No Link between Steroids and Chronic Traumatic Encephalopathy, Study SuggestsThe experimental findings do not support the theory that steroid use by athletes plays a role in the long-term damaging effects of anabolic steroids brain injury traumatic brain injury MTBI. The lead author was Anabolic steroids brain injury. Mills of West Virginia University, Morgantown. Mills and colleagues designed an experiment to determine whether treatment with anabolic steroids makes any difference in the effects of brain trauma in rats. It has been suggested that steroids may contribute to the abnormalities seen in brian traumatic encephalopathy Gnc testosterone reviews
No Link between Steroids and Chronic Traumatic Encephalopathy, Study Suggests
Conceived and designed the experiments: Concussion is a serious health concern. Concussion in athletes is of particular interest with respect to the relationship of concussion exposure to risk of chronic traumatic encephalopathy CTE , a neurodegenerative condition associated with altered cognitive and psychiatric functions and profound tauopathy.
However, much remains to be learned about factors other than cumulative exposure that could influence concussion pathogenesis. How acute, chronic, or historical AAS use may affect the vulnerability of the brain to concussion is unknown. AAS treatment induced the expected physiological changes including increased body weight, testicular atrophy, aggression and downregulation of brain 5-HT1B receptor expression.
The global annual incidence of TBI is estimated to be approximately per , persons [ 1 ]. In the United States, the overall incidence of TBI is estimated to be per , persons, which represents at least 1. The past decade has witnessed a tremendous surge of interest in concussion in youth and young adults, as this age group represents a major peak of TBI incidence for whom decreased educational and occupational achievement could have profound long-term consequences. Concussions in youth and young adults are attributed mostly to falls, motor vehicle accidents, and participation in sports.
As current diagnosis relies on subjective symptom reporting, there is great of interest in characterizing the cellular and histopathological changes that occur after concussion to aid in the development of evidence-based objective, sensitive and specific metrics of concussion diagnosis, prognosis and recovery in young people.
Although PCS typically resolves within 3 months of injury for the majority of patients, some develop persistent PCS [ 6 , 7 ]. However, the symptoms of PCS are nonspecific and also found in patients with traumatic injuries to areas of the body other than the head [ 8 , 9 ]. Factors that influence symptom prevalence include concurrent components such as pain, anxiety, depression, post-traumatic stress, pre-existing psychiatric conditions and litigation [ 11 , 12 ].
With respect to factors that predict symptom reporting, a recent investigation of close to 32, uninjured high school athletes without concussion for 6 months found that symptom reporting was more common in girls, especially those with prior treatment of a psychiatric condition or substance abuse and attention deficit-hyperactivity disorder [ 13 ].
For boys, prior treatment of a psychiatric condition was the strongest independent predictor for symptom reporting, followed by a history of migraines [ 13 ]. Intriguingly, the weakest independent predictor of symptom reporting for both sexes was history of prior concussion [ 13 ]. Given the challenges associated with monitoring concussion recovery using subjective and nonspecific symptoms, objective and quantifiable biomarkers are highly desirable.
Diffuse axonal injury DAI and white matter neuroinflammation are commonly found upon histopathological examination of TBI brain tissue. Deformation of white matter at the moment of traumatic injury is believed to lead to mechanical failure and calcium dependent proteolysis of the axonal cytoskeleton, leading to several markers of axonal damage that can include silver uptake as well as accumulation of amyloid precursor protein APP , neurofilament and proteolytic fragments of alpha II spectrin [ 14 ].
Neuroinflammation is another well-recognized response to TBI in both humans and experimental models. As the activation of immune and non-immune cells that occurs in the days to weeks after injury can influence many post injury symptoms, the term Post-Inflammatory Brain Syndrome has recently been suggested [ 15 ].
Neuroinflammation is also a major component of many neurodegenerative diseases of aging, and a dysregulated neuroinflammatory response after TBI could contribute to the increased risk of chronic consequences of TBI. One such consequence of interest to those who participate in high contact sports is chronic traumatic encephalopathy CTE , a progressive neurodegenerative disease clinically characterized by alterations in mood and behavior, motor disturbances and, in severe cases, progressive dementia [ 16 , 17 ].
Originally described as dementia pugilistica [ 18 ], recent studies have revealed that similar clinical symptoms and neuropathology are also observed in other contact sports such as American football, boxing, hockey, and association football soccer [ 19 — 21 ]. The primary neuropathological feature of CTE is extensive deposition of hyperphosphorylated tau [ 20 , 24 ].
AAS are synthetic derivatives of testosterone used by both elite and recreational athletes [ 26 , 27 ] for their ergogenic effects including increased muscle strength, endurance and power, increased lean body mass, and enhanced recovery between workouts and from injury [ 28 ]. A recent meta-analysis of studies found the global prevalence rate of AAS use among elite and recreational athletes to be Further, athletes younger than 19 years have a higher prevalence of AAS 2.
AAS use in athletes is driven by perceived benefits to both athletic performance and appearance. While AAS may enhance sports performance, chronic AAS use is associated with several adverse psychiatric effects including increased aggression, depression, mood and anxiety disorders, irritability and suicidal tendencies [ 27 , 30 ].
Chronic AAS use also induces widespread adverse physiological alterations involving multiple organ systems, particularly of the hypothalamic-pituitary-gonadal axis [ 31 ]. Moreover, preclinical and clinical studies indicate altered structural remodeling and neurotransmitter physiology in adolescent brains following AAS exposure, which also may affect behavior [ 32 ].
AAS-related aggressive behaviors may also increase the probability of experiencing head trauma both on and off the field. Ethical constraints clearly pose considerable challenges for controlled clinical studies of how AAS exposure affects both TBI risk and pathogenesis in athletes.
To our knowledge, only one preclinical study to date has investigated the effects of a week exposure to a single AAS, nandrolone, on axonal injury in rats subjected to a single weight drop TBI [ 33 ]. In this study, axonal injury as assessed with amyloid precursor protein APP immunohistochemistry was the sole TBI outcome examined. No alteration in axonal damage was observed after TBI in AAS-treated compared to control rats when assessed 30 d post-injury. CHIMERA is designed to reliably replicate the biomechanical conditions encountered in human mTBI, namely, impact to a closed skull with free head movement after impact [ 34 ].
We first assessed the physiological effects of chronic AAS treatment by recording body weight weekly for 6 weeks during an 8-week total treatment period. C Macroscopic size comparison of seminal vesicle, testes and brain. Body weight data are analyzed by two-way repeated measures ANOVA followed by a Bonferroni post hoc test, tissue weight data were analyzed by two-tailed unpaired t test. LRR duration remained stable over two impacts time effect: Injured mice showed anxiety-like behavior as indicated by significantly increased thigmotaxis in an open field test performed 6 d post-rTBI Fig 2D , TBI effect: AAS treatment did not significantly alter thigmotactic behavior.
Open field thigmotaxis was not affected by gross motor activity as no significant differences in total distance traveled Injury effect: Finally, aggressive behavior was exacerbated in AAS-treated resident mice prior to injury as indicated by significantly decreased latency to fight with intruder mice as assessed by RIT at the 5 th and 6 th week of AAS treatment Fig 2E , treatment effect: C Motor performance was assessed on an accelerating rotarod at 1, 2, and 7 d post-rTBI.
D Thigmotaxis was quantified at 1 and 6 d post-rTBI and is represented as thigmotaxis index. Graphs represent latency to initiate fighting by the resident mouse. Data are analyzed by repeated measures general linear model.
Legends and cohort sizes are consistent across all graphs. Using the same experimental conditions as in this study, we previously showed that endogenous murine tau shows a transient increase in phosphorylation that resolves to baseline 7 days after CHIMERA rTBI [ 34 ].
To determine the effect of AAS exposure, we assessed the phosphorylation levels of endogenous murine tau in half-brain homogenates collected at 7 d post-rTBI using two antibodies directed against different tau phosphorylation sites, namely CP13 pSer and RZ3 pThr Total murine tau levels were determined by the antibody DA9.
Simple Western analysis showed that, as expected from our previous study, tau phosphorylation levels were not significantly different in the rTBI group compared to sham controls at 7 d post injury Fig 3. AAS treatment also had no significant effect on tau phosphorylation levels at this time point Fig 3.
Tau phosphorylation was assessed using the Simple Western system Protein Simple. Graphs in the middle column C and D depict quantitation of phosphorylated tau as a proportion of total tau DA9. Representative digital immunoblots of phosphorylated and corresponding total tau are depicted in the right column E and F. To confirm the neurochemical effects of AAS treatment in the present study, we assessed serotonin receptor 5-HT1B expression in brain tissues by immunohistochemistry and found significantly decreased staining intensity selectively in the substantia nigra Fig 4 , treatment effect: Immunohistochemistry was used to assess 5-HT1B receptor expression levels.
Representative images of whole-mount sections for AAS-treated and control brains are depicted in Panel A. Graph in Panel B depicts mean staining intensity in arbitrary units.
As we have previously reported [ 34 ], CHIMERA-injured brains revealed widespread multifocal axonal injury, as indicated by intense punctate and fiber-associated argyrophilic structures in several white matter tracts including the corpus callosum, external capsule, septal-fimbrial area, and optic tract Fig 5.
Axonal injury was observed at both coup corpus callosum and contrecoup optic tract regions, indicating a diffuse injury pattern. With the exception of septal-fimbrial area in the VH-rTBI group, quantitative analysis of silver stained images revealed significantly increased silver uptake in all of the above-mentioned white matter regions for both rTBI groups Fig 6.
Post-rTBI axonal damage was assessed with silver staining. Representative 40X-magnified images of corpus callosum, external capsule, septal-fimbrial area and optic tract of sham left column and VH- middle column and AAS-treated right column rTBI brains are depicted. Silver stained images were quantified by calculating the percent of region of interest ROI in the white matter tract area that was stained with silver.
Graphs indicate percent of the ROI showing positive signal in the respective white matter regions. Using Iba1 immunohistochemistry, we observed significant microglial activation throughout several white matter regions including the olfactory nerve layer, corpus callosum, brachium of superior colliculus and optic tract of injured brains compared to sham controls Fig 7.
Microglia in sham brains displayed high fractal dimensions consisting of highly complex, extensively branched and ramified morphology indicative of the resting state Fig 8A—8D. By contrast, microglia in injured brains from both VH and AAS-treated groups showed significantly reduced fractal dimensions in the above white matter regions indicative of an activated state Fig 8A—8D. Post-rTBI microglial activation was assessed with Iba1 immunohistochemistry at 7 d.
Representative 40X-magnified images of white matter regions show resting microglia in sham brains left column and activated microglia in injured brains second and third columns. Microglial morphology was quantitatively assessed using fractal analysis. Graphs in the left column represent fractal dimension for microglial morphology in A olfactory nerve layer, B corpus callosum, C brachium of superior colliculus and D optic tract.
Graphs in the right column E-H show number of Iba1-positive cells per mm 2 in the same white matter regions. Prior to rTBI, AAS-treated mice exhibited the expected morphological changes in body, seminal vesicle and testicular weights, as well as the expected increase in aggressive behavior.
In the acute period of 7 d after rTBI, AAS-treated mice displayed more severe axonal injury and significantly increased microgliosis in several white matter tracts, but did not exhibit significantly worsened behavioral deficits or sustained phosphorylation of murine tau under the experimental conditions used here. Future studies will be needed to determine if more severe injury, a greater cumulative number of injuries, or additional time points of analyses unveil significant behavioral outcomes between AAS-treated and control mice after rTBI.
In addition, studies using transgenic mice will be needed to investigate whether AAS exposure influences additional neuropathological changes including tau-containing neurofibrillary tangles. Using the Marmarou method of dropping a weight onto a metal disk affixed to the exposed skull of rats, Mills et al reported no difference in axonal injury as measured by APP immunohistochemistry when analyzed at 30 d after TBI [ 33 ]. No other outcome was reported. Our study has several important differences compared to Mills et al that could explain why we observed exacerbated axonal pathology and inflammation whereas Mills et al did not.
Compared to the Marmarou weight drop model, CHIMERA is nonsurgical and allows completely free and reliable head motion after impact, thus recapitulating the biomechanical responses of the head after impact in humans [ 34 ]. Head motion has previously been shown to be an important parameter of injury in blast TBI [ 38 ] and is believed to be a major contributor to the biomechanical strain that underlies DAI [ 39 ].
Additionally, many weight-drop TBI models are susceptible to large experimental variability [ 40 ]. The post-rTBI behavioral and neuropathological assessments data from the present study are in close agreement with our previously-reported observation [ 34 ] indicating excellent reproducibility of CHIMERA model. We confirmed that AAS treatment produced the expected physiological effects and also demonstrated decreased 5-HT1B receptor expression in the substantia nigra. Serotonin 5-HT is well-studied neurotransmitter that is consistently shown as an inhibitor of aggression [ 41 , 42 ].
On the other hand, 5-HT receptor agonists reduce aggression in experimental settings [ 46 , 47 ]. Several studies have shown that chronic AAS exposure reduces the levels of serotonin and its metabolites [ 48 , 49 ], 5-HT-immunoreactive neuronal fibers [ 50 ] as well as receptor expression [ 51 , 52 ]. Regionally, the substantia nigra has the highest 5-HT1B receptor expression [ 53 , 54 ]. These observations suggest that AAS exposure may cause subtle changes in post-rTBI motor deficits during the acute phase of TBI that may or may not resolve over a further period of recovery.
We did not evaluate the effect of AAS exposure on cognitive outcomes, which are not feasible to examine during this very acute phase post rTBI. Future studies will be needed to understand how AAS exposure may affect the trajectory of post-TBI behavioral deficits over the long term. Using silver staining, we observed increased argyrophilic fibers and punctate structures in several white matter tracts throughout the brain that were significantly exacerbated by chronic AAS exposure when assessed at 7 d post-rTBI.