damaged resulting in the degradation of neuronal circuitry. This loss of axonal branches alters neurotransmission and can be a major contributor to functional loss. The actual axonal damage is thought to be a result of secondary injury involving signaling cascades (Smith et al., 2003). This damage can be very significant even in mild injuries and is not detectable until hours or days following injury.
It is the secondary injury that provides targets for the treatment of traumatic brain injury. If sufficient neurons can be preserved then the cognitive deficit will be reduced along with prolonged symptoms observed in patients and this may eliminate the need for more invasive treatments in the future. One pharmacological target is the regulation of transcription factor signaling, which is altered by the secondary injury. Modest alterations of these factors can significantly alter downstream signaling to reduce detrimental signaling and promote pro-survival pathways allowing for neuronal preservation (Kane and Citron, 2009). Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is one transcription factor of interest and it is known to activate downstream antioxidant and oxidative stress genes. Nrf2 is a transcription factor that is sequestered in the cytoplasm and tethered to Kelch-like ECH-associated protein 1 (KEAP1). When Nrf2 is activated, it disassociates and translocates to the nucleus where it induces downstream targets by binding canonical antioxidant response elements (Rushmore et al., 1991, Lee et al., 2003, Johnson et al., 2008). Nrf2-deficient mice display an increase in apoptosis and inflammatory signaling following moderate closed head TBI (Jin et al., 2009). The downstream gene heat shock protein 70 (HSP70) acts through several mechanisms by which it protects protein function and stability in a cell. To elicit the effects of Nrf2 a chemical activator, tert-butylhydroquinone (tBHQ) can be administered (Shih et al., 2005). This compound is a metabolite of the antioxidant butylated hydroxyanisole, can cross the blood-brain barrier, and has produced neuroprotection in several models of disease (Kraft et al., 2004, Jakel et al., 2007, Shih et al., 2007).
The health impacts of TBI are extreme and the economical burden of these injuries is an estimated 60 billion dollars in the US alone (CDC, 2011). The need for effective treatments is clear. We show that the use of tBHQ, an activator of Nrf2, as a treatment following traumatic brain injury can improve visual memory in mice. This may be the result of an increase in HSP70 and the decrease in activated caspase-3.
Section snippets
Animals
Male ICR mice were originally purchased from HSD Jerusalem or Harlan Indianapolis, IN and were bred and raised in our vivarium. The mice weighing 30–40 g were housed five per cage under a constant 12-h light/dark cycle. The mice were maintained at 22 °C ± 0.5 °C. Food and water were supplied ad libitum. All experiments were performed in accordance with local and national guidelines.
Treatment
Treatment solution: 100 mg tBHQ (tert-butylhydroquinone) was dissolved in 1 ml of 100% dimethyl sulfoxide (DMSO) and was
Cognitive performance and tBHQ treatment
Mice were subjected to a mild closed head injury at the right temporal region of the skull and treated 30 min post injury by inner peritoneal injection of either tBHQ or vehicle (1% DMSO). Mice were treated once per day for 6 days following injury and behavioral tests were performed to measure cognitive ability at 7 and 30 days post injury (Fig. 1).
The object recognition test demonstrated that mice subjected to a closed head injury spent significantly less time exploring the “new” object than sham
Discussion
The transcription factor Nrf2 makes an ideal candidate to be altered for treatment as it can be chemically modulated by a compound that is currently in use in the food industry, can be administered by several methods, and is able to cross the blood-brain barrier. Using an array of tests we measured cognitive impairment, motor function, and anxiety following TBI. This model of mTBI only displayed cognitive deficits while motor function and anxiety were not affected. This battery of cognitive
Conclusions
Modulation of Nrf2 activity is a promising treatment for mTBI. Administration of an activator after injury resulted in improved cognitive performance. The neuroprotection observed by Nrf2 is likely in part through the reduction in the activation of caspase-3 in the hippocampus. The specific mechanism by which tBHQ reduces activated caspase-3 is yet to be determined but could result from an increase in oxidative stress response genes such as HSP70. The method of administration and optimal dosage
Author disclosure statement
No competing financial interests exist. The contents do not represent the views of the Department of Veterans Affairs or the United States Government.
Acknowledgments
The authors thank Andrea Smith for expert assistance with animal studies. This study was supported by the Department of Veterans Affairs (Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development), the Florida Department of Health James and Esther King Biomedical Research Program, the Bay Pines Foundation, and the University of South Florida Signature Interdisciplinary Program in Neuroscience.
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2020, ToxicologyCitation Excerpt :With the increase in caspase-3 level, more spermatogenic cells are damaged leaving fewer viable cells as demonstrated with the increase in the percentage of seminiferous tubules with germ cell loss and decreased PCNA-positive cells in Cis group in the present study. Our findings are supported by previous studies where caspase-3 was reportedly upregulated in the testis following Cis treatment (Aksu et al., 2017; Fouad et al., 2017), corroborated by increase in TUNEL-positive spermatogenic cells (Köroğlu et al., 2019), and decreased Ki-67 and PCNA immunoexpressions (Almeer and Abdel Moneim, 2018; Köroğlu et al., 2019; Saad et al., 2020). Consistent with its effects on oxidative stress and inflammation, pre-treatment of Cis administered rats with tBHQ attenuated apoptosis and increased PCNA-positive spermatogenic cells in the testis.