Browsing by Author "Schreihofer, Derek"
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Item Acute sex difference in response to repeated mild traumatic brain injury in mice(2023) Kuo, Aaron; Schreihofer, Derek; Sumien, Nathalie; Vann, Philip; Ahmed, AffanAcute sex difference in response to repeated mild traumatic brain injury in mice Aaron Kuo, Philip Vann, Nathalie Sumien, Ahmed Affan, Derek Schreihofer Background: Repetitive mild traumatic brain injury (rmTBI), such as that occurring in contact sports, is associated with the development of neurodegenerative diseases of aging. Severe TBI and repetitive concussions are associated with chronic traumatic encephalopathy (CTE), Alzheimer’s disease (AD), and Parkinson’s disease (PD), among others. However, less severe injuries may also lead to delayed neurological disfunction if repeated often and without sufficient rest time between injuries. Previously, we found that the progression of behavioral deficits in males and female mice differed from 5 to 15 weeks after 25 rmTBI. Both sexes showed motor deficits at 5 weeks, but only males showed affective and cognitive deficits at 15 weeks. Purpose: This study tested the hypothesis that rmTBI neurological deficits in male mice will appear earlier after rmTBI than in female mice. Methods: C57BL/6 male and female mice (8 wk old) were assigned to sham and rmTBI groups (n=20/group). Lightly anesthetized mice received 7 mild head injuries, once a day (M-F) using a weight drop model (75 g from 1 meter) that included a free fall with rotational injury. Five minutes after the final injury, mice were tested on a balance beam. Additional behavioral assessments began the following day. Results: No sex differences in balance beam performance were observed 5 minutes after the final injury. There were no significant effects of rmTBI on vestibular motor function assessed with a rotarod; cognition assessed with the Morris water maze; or affective behavior assessed with the elevated plus maze. However, in the open field test there was a significant increase in total distance traveled in rmTBI mice (F1,35 = 6.47, P=0.016). Post-hoc analysis revealed that this effect was only significant in male mice (Fisher LSD, P<0.05), supporting the hypothesis that males exhibit earlier deficits than females. Conclusion: At extended time points following rmTBI, both male and female mice develop motor deficits. However, up to 15 weeks after injury, only male mice experience cognitive and affective deficits. The current study reveals that male mice also display hyperactivity in the week after rmTBI that is not observed in female mice. Thus, sex differences in response to rmTBI are apparent both in the acute and chronic phase of injury and suggest that interventions to reduce brain injury may require different timing for males and females. Ongoing studies are examining potential differences in biochemical and histological responses in the brains of male and female mice. AUP: 2021-0035Item Androgen Receptors in the Middle Aged Male Rat Brain: Influence of Testosterone Deprivation on Expression(2017-03-14) Contreras, Jo; Cunningham, Rebecca; Fort*, Callie; Cuellar*, Elric; Lopez, Gladys; Metzger, Daniel; Oppong-Gyebi, Anthony; Schreihofer, Derek; Smith, Charity B.S.Purpose: 1) To determine whether long-term testosterone deprivation (LTTD) alters the levels and/or distribution of androgen receptors in the middle-aged male rat brain and 2) to determine whether testosterone replacement after LTTD influences androgen receptor levels. Methods: Twelve-month old male Fischer 344 rats were left intact or castrated for 2 weeks and replaced with subcutaneous implants containing testosterone (STTD). Additional groups were castrated for 10 weeks before being treated with testosterone (LTTD+T) or cholesterol (LTTD). Four weeks later, rats were euthanized and brains were collected for immunoblotting and immunohistochemistry (IHC) for androgen receptors (AR) using antibodies targeting the N-terminus or C-terminus of the protein. The cerebral cortex, hippocampus, thalamus, hypothalamus were examined, and testes were used as positive control tissue. Results: Contrary to expectations, the full-length AR (116 kDa) was barely detectable in the hippocampus and cerebral cortex by immunoblotting with antibodies directed to either end of the AR. Rather, smaller fragments were readily detected. Examination of the size of these fragments (~30, 37, 50, and 80 kDa) and consultation of the literature for the human AR, suggested that they represented calpain-dependent cleavage fragments. A series of control experiments was performed in an attempt to extract the full-length AR using rat testes as a positive control tissue. Protease inhibitors, EDTA, and the AR agonist dihydrotestosterone failed to reduce the appearance of fragments. Interestingly, the pattern of fragments from the hippocampus (80 [greater than] 37 kDa) differed from that from the cortex (37 [greater than] 80 kDa) suggesting differential processing. IHC of coronal brain sections though the forebrain revealed nuclear AR staining consistent with full-length AR in regions of high expression, including the hypothalamus. In agreement with immunoblotting AR staining in the cortex and hippocampus appeared to be cytoplasmic, rather than nuclear. No significant differences were observed between treatment groups. Conclusions: These data suggest that AR protein in some areas of the middle-aged male rat brain is rapidly degraded into fragments with altered localization and potential for transcriptional activity and/or signaling functions. Although no differences in expression were apparent between treatment groups, the differential processing of AR in the rat brain is a novel finding warranting further investigation.Item Assessment of Sex Differences Following Repeated Mild Head Injuries(2022) Duggal, Aakaash; Vann, Philip; Metzger, Daniel; Ahmed, Affan; Sumien, Nathalie; Schreihofer, DerekBackground: Traumatic brain injury (TBI) is a major cause of disability, morbidity, and mortality in the U.S. Although there is a growing understanding of the effects of moderate and severe TBI, less is understood about the effects of repetitive mild TBI (rmTBI). Nevertheless, some studies show that long term participants in contact sports have an increased risk for neurodegenerative disease. In addition, there is limited information about sex differences in TBI, despite some studies suggesting females participating in contact sports experience more head injuries than males. With an increasing number of females participating in contact sports, it's important to explore the effects of rmTBI in females. Purpose: This study will test the hypothesis that rmTBI will lead to more severe neurological deficits in female mice than in male mice. Methods: C57BL/6 female mice were assigned to sham and rmTBI groups (n=30/group). Lightly anesthetized mice received 25 mild head injuries, once a day (M-F) over 5 weeks using a weight drop model that included a free fall with rotational injury. Acute effects of injury were assessed by righting reflex and balance beam tests weekly. Chronic effects were tested with rotarod, Morris water maze (MWM), elevated plus maze (EPM), and T-maze beginning 5 or 25 weeks after the last injury. Effects in female mice will be compared to previously collected data in male mice. Inflammation and white matter injury will be assessed with western blotting and immunohistochemistry, respectively. Results: Acutely, rmTBI female mice performed worse than sham injured mice on the balance beam (F (1,28) =4.309, P=0.0472) whereas there was no difference in males. Five weeks after injury, both male and female mice in the rmTBI group performed significantly (T-test P< 0.01) worse on the Rotarod. Neither males nor females displayed deficits in cognition on the T-Maze or learning phase of the MWM, although males had a significant impairment on MWM memory (Probe T-test P< 0.05). Neither sex showed deficits in the EPM. Fifteen weeks after injury, male mice displayed significant deficits in learning in the MWM (T-test P< 0.05) and EPM (T-test P< 0.05). Male mice in the rmTBI group also showed increased astrogliosis and Tau phosphorylation in the cerebral cortex compared to sham injured mice. Additional assessments of white matter injury are planned, but assessment of female mice 15 weeks after injury is incomplete at this time. Conclusion: Acutely, female mice showed balance deficits that were not apparent in males. Five weeks after injury, both sexes continued to show motor deficits on the rotarod, but only males had mild deficits in cognition. Ongoing studies will assess whether these differences persist or new differences between males and females appear chronically. AUP: 2021-0035Item Astrocytes & Ischemic stroke(2014-08-01) Roy Choudhury, Gourav; Yang, Shaohua; Singh, Meharvan; Schreihofer, DerekAlthough less appreciated, recent findings introduced critical contributions of astrocytes to numerous CNS functions, like neurogenesis, synaptogenesis, ion homeostasis, neurotransmission, and blood brain barrier formation. Their active participation in the progression of specific CNS pathologies has garnered major attention and culminated in thorough investigation of astrocyte function in brain. Reactive astrogliosis, characterized by increases in glial fibrillary acidic protein (GFAP) and cellular hypertrophy, describes the extensive structural and functional changes that astrocytes undergo in response to tissue injury. Despite of extensive investigation, the molecular mechanism of reactive astrogliosis in ischemic stroke still remains elusive. p38 MAPK is a well studied signal transducing pathway known to be involved in modulating cell type specific responses to ischemic injury. The first study presented in the dissertation delineates the involvement p38 MAPK signaling pathway in reactive astrogliosis after ischemic stroke. Results showed that astrocyte specific deletion of p38 MAPK attenuated oxygen-glucose deprivation (OGD)-induced increase in GFAP expression in primary astrocytes in vitro. Additionally, inhibition of p38 MAPK (SB239063/genetic deletion) slowed astrocyte migration without affecting astrocyte proliferation. In vivo deletion of p38 MAPK from astrocytes attenuated reactive astrogliosis after permanent middle cerebral artery occlusion in mice. These findings strongly indicated that p38 MAPK plays a critical role in reactive astrogliosis after ischemic stroke. During ischemic stroke, astrocyte dysfunction causes extensive cell death through excitotoxicity, disruption of ion and water homeostasis. Restoration of astrocyte function thus may be beneficial to ischemic tissue in the long term. Methylene blue (MB), a metabolic enhancer, has been well studied and known to improve cellular respiration, glucose metabolism and attenuate superoxide production by efficient electron transport in mitochondria. In the second part of this dissertation we determined the effect of MB in astrocytes under oxygen glucose deprivation (OGD) and reoxygenation stress and the underlying protective mechanisms. Our studies demonstrated that MB improved astrocyte bioenergetics and promoted astrocyte survival following OGD and reoxygenation. In conclusion both the studies presented, provide a unique perspective of the importance of astroglial response in ischemic injury and how its modulation can benefit the healing and recovery of the brain following ischemic injury.Item Chronic Intermittent Hypoxia Advances Hormonal Aging: Implications for Parkinson’s Related Sexual Dysfunction(2017-03-14) Schreihofer, Derek; Cunningham, Rebecca; Anderson, MarcPurpose: Chronic intermittent hypoxia (CIH) is an established model for sleep apnea and a common comorbidity in Parkinson’s disease (PD). Further, CIH is a known inducer of oxidative stress (OS), which is a key characteristic of PD and aging. Interestingly, in men both sleep apnea and PD are strongly linked with sexual dysfunction. However, it is unknown if CIH induces sexual dysfunction. Therefore, we examined the role of CIH on steroid hormones, sex behaviors, neuropeptides associated with social behaviors, and OS generation in young and old rats. Methods: Young (3-months) and old (12-months) male F344/BNF1 rats, were exposed to either mild CIH or normoxic conditions. CIH consisted of cycling oxygen levels from 21% to 10% over a span of 6 minutes during the rat’s sleep phase for a total of ten days. Sex behavioral tests were conducted to examine the influence of CIH. Specifically, the frequency and latencies of mounts, intromissions, and ejaculations were quantified. At the end of testing, plasma was collected and assayed for testosterone (T), corticosterone (C), vasopressin (AVP), oxytocin (OXY), and advanced oxidation protein products (AOPP). Results: Old rats had impaired sex behaviors compared to young rats. However, CIH induced sexual dysfunction in young rats, consistent with behaviors in old rats. Accordingly, in young rats CIH decreased T, increased C, and increased OS, as indicated by AOPP. CIH did not alter OXY and AVP in young rats. Interestingly, in old rats CIH had no effect on sexual behavior, T, C, OXY, or AVP, indicating that age may have a ceiling effect. Conclusions: Results show that mild CIH advances hormonal aging. Hormonal aging is an understudied phenomenon in PD and in sleep apnea. Therefore, PD progression may be halted by examining the influence of sleep apnea induced hormonal aging.Item Chronic testosterone deprivation sensitizes the middle-aged rat brain to damaging effects of testosterone replacement(2020) Vann, Philip; Wong, Jessica; Sumien, Nathalie; Cunningham, Rebecca; Metzger, Daniel; Schreihofer, Derek; Oppong-Gyebi, Anthony; Contreras Garza, Jo; Kasanga, Ella; Smith, CharityHypothesis: We hypothesized that a delay in testosterone replacement therapy (TRT) following castration in middle-aged male rats would result in increased oxidative stress and a reduction in the neuroprotective effects of testosterone following stroke. Background: Levels of the hormone testosterone (T) fall in aging men. Recently, the number of men obtaining TRT has increased dramatically. However, other consequences of aging, such as oxidative stress, may result in detrimental effects when combined with TRT, including stroke risk. Methods: Twelve-month old male Fischer 344 rats were divided into 5 groups (n=9-14): 1) gonad Intact sham stroke (SH), 2) Intact stroke (IN), 3) short term castrate + T (ST), 4) long term castrate (LC), and 5) long term castrate + T (LCT). Rats were castrated 2 weeks (ST) or 10 weeks (LT, LCT) prior to T. Middle cerebral artery occlusion (Stroke) done via stereotaxic injection of endothelin 1 (ET1). Three, 7, and 14 days after stroke several behavior tests were done. Rats were humanely euthanized, and blood/brains were collected. Results: Plasma oxidative stress measured by Advanced Oxidative Protein Products (AOPP) was significantly negatively correlated with T levels. Long-term hypogonadism in middle-aged male Fischer 344 rats TRT exacerbated the detrimental behavioral effects of experimental focal cerebral ischemia. Conclusion: Data suggest that TRT after long-term hypogonadism can exacerbate functional recovery after focal cerebral ischemia.Item Effect of dietary genistein on functional recovery and chronic post-stroke inflammation in ovariectomized middle-aged rats(2021) Oppong-Gyebi, Anthony; Metzger, Daniel; Vann, Philip; Sumien, Nathalie; Schreihofer, DerekPURPOSE: Increasing age increases stroke risk in women after menopause. A drop in circulating estrogens after menopause has been described as a key reason for this age-related risk, considering that estrogen has shown neuroprotection preclinically. However, using estrogen therapy for chronic prevention of cardiovascular diseases is limited by inconsistent beneficial and detrimental outcomes. For this, other agents are investigated as alternatives to protect women against changes that come with aging and low estrogen concentrations. In this study, we hypothesized that genistein, a neuroprotective plant-derived estrogen will confer neuroprotection following hypogonadism and experimental stroke. METHOD: We used ovariectomized proven retired breeder Sprague-Dawley rats (aged ~9months old), categorized into two hypogonadal time points (2weeks=short-term deprivation(STD) and 12 weeks=long-term deprivation(LTD)) and treated with isoflavone-free diet, genistein diet(GEN) or 17-β estradiol(E2) implant. Animals were subjected to intraluminal middle cerebral artery occlusion or sham surgery followed by motor and cognitive behavioral tests and biochemical analyses for chronic post-stroke inflammation. RESULTS: Sham-operated animals showed locomotor symmetry after both STD and LTD. Both GEN and E2 improved locomotor symmetry after LTD. GEN but not E2 improved cognitive flexibility after STD. Both GEN and E2 reduced activated calcium-binding adaptor molecule 1(Iba1) after STD. GEN but not E2 increased transforming growth factor-β1 and growth-associated protein at the contralateral hemisphere of stroke after STD. CONCLUSION: Dietary Genistein may improve locomotor function in the acute phase of stroke following LTD, improve aspects of cognition and reduce inflammation after STD.Item Effect of dietary genistein on functional recovery following cerebral ischemia in ovariectomized middle-aged rats(2020) Sumien, Nathalie; Vann, Philip; Metzger, Daniel; Schreihofer, Derek; Sun, Fen; Oppong-Gyebi, AnthonyPURPOSE: Advancing age increases women's susceptibility to stroke compared to men, after the menopausal transition, which has been attributed mainly to a drop in estrogen concentrations postmenopause. However, time-dependent mixed benefits and detriments of estrogen therapy for prevention of stroke and cardiovascular diseases after menopause have contributed to widespread mistrust of estrogen use. This has led to the use of other agents like soy isoflavones as alternatives to estrogen therapy. In this study, we hypothesized that the neuroprotective effects of genistein, a soy isoflavone following cerebral ischemia are less sensitive to length of hypogonadism than estrogen. METHOD: Proven retired breeder Sprague-Dawley rats (aged ~9 months old) were ovariectomized, grouped into two hypogonadal time points (2weeks= short-term and 12 weeks= long-term) followed by treatment with isoflavone-free diet or genistein diet (GEN). A group that received 17-β estradiol (E2) was introduced as a control for hormone supplementation. All animals underwent middle cerebral artery occlusion or sham surgery followed by behavioral tests including neurological function, motor function with cylinder and rotarod, cognition with Morris Water Maze (MWM) and gait performance with Catwalk test. RESULTS: We observed effect of stroke on motor and gait performances. Effect of treatment and length of hypogonadism were observed on motor, cognitive and gait performances. GEN but not E2 improved spatial learning on the reversal phase of MWM test. CONCLUSION: Dietary GEN may improve forelimb asymmetry in the acute phase of stroke following long-term hypogonadism and aspects of cognitive functions post-stroke.Item Graded Mild Head Injury as a Model for Sports Injury(2017-03-14) Metzger, Daniel; Oppong-Gyebi, Anthony; Vann, Philip; Sumien, Nathalie; Luedtke, Robert R.; Schreihofer, Derek; Sun, FenPurpose: To develop a graded model of mild head injury that produces graded behavioral deficits in the mouse. This model will be used to test neuroprotective effects of novel compounds. This study was designed to determine the severity of injury required to cause different behavioral deficits in motor function and cognition. Methods: Young adult male C56/B6J mice were anesthetized daily with isoflurane (20 sec) and 15 sec later were subjected to a weight drop head injury using a tethered steel bar (43 grams) dropped through an acrylic tube from a height of 28 inches. Mice were placed prone on a scored aluminum foil stage 2 cm below the end of the tube. The blow was directed to a 5 mm midline area of the head rostral to the aural canals. The blow causes a break in the scored aluminum foil and allows the mouse to flip 180 degrees and land supine on a foam cushion. This model was chosen to model a hit to the head followed by rotational acceleration indicative of closed head injuries occurring in contact sports. Five groups of mice were randomized to receive 0, 5, 10, 15, 20, or 25 blows, 1 per day M-F. Five days after the final hit, mice then underwent cognitive and behavioral testing consisting of an accelerating Rotorod, Morris water maze, and active avoidance T-maze. Following testing brains will be examined for cell death and inflammation. Results: A total of 30 mice (5 per group) were used for this study. Body weight did not differ among the groups over the course of the study, however waking time after anesthesia was increased in all groups subjected to injury compared to mice anesthetized and not injured. Coordinated movement on an accelerating Rotorod revealed a linear trend for decreased performance with increasing number of head impacts suggesting that a graded approach is possible with this model. Time to fall was significantly shorter than controls at 15 and 25 hits. Water maze and T-maze tests are ongoing. Conclusions: These data suggest that a graded injury regimen can lead to graded behavioral responses in the young male mouse and will provide a useful model for testing the effectiveness of neuroprotective compounds that have the potential to be used as prophylactic agents for those involved in contact sports.Item Influence of ovarian hormone deprivation length on the neuroprotective effects of genistein in stroke(2019-03-05) Metzger, Daniel; Sun, Fen; Sumien, Nathalie; Schreihofer, Derek; Oppong-Gyebi, AnthonyPURPOSE: Advancing age increases women’s susceptibility to stroke compared to men, especially after the menopausal transition. Among the reasons proposed for high stroke incidence in postmenopausal women is a significant decrease in estrogen (E2) concentration, based on well-established evidence that E2 is neuroprotective during ischemia in animal studies. While E2 treatment can be beneficial, extended delays in its replacement can result in detrimental actions on the brain which contributes to widespread mistrust of menopausal hormone therapy. Interest in the beneficial effects of soy isoflavones has grown as a viable alternative for E2. However, results from clinical trials have been inconsistent as there seems to be no consensus on the benefits of soy isoflavones in menopausal women. Notwithstanding, evidence suggests a time-dependent benefit of soy isoflavones, even though there is no systematic assessment in preclinical studies to identify the window of opportunity for their proposed optimal benefits. Hypothesis: After long-term hormone deprivation, the soy isoflavone genistein will maintain the ability to provide neuroprotection in the brain following aging and the loss of endogenous E2 in an experimental stroke model. METHODS: Young adult and retired proven breeder Sprague-Dawley rats ( [greater than] 9 mo) were bilaterally ovariectomized, divided into 2 post-ovariectomized time points (2 and 12 weeks) and fed with an isoflavone free (IF) diet. At the end of each time point, rats were continued on IF diet or switched to genistein diet. Two weeks later, rats underwent transient middle cerebral artery occlusion for 60 mins. After stroke rats were subjected to a series of behavioral tests including neurological function, cylinder test, rotarod, and the Morris Water Maze (MWM). RESULTS:Our results demonstrated a significant effect (p CONCLUSION: Dietary genistein had little effect on the sensorimotor outcomes but holds a promise in improving cognitive function post-stroke in the long term.Item Influence of ovarian hormone deprivation length on the neuroprotective effects of genistein in stroke (2018)(2018-03-14) Metzger, Daniel; Smith, Charity B.S.; Doan, Trinh; Han, Jordan; Schreihofer, Derek; Oppong-Gyebi, AnthonyPurpose: Estrogen deprivation increases the risk of stroke, cardiovascular disease, and cognitive decline in women. Studies in rats demonstrate that the beneficial effects of estrogen in the brain are lost 10 weeks after Ovx. Similar effects are seen in women after several years without estrogen. Thus, most benefits require early intervention with hormones after menopause. Unfortunately, estrogens also have undesirable effects (such as breast cancer) that lead women to alternative treatments for menopause, including plant estrogens such as genistein. Natural products are perceived to be safe even though their benefits are not well established. This project sought to investigate genistein’s ability to protect the brain at varying lengths of ovarian hormone deprivation. Hypothesis: Dietary genistein will maintain the ability to provide neuroprotection in the brain and improve functional recovery after long-term hormone deprivation associated with ovariectomy (Ovx). Method: Adult female Sprague-Dawley rats (n=6-8) were bilaterally ovariectomized and randomly assigned to 2- (Short, ST) or 12-weeks (Long, LT) estrogen deprivation before starting diets with no isoflavone or genistein (500 ppm) for the rest of the study. After 6 weeks on diets, all rats were subjected to 60 minutes transient middle cerebral artery occlusion (MCAO) or sham surgery. Neurological (neuroscore), motor (rotarod) and cognitive function (Morris water maze, MWM) were used to assess post-MCAO function over 21 days. Rats were humanely euthanized for biochemical and histological follow-up. Data was assessed with 2-way ANOVA and significance set at p Results: Neuroscore showed a significant effect of stroke, but not diet, in both ST and LT with the LT group performing worse than the ST group (P=0.06). Rotarod showed a significant effect of stroke, but not diet, on learning phase performance for the ST group and a significant interaction between diet and stroke on the learning phase in the LT group. Comparison between ST and LT stroke subgroups showed a significant effect of diet in the learning phase of rotarod. MWM tests are ongoing and suggest that genistein improves performance in the ST groups. Conclusion: Results from these preliminary studies suggest that long-term estrogen deprivation enhances the detrimental behavioral effects of stroke. In sensory-motor assessments, dietary genistein had little effect but may be beneficial for post-stroke cognitive behavior.Item Influence of Testosterone Deprivation and Replacement on Cognition and Oxidative Stress in Middle-Aged Male Rats(2017-03-14) Contreras, Jo; Metzger, Daniel; Oppong-Gyebi, Anthony; Kasanga, Ella; Vann, Philip; Sumien, Nathalie; Cunningham, Rebecca; Schreihofer, Derek; Smith, Charity B.S.Purpose: Data from aged men suggests a negative correlation between testosterone levels and cognitive function, including the development of mild cognitive impairment and Alzheimer’s disease. The purpose of this study was to 1) determine whether long-term testosterone deprivation (LTTD) impairs cognition and increases oxidative stress in the middle-aged male rat brain and 2) determine whether testosterone (T) replacement after LTTD can reverse these effects. Methods: Twelve-month old male Fischer 344 rats (13 per group) were left intact or castrated for 2 weeks and replaced with subcutaneous implants containing T (short-term T deprivation; STTD). Additional groups were castrated for 10 weeks before being treated with T (long-term T deprivation; LTTD+T) or cholesterol (LTTD). Rats underwent cognitive testing with the Morris water maze (MWM). A 4-day acquisition phase was used for rats to learn the location of a hidden platform. A retention day was used to determine whether rats remembered the platform location after it was removed. A 2-day reversal trial in which the platform was moved to a new location was used to examine mental flexibility. These tests require both hippocampal and cortical areas of the brain. Following MWM rats were euthanized and brains were collected for immunoblotting for markers of cell death (Spectrin) and oxidative stress responses (NFkB, COX2, NOX2) in the hippocampus and cerebral cortex. Plasma advanced oxidative protein products (AOPP) were used as a peripheral marker of oxidative stress. Total testosterone was measured by ELISA. Results: Castration reduced total testosterone to 40% of intact levels whereas testosterone implants increased levels back to those of intact males. Overall, intact rats performed significantly worse on the MWM than STTD and LTTD with or without T replacement. We saw no significant changes in blood AOPP among treatment groups. Similarly, there were no significant differences in the expression of oxidative stress regulated genes or Spectrin cleavage in the hippocampus. Cortical measurements are on-going. Conclusions: These data suggest that castration with or without T replacement improves cognitive function in middle-aged rats, but does not significantly alter oxidative stress in the brain or periphery. These data support the safety profile of testosterone replacement to physiological levels and do not recapitulate correlative data observed in men.Item Membrane androgen receptor-induced oxidative stress: mechanism involved in neurodegeneration(2019-05) Tenkorang, Mavis A. A.; Cunningham, Rebecca L.; O'Bryant, Sid; Schreihofer, Derek; Barber, Robert C.Oxidative stress-associated neurodegenerative diseases, such as Parkinson's disease (PD), affect millions of people worldwide. Although aging is the greatest risk factor for PD, other significant factors may be implicated, such as sex hormones that can mediate sex differences. Men have a higher incidence and prevalence of PD than women. Therefore, testosterone, a primary male sex hormone and a known oxidative stressor, is implicated in PD pathophysiology. Since androgens can have negative effects on dopaminergic cells, it is imperative to understand the underlying mechanisms in order to determine what mediates the observed sex differences in PD prevalence. NADPH Oxidase 1 and 2 are major oxidative stress generators in the brain, thus potential targets for testosterone-induced oxidative stress and cell death. This dissertation project therefore investigates the role of androgens and membrane androgen receptor activation on NOX1/2. We hypothesize that in dopaminergic cells, testosterone activates the membrane androgen receptor (AR45) that is complexed with NOX1/2 to increase oxidative stress. In an oxidative stress environment, androgen activation of this AR45-NOX complex leads to cell death. Results indicate that classical androgen receptor (AR) antagonists do not block testosterone's negative actions in an oxidative stress environment. The effects of AR45-NOX complex on cell viability can be blocked by either degrading AR45 protein or blocking NOX activation by apocynin. Further, these results show that testosterone's detrimental effect on cells is via a non-genomic mechanism, specifically via a novel membrane androgen receptor, AR45. The findings of this study help identify key players in testosterone-induced neurodegeneration, which could serve as potential therapeutic targets for PD. Ultimately, this project provides novel mechanisms to explain thought provoking questions on male sex bias in PD.Item Neuroprotection by Novel Sigma 1 ligands(2024-03-21) Kinariwala, Kush; Taylor, Michelle; Schreihofer, DerekPurpose: Identify neuroprotective compounds that could potentially be used for conditions like Alzheimer’s disease. Sigma 1 receptors are an intracellular chaperone protein involved in endoplasmic reticulum stress response. Ligands of sigma 1 receptor have been shown to be acutely neuroprotective in a number of in vitro and in vivo brain injury models including stroke and traumatic brain injury. We are examining potential for novel sigma 1 compounds to protect the mouse hippocampal cell line HT22 from oxidative stress and endoplasmic reticulum stress. Among the compounds tested are haloperidol, cutamesine, oxeladin which are neuroprotective in stroke, and additional proprietary ligands derived from substituted haloperidol. Methods: Cell death in HT22 cells was determined using Cell Counting Kit 8. Cells were plated in quadruplicate in 96 well plates for 24 hours in DMEM/10%FBS. Cells were then treated with either hydrogen peroxide (H2O2, 0.5 mM) or Tunicamycin (50 ng/mL) for 24 hours with or without sigma 1 ligands (100 nM). Results: Haloperidol, a mixed sigma 1 and dopamine agonist, dose dependently protected cells from both H2O2 and Tunicamycin induced cell death. However, the sedative actions of Haloperidol make it unsuitable for use against neuroprotective diseases in vivo. We tested 3 sigma 1 compounds without dopamine activity to determine whether sigma 1 activity would protect cells against these insults. Preliminary results suggests that all sigma 1 compounds tested show some efficacy against oxidative stress and ER stress dependent cell death. Average percent live cells after treatment with 0.5 mM hydrogen peroxide was 59.55% (n = 3) and average percent live cells after treatment with 0.5 mM hydrogen peroxide and 100 nM Haloperidol was 101.06% (n = 5). Average percent live cells after treatment with 50 ng/mL Tunicamycin and 100 nM Haloperidol was 85.8% (n = 2). Average percent live cells after treatment with 50 ng/mL Tunicamycin and 100 nM Oxeladin was 49.7% (n = 2). Average percent live cells after treatment with 50 ng/mL Tunicamycin and 100 nM Cutamesine was 74.4% (n = 2). Average percent live cells after treatment with 50 ng/mL Tunicamycin and 100 nM of a novel sigma 1 agonist was 85.7% (n = 2). Ongoing studies are examining the intracellular pathways responsible for neuroprotective effects. Conclusion: Novel sigma 1 agonists may be suitable for protecting neurons against neurodegenerative diseases like Alzheimer’s. In terms of examining mechanisms, we are exploring endoplasmic reticulum stress pathways and more traditional apoptotic signaling.Item Observations of impaired autonomic regulation of arterial pressure in obese Zucker rats: focus on changes in the nucleus of the solitary tract.(2015-05-01) Fu, Guangchao; Schreihofer, Ann M.; Cunningham, J. Thomas; Schreihofer, DerekLike obese humans, obese Zucker rats (OZR) develop hypertension and impaired baroreflexes. In adult OZR, brainstem nucleus tractus solitarius (NTS) becomes less responsive to baroreceptor inputs. We hypothesized NTS neurons of male OZR undergo abnormal neuroplasticity (ΔFosB expression) versus lean Zucker rats, and these changes would be absent in females with normal baroreflexes. We saw increased NTS ΔFosB at 7-8 wks in male but not female OZR. Unlike male OZR, females had normal baroreflexes and NTS function. We were unable to identify the NTS neurons with ΔFosB. They do not project to caudal ventrolateral medulla (baroreflex pathway), are not A2 neurons, and do not make nitric oxide. These data suggest the NTS of male OZR undergoes abnormal neuroplasticity with the onset of cardiovascular deficits and females are protected. Future studies will identify the NTS neurons and determine what causes changes in them to contribute to impaired cardiovascular regulation in OZR.Item Oxidative stress and androgen replacement as a model for neurodegeneration in the SN56 cell line(2016-03-23) Smith, Charity; Cunningham, Rebecca L.; Schreihofer, Derek; Warren, MarshallBackground: Sleep apnea has been linked to oxidative stress (OxS) in the form of reactive oxygen species. The presence of oxidative stress has been shown to determine either the protective or toxic property of androgens in the N27 dopaminergic cell line, which has been used as a model of Parkinson’s disease. Androgen treatment prior to OxS protects N27 cells from injury, whereas androgen treatment after OxS increases the toxicity of OxS. We sought to determine whether similar oxidative stress dependent androgen effects also occurred in the SN56 cholinergic cell line, which has been used to model Alzheimer’s disease using intermittent hypoxia as an oxidative stress. Hypothesis: We hypothesize that testosterone given prior to oxidative stress is neuroprotective and testosterone given after oxidative stress is neurotoxic. Materials and Methods: The pre-treatment group was exposed to 100 nM of testosterone followed by 17-40 cycles of intermittent hypoxia in a chamber with fluctuating levels of oxygen. One cycle of IH involved reducing the chamber from 20% to 1.5% oxygen for 30 seconds followed by re-oxygenation for 4 minutes. The post-treatment group was first exposed to 17 cycles of IH and then treated testosterone. The control group used SN56 cells that were not exposed to androgen or IH. Cells were lysed at 6 & 24-hour intervals. Western blots were performed for two markers of apoptosis, Poly ADP ribose polymerase (PARP) and alpha spectrin, and were normalized for beta actin levels. Additional experiments used a cell viability assay to determine cell survival 24 hours after the same treatments and 30 or 40 cycles of IH. Results: Cleavage of PARP and alpha-spectrin was significantly increased in the post-treatment group that was lysed at 6 hours, but not in the pretreatment groups. Conversely, both pre and post treatment with testosterone appeared to reduce cell death induced by IH. Conclusions: The results of the cells lysed at 6 hours confirm the original hypothesis that androgens given prior to oxidative stress are neuroprotective while androgens given after oxidative stress are neurotoxic in SN56 cells. However, at longer time points, testosterone appeared to only be protective to SN56 cells. These data suggest that IH, such as occurs with sleep apnea, may interact with androgens in both detrimental and beneficial ways in cholinergic cells, as they do in dopaminergic cells.Item The Effects of Oxidative Stress and Testosterone on Dopamine Neuron Viability: Implications for Parkinson’s Disease(2015-12-01) Holmes, Shaletha S.; Cunningham, Rebecca L.; Singh, Meharvan; Schreihofer, DerekParkinson’s disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. Oxidative stress, mitochondrial dysfunction, inflammation and apoptosis are mechanisms implicated in Parkinson’s pathology. Interestingly, males have a higher incidence of PD than females. Therefore, the major male sex hormone, testosterone may play a role in oxidative stress-induced dopamine neurodegeneration and thus underlie the sex bias observed in PD. Oxidative stress, the imbalance of antioxidant mechanisms and reactive oxygen species, mediates downstream signaling of mitochondria dysfunction, inflammation and apoptosis. Oxidative stress can induce mitochondria dysfunction via calcium neurotoxicity, and oxidative stress can stimulate the pro-inflammatory mediators of NFkB and COX2. This activation of mitochondrial dysfunction and inflammation can trigger apoptosis in dopaminergic neurons. Therefore, it is hypothesized that under oxidative stress conditions, testosterone will induce dopaminergic neurodegeneration by increasing mitochondrial dysfunction and inflammation, leading to apoptosis in dopamine neurons. To test this hypothesis, a N27 dopaminergic cell line was treated with tert-butyl hydrogen peroxide followed by exposure to physiologically relevant concentrations of testosterone to assess cell viability, mitochondria function, calcium influx, inflammation, oxidative stress and apoptosis. These results show that testosterone, alone, increase calcium influx and acts as an oxidative stressor without affecting cell viability. However, under conditions of oxidative stress, testosterone decreases cell viability and exacerbates inflammation, resulting in increased apoptosis. These results indicate that testosterone, only in an oxidative stress environment, can increase pathological features associated with dopamine neurodegeneration in PD. In conclusion, these results suggest that a testosterone mediated mechanism may underlie the increased risk of PD for men compared to women.Item The Role of Sleep Apnea Induced Oxidative Stress in Stroke Pathogenesis and Recovery(2015-03) Bullock, Monica; Schreihofer, Derek; Cunningham, RebeccaPurpose: Obstructive sleep apnea (OSA) is a common, but under-diagnosed comorbidity among patients with a number of age-related disorders, including stroke, Alzheimer’s disease, and Parkinson’s disease. Many of the risk factors for sleep apnea, such as obesity, are modifiable and treatment of sleep apnea itself can limit its systemic effects. Because of these facts, understanding the role of OSA in more serious diseases may promote awareness and early diagnosis, thus preventing serious adverse health outcomes. Given the knowledge that sleep apnea increases oxidative stress, in order to investigate the effects of sleep apnea on the pathogenesis of and recovery from stroke, we used chronic intermittent hypoxia (CIH) as an animal model of sleep apnea in rats. Methods: 12 rats underwent behavioral testing and were then randomly assigned to chambers with either a constant normoxic environment or one that simulates the chronic intermittent hypoxia of sleep apnea. Cerebral ischemia was induced in rats by occlusion of the middle cerebral artery. After a day of recovery, cognitive impairment, oxidative stress, and the size of the ischemic lesion was measured. Results: The experiment showed that CIH increased the size of the stroke lesion in the brain. In this setting, CIH did not appear to alter circulating oxidative stress protein measures or acute stroke behaviors. Conclusion: Based on these results, sleep apnea co-morbidity can have deleterious effects on stroke outcomes.Item The Role of Testosterone Deprivation and Replacement on Stroke Outcome in Middle-Aged Rats(2018-03-14) Han, Jordan; Doan, Trinh; Metzger, Daniel; Oppong-Gyebi, Anthony; Schreihofer, Derek; Smith, Charity B.S.Background: Circulating levels of the steroid hormone testosterone fall in aging men, and in the last decade the number of men obtaining prescriptions for testosterone replacement therapy (TRT) has increased dramatically. However, other consequences of aging, such as increased oxidative stress, may result in detrimental effects when combined with TRT. This include increased risks of thromboembolism and stroke. In women, a delay in hormone therapy (estrogen/progesterone) after menopause results in a loss of benefit for the brain and an increase in risk for stroke and cognitive decline. Whether such a delay would alter the effects of TRT is not known. Hypothesis: In this study, we hypothesized that a delay in TRT following castration in middle-aged male rats would result in increased oxidative stress and a reduction in the neuroprotective effects of testosterone following stroke (transient cerebral ischemia). Methods: Twelve-month old male Fischer 344 rats were obtained from the National Institutes on Aging. Rats were divided into 5 groups as follows: 1) gonad Intact sham stroke (SHAM), 2) Intact stroke (INT), 3) short term castrate + TRT (STT), 4) long term castrate (LT), and 5) long term castrate + TRT (LTT). Rats were castrated 2 weeks (STT) or 10 weeks (LT, LTT) prior to TRT by subcutaneous silastic capsules containing T. L3T rats were treated with the antioxidant TEMPOL in drinking water starting 2 weeks before TRT. Middle cerebral artery occlusion (Stroke) was accomplished under gas anesthesia by stereotaxic injection of the vasoconstrictor endothelin 1 (ET1) adjacent to the left middle cerebral artery. One, 3, 7, and 14 days after stroke, rats were assessed for neurological deficits using a standardized scoring system. Forelimb bias to the ipsilateral left side was assessed using the cylinder test, and coordinated walking was assessed with an automated ladder walk. Following behavior assessments, rats were humanely euthanized and blood and brains were collected. The effects of stroke and treatments were compared to intact sham stroke (SHAM). Results: Peripheral oxidative stress measured by Advanced Oxidative Protein Products (AOPP) was significantly negatively correlated with T levels, similar to men. ST rats experienced the smallest neurological deficits following stroke, suggesting thatItem UNTHSC’s MedSci Program Prepares Students for Medical School Success(2024-03-21) Stafford, Suzanne; Schreihofer, DerekPurpose: It is well known that a medical school acceptance is difficult to obtain. The Association of American Medical Colleges’ most recent report of medical school acceptances shows that in 2021, a total of 62,443 students applied to U.S. medical schools, and 23,711 were accepted1. With a greater demand for seats than are available, admissions committees must determine who is or is not eligible for attending their program. The Texas College of Osteopathic Medicine (TCOM) has a class size generally seating 230–240 students from a pool of about 4,500–5,000 applicants2. For students whom the office of admissions feels could benefit from additional curriculum preparation, the University of North Texas Health Science Center (UNTHSC) offers a postbaccalaureate Master of Science in Medical Science. UNTHSC had its first Post-Baccalaureate Premedical Certification Program class in the fall of 2000 with a total of 7 students. The Medical Science master’s program was offered in 2005. The intent was to provide qualified, yet ultimately not accepted, applicants an opportunity to prepare for the rigors of medical school while improving their medical school application. In 2017, about 82% of the students who completed the program were successful in receiving either admission to a medical or dental school in the United States3. Currently, the program has grown to over 300 students and includes face-to-face and online cohorts. Methods: Our study examines the development and changes to the program over a 20-year period including curriculum and cohort size. We also examine the academic readiness of the TCOM-attending “MedSci” students to their non-MedSci classmates by comparing individual TCOM course GPAs and both COMLEX and STEP medical board examination passing scores. Results: Over the span of the program, the required courses have become more similar to the year 1 curriculum that TCOM offers. In 2021, the average TCOM GPA of former MedSci students was 86.61. The average GPA of their non-MedSci counterparts was 85.45. The general trend from 2015–2021 shows comparable scores for the MedSci students. For the 2021 COMLEX exam, 94.12% of MedSci students passed on their first attempt compared to the 97.62% of non-MedSci students. The average COMLEX 1 score for MedSci students was 559.83 compared to the non-MedSci average score of 546.92. Although not required, some students chose to take STEP 1, the first USMLE board examination. Of the MedSci students, 46 of 51 chose to take the exam with a first-time passing rate of 97.83% and an average score of 230.81. Of the non-MedSci students, 133 of 168 chose to take the exam with a first-time passing rate of 96.99% and an average score of 226.15. Conclusion: The MedSci program offered by UNTHSC allows students who were originally not accepted to medical school to match or exceed the academic expectations for medical students. 1Association of American Medical Colleges. (2021, December). 2021 Fall Applicant, Matriculant, and Enrollment Data Tables. 2TCOM admissions statistics. Texas College of Osteopathic Medicine. (2023, May 18). https://www.unthsc.edu/texas-college-of-osteopathic-medicine/admissions-and-outreach/tcom-admissions-statistics/ 3Medical science. School of Biomedical Sciences. (2024, February 2). https://www.unthsc.edu/school-of-biomedical-sciences/medical-sciences