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    Altered gene expression in HIV gp120 transgenic mouse brains: Effects of methamphetamine treatment.
    (2018-03-14) Ghorpade, Anuja; Molles, Brian
    Purpose: Antiretroviral therapy (ART) has extended lifespans by decades for HIV-infected individuals. However, the prevalence of HIV-associated neurocognitive disorders (HAND) continues to be high despite successful ART therapy. Brain astrocytes can harbor provirus and express neurotoxic HIV proteins such as gp120 and TAT, potentially contributing to HAND. In addition, neurocognitive decline is exacerbated in individuals who use methamphetamine (METH). We hypothesize that METH treatment in gp120+ mice will cause changes in gene expression and damage/death to specific cell populations. Methods: To examine the role of METH in HAND, we used a transgenic mouse line expressing GFAP-controlled HIV gp120 protein (gp120+). Mice were injected intraperitoneally with either 0.9% saline vehicle or successive weekly escalating doses of 1, 5, 10, or 30 mg/kg METH, and their brains were harvested 7 days post-injection for qPCR, immunohistochemistry and protein analyses. Results: In qPCR experiments, gp120+ mice showed dramatically increased levels of GFAP mRNA, suggesting chronic gp120 expression causes astrocyte activation. However, a qPCR probe designed to distinguish the transgene versus the endogenous GFAP transcripts showed lower levels of GFAP activation, suggesting that some of the GFAP mRNA expression is read-through from the transgene construct. GFAP expression in the rostral portion of the brain (anterior to ~0 bregma) was lower than the caudal portion in the gp120- mice, whereas GFAP was higher in the rostral portion in the gp120+ mice, suggesting enhanced astrocyte activation in the rostral portion of the brain encompassing the striatum and frontal cortex. Tissue inhibitor of metalloproteinase-1 (TIMP1) and interleukin 1-beta (IL1b) mRNA levels were increased in gp120+ mice compared to gp120- mice. Gene expression levels of excitatory amino acid transporter-2, tyrosine hydroxylase, and dopamine transporter were not changed in gp120+ mice. None of the METH treatments changed in gene expression in either mouse group at 7 days post-injection. Conclusions: Long-term expression of gp120 in brain leads to altered gene expression of neuroinflammatory mediators. Single-dose METH treatment did not alter gene expression for the targets studied in either the gp120- or gp120+ mouse lines. Future experiments will focus on changes in protein expression and functional properties of specific cell populations in the brain at different times post-METH treatment.
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    (2018-03-14) Yang, Shaohua; Hersh, Jessica
    Purpose: In post-ischemic stroke, T-lymphocytes enter the brain. Presently, the role of these T-cells in the progression of cerebral infarction or repair mechanisms is unclear. Our goal is to analyze the function of T-cells in regions of cerebral infarction by examining the pro- and anti-inflammatory interaction between T-cells and brain cells within these lesions. Methods: Ischemic stroke was induced by middle cerebral artery occlusion in young adult C57/B6 male mice. Mice were sacrificed at 3 days or 1-month post-ischemic stroke. T-cells were harvested from the brain by digestion; percoll enriched, and incubated with anti-CD3 and CD25 antibodies. T-cells were sorted via flow cytometry. The cytokine expression profile of brain infiltrated T-cells was compared to spleen T-cells using q-RTPCR. Results: In this in vivo study the following cytokines, poststroke, were found to be elevated: IFN-γ, IL-10, IL-17, TNF, Perforin, T-bet, and RORc. Conclusions: Our data suggests that understanding the interaction between T-cells and astrocytes could open new therapeutic strategies for stroke patients.
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    Cholesterol sulfate alters astrocyte metabolism and offers some neuroprotective effects
    (2018-03-14) Yang, Shao-Hua; Prah, Jude
    Introduction: Cholesterol sulfate (CS) is one of the most important known sterol sulfates in human plasma and present as a normal constituent in a variety of human tissues. In both the brain and periphery, CS serves as a substrate for the synthesis of sulfonated adrenal steroids such as pregnenolone sulfate and Dehydroepiandrosterone (DHEA) sulfate and as a constituent of many biological membranes including red blood cells where it functions as a stabilizing agent. It also acts as endogenous regulator of cholesterol synthesis. It is known that CS serves as a substrate for synthesizing other sterol sulfates in the brain. However, the role of CS in neurological insult and brain metabolism is unknown. Our goal in this study is to investigate the neuroprotective action of CS as well as its effect on brain energy metabolism. Materials and Methods: Primary astrocytes were prepared from the cortex of postnatal day 0-2 C57BL/6 pups and seeded in Dulbecco’s modified eagle medium (DMEM) with 10% FBS under normal glucose (5.5 mM). HT-22 cells were maintained in high glucose (25 mM) DMEM supplemented with charcoal stripped FBS. The neuroprotective effect of CS and its role on cell metabolism were determined in primary astrocyte and HT-22 cells using Calcien AM cell viability assay, flow cytometry, seahorse extracellular flux analysis, and metabolism assay kits. Results: CS protects HT22 cells against glutamate toxicity and impact astrocyte metabolism by increasing ATP, and glycogen contents. Conclusion: Our study demonstrated that CS have neuroprotective effect and modulate brain energy metabolism. Further studies are needed to determine the mechanisms underlying the neuroprotective action of CS and its action on brain energy metabolism.
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    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 that
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    Moyamoya Disease Presenting as Subarachnoid Hemorrhage without Cerebral Aneurysm
    (2018-03-14) Mirzadeh, Angela
    Abstract Background: Moyamoya Disease (MMD) is a rare cerebrovascular disease caused by progressive stenosis or occlusion of the distal portion of the internal carotid arteries and/or the major cerebral arteries that arise from it. The stenosis triggers the development of an abnormal network of compensatory vessels to maintain cerebral blood flow. Subarachnoid hemorrhage (SAH) is a rare presentation of MMD, and non-aneurysmal subarachnoid hemorrhage is extremely rare, with only six previously documented cases worldwide. Our case report is an example of this extremely rare case presentation. Case Description: In this report, we describe the case of a 52-year-old male who suddenly developed severe headache and loss of consciousness after sexual intercourse. CT scan revealed SAH over the left frontal and temporal cortex. Cerebral angiogram demonstrated no aneurysm, but high grade narrowing of the left middle cerebral artery (MCA) and collateral blood flow from surrounding cerebral arteries consistent with moyamoya disease. The patient was managed medically throughout his hospital course and remained neurologically intact with no further hemorrhagic events. He was referred to neurosurgery outpatient for evaluation of revascularization surgery. Conclusions: Non-aneurysmal SAH in moyamoya disease is extremely rare. The evidence from this case, as well as the literature, supports the hypothesis that it is due to rupture of fragile transdural anastomotic vessels on the brain surface as they traverse through the subarachnoid space. An understanding of this unique mechanism of disease is valuable not only for tailored management, but also for considerations in surgical approach to revascularization.
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    Influence of exogenous oxidative stress on the resilience of aged glutathione-deficient mice
    (2018-03-14) Sumien, Nathalie; Mock, J.; Davis, Delaney
    Influence of exogenous oxidative stress on the resilience of aged glutathione-deficient mice  Delaney L. Davis, J. Thomas Mock and Nathalie Sumien    Introduction  With age, individuals become less resilient against stressors, rendering them more susceptible to diseases and leading to exacerbated brain impairments. Aging also involves a shift in redox state that can be associated with the key molecule, glutathione. To determine the importance of glutathione in stress resilience, we sought to study the effect of an oxidative stressor (paraquat) in an animal model of chronic glutathione deficiency. Glutathione deficiency can be achieved by the genetic knockout of the modifier subunit of the enzyme, glutamate cysteine ligase (gclm). Our hypothesis was that chronic glutathione deficiency will render mice less resilient to the oxidative stress induced by paraquat and will cause further impairments in motor and cognitive function. Methods   Old (18 months) male and female gclm+/+ and gclm-/- mice received one to two intraperitoneal injections of 10 mg/kg of paraquat (PQ) or saline for one week. Four days following the last injection, animals underwent behavioral tests measuring affective, cognitive and motor function (locomotor activity, elevated zero maze, bridge walking, wire suspension, fear conditioning, rotorod, and active avoidance). After the completion of behavioral testing, brain regions, skeletal muscle, liver, heart, and kidney tissues were harvested and will be used to examine biochemical changes in oxidative damage and redox signaling. The data were analyzed using 2 or 3-way ANOVAs followed by pairwise comparisons. Results  Body weights in male and female mice dropped immediately after PQ injection, with the gclm-/-treatment group having a greater decrease in percentage of body weight than the gclm+/+. PQ injections appeared to decrease overall activity of both gclm+/+ and gclm -/-. PQ-treated mice seemed to spend less time in open arms, indicating increased anxiety levels. Overall, PQ-treated mice appeared to perform better on motor skills and worse on cognitive tests. Conclusions These preliminary data suggest a trend that paraquat may have differential effects depending on the task (cognitive vs. motor), and gclm -/- mice may be more susceptible to an oxidative stressor.
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    Inline flow sensor for ventriculoperitoneal shunts: Experimental evaluation in swine
    (2018-03-14) Qin, Chuchu; Williams, Arthur Jr.; Dasgupta, Pernandu; Mallet, Robert T.; Yurvati, Albert; Eskildsen, Dane
    Background: Hydrocephalus is a potentially life-threatening disorder in which cerebrospinal fluid (CSF) fails to circulate properly, causing a dangerous buildup of pressure in the cerebral ventricles and the surrounding brain tissue. It is seen most often in infants with congenital abnormalities of the CSF tract, and in patients with traumatic brain injury. The only treatment in most cases is the placement of a tubing to drain the excess CSF from the brain to the abdomen. These shunts are prone to blockage that requires invasive replacement surgery, so a reliable flow sensor is needed to detect shunt failure at its early stages. Currently available flow sensors often fail to detect blockage. Objective: The purpose of this project is to evaluate an advanced in-line electronic flow sensor capable of monitoring CSF flow over time for use in the treatment of hydrocephalus. This project evaluated the performance of this sensor in domestic swine. Methods/Materials: Ventriculo-peritoneal shunts were installed in the third cerebral ventricle of juvenile Yorkshire pigs, and routed to the peritoneal space in the abdomen. The flow sensor was positioned halfway between the cephalic and peritoneal ends of the shunt. Data were acquired on a laptop computer. Shunt flows were obtained at 30 s intervals. The sensor alternately heated the shunt fluid for 5 s and then monitored temperature decline, the rate of which was proportional to flow, for 25 s. The fluid was diverted into pre-weighed vials for 1- or 5-min to determine flow gravimetrically. At regular intervals, 5-20 ml boluses of artificial CSF were injected into the third ventricle. Flows reported by the sensor were compared to concomitant gravimetric flows by linear regression. Results: Over 4300 sensor measurements of flow were obtained in 6 experiments. The flow sensor reliably reports shunt flows up to 35 ml/min, the highest rate produced by 20 ml CSF injections. Four experiments showed strong linear correlations (r2 ³ 0.90) between gravimetric and sensor flows. The slope of the linear regression between the two flows was 1.05 ± 0.14 in the 6 experiments, indicating that the sensor accurately reported flows of up to 35 ml/min. Conclusions: The results of this experiment indicate that the flow sensor can report accurately ventriculo-peritoneal shunt flows over a wide range in a large animal model. Studies are planned to evaluate performance of chronically implanted shunts in ambulatory pigs.
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    Corticotropin-Releasing Hormone Receptor 2 in the Nucleus of the Solitary Tract Contributes to Sleep Apnea Induced Hypertension
    (2018-03-14) Nguyen, Dianna; Cross, Stephanie; Mifflin, Steve; Wang, Lei
    Purpose: This study tested the hypothesis that corticotropin-releasing hormone (CRH) derived from the paraventricular nucleus of the hypothalamus (PVN) acts in the nucleus of the solitary tract (NTS) to facilitate sleep apnea induced hypertension. Materials and Methods: To model sleep apnea induced hypertension, we exposed rats to intermittent hypoxia (IH) for 7 days. To detect CRH type 1 and type 2 receptors (CRHR1 and CRHR2) in the NTS, we conducted in situ hybridization. To study the signaling pathway of CRH, we performed calcium imaging on NTS slice preparation using Fura-2-acetoxymethyl ester. To test whether central CRH contributes to IH-induced hypertension, we implanted telemetry transmitters and osmotic mini pumps to infuse CRH receptor agonist/antagonist into the 4th ventricle of rats. To selectively excite CRH-producing pathways from the PVN to the NTS, we optogenetically stimulated the PVN and the NTS of CRH-Cre mice that received intra-PVN injections of Cre-inducible viral constructs expressing Channelrhodopsin 2 (ChR2). Results: The mRNA level of CRHR2 was significantly higher than CRHR1 in the NTS. CRH induced a transient increase of intracellular calcium level in NTS neurons that was abolished by the voltage-dependent calcium channel blocker nifedipine. CRH-induced calcium influx was attenuated by the CRHR2 antagonist K41498 but not by NBI-35965, an antagonist for CRHR1. Calcium influx was induced by the CRHR2 agonist Urocortin II but not by the CRHR1 agonist Stressin I. More importantly, IH decreased the CRHR2 mRNA level and attenuated the CRH-induced calcium influx in the NTS. Further in vivo studies revealed that IH-induced hypertension was significantly attenuated by chronic intra-4th ventricle infusion of the CRHR2 antagonist K41498, but was significantly exacerbated by chronic intra-4th ventricle infusion of the CRHR2 agonist Urocortin II. Optogenetic stimulation of either CRH somas in the PVN or CRH fibers in the NTS that originated from the PVN significantly increased blood pressure (somas, 12.3 ± 1.13 mmHg; fibers, 3.54 ± 0.69 mmHg), suggesting that activation of CRH projections from the PVN to the NTS increases blood pressure. Conclusions: These results suggest that CRH derived from the PVN activates CRHR2 in the NTS, which may contribute to sleep apnea induced hypertension; down-regulation of CRHR2 and CRHR2-mediated calcium influx in the NTS may serve as compensatory responses to protect against sleep apnea induced hypertension.
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    Tissue Plasminogen Activator-Porous Magnetic Nanorods for Targeted Thrombolytic Therapy after Ischemic Stroke
    (2018-03-14) Jin, Kunlin; Hu, Jiangnan
    Purpose: Stroke is the 5th leading cause of death in the US. The only FDA-approved treatment is the intravenous administration of tissue plasminogen activator (tPA). However, due to tPA’s inability to lyse the clot fully, about 90% of these patients still live with speech or motor impediments. Moreover, the large dose of tPA administered increases the susceptibility to global tPA-mediated hemorrhage. Therefore, the purpose of the study was to increase tPA’s thrombolysis rate and reduce total tPA administered using a novel nanomaterial, tPA-loaded Fe3O4 nanorods (tPA-NRs). We hypothesize that tPA-NRs will be more successful at thrombolysis and minimize the off-target effects of tPA. Methods: Fe3O4 nanorods were fabricated by oblique angle deposition technique and loaded with tPA using glutaraldehyde as the cross-linker. To determine the thrombolysis efficiency of tPA-NRs in vitro, PE 50 catheters containing blood clots were used as the vascular thrombosis model to mimic in vivo thrombotic conditions. Such PE 50 catheters containing blood clots were placed vertically in the center of a rotating magnetic field and the blood clot lysis time was recorded. To examine the proposed approach in vivo, a FeCl3-induced distal middle cerebral artery occlusion (dMCAO) model was used. Animals will randomly be assigned to four groups and treated with tPA-NRs (1 mg/kg), NRs (1 mg/kg), tPA (10 mg/kg) or vehicle accordingly via internal carotid artery injection after ischemic stroke. A custom-made rotational magnetic field was applied above the head of mouse (near infarction region) during and after injection for 60 min, and the thrombolysis process was observed under microscope. Results: In vitro results demonstrated that tPA-NRs could achieve a mass loading ratio as high as 12.9% and the loaded tPA can be released when stimulated by an external rotating magnetic field. Furthermore, PE50-catheter thrombolysis results demonstrated that tPA-NRs had a significant enhancement of thrombolysis efficiency in comparison with high-dose tPA group (P In vivo results unequivocally showed that: 1) intra-arterial injection of tPA-NRs could target the site of the clot under magnetic guidance; 2) the mechanical force generated by the spinning of the tPA-NRs under the external rotational magnetic field could significantly decrease dMCA blood flow recanalization time from 85 min with high dose tPA (10 mg/kg) to 25 min with low dose tPA-NRs (1 mg/kg) (p Conclusions: In summary, this study provides a proof of concept for developing novel, biocompatible, magnetically guided tPA-NRs delivery system to enhance thrombolysis after ischemic stroke. This approach is significant in that it could not only revolutionize for the treatment of ischemic stroke but also have major impacts on treatments for other deadly thrombotic diseases such as myocardial infarction and pulmonary embolism.
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    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, Anthony
    Purpose: 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.
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    Hormone treatments reverse stroke-associated declines in cognitive function in a rat model of menopause
    (2018-03-14) Davis, Delaney; Li, Wenjun; Liu, Ran; Winters, Ali; Forster, Michael; Yang, Shaohua; Sumien, Nathalie; Vann, Phillip
    Purpose This study addresses the critical questions important to the future of hormone therapy. The purpose of this study was to provide information on how different durations of hormone deprivation can alter the responsiveness of the brain to ischemic injuries and hormonal therapies. Ultimately, these studies will identify a window of opportunity for treatment with hormones preventing brain dysfunction associated with menopause. Methods Eighty-two Sprague-Dawley retired breeder females rats were ovariectomized (ovx). Twelve or two weeks post-surgery, the rats were implanted with hormone pellets containing cholesterol (vehicle), estrogen (E2) or progesterone (P4), which were replaced every 2 weeks. Two weeks post implantation, the rats received either a sham or ischemic stroke (transient Middle Cerebral Artery Occlusion) surgery. After a one week recovery period, the rats were subjected to a behavioral battery of tests measuring affective (plus maze), motor (rotorod) and cognitive (Morris water maze) function. The rats were then euthanized and brain regions were collected for further biochemical analyses. Data were analyzed using 2- or 3-way ANOVAs followed by pairwise comparisons. Results Treatment with E2 or P4 decreased the time spent in the open arms in both 2 and 12 weeks post-ovx groups. There was no effect of stroke or hormone treatment on the rotorod. For spatial learning and memory, stroke impaired the rats in their ability to learn and retain the location of the platform and impairments were worst in the 12-weeks post-ovx group. E2 and P4 treatment improved performance of the stroke rats in both 2 and 12-weeks post-ovx groups. Conclusions These data suggest that the outcome of stroke is worst as a function of time post-ovx, especially on spatial learning and memory. Hormonal treatment with E2 and P4 were successful in reversing the deleterious effects of stroke on cognitive function. Further studies to identify the mechanisms underlying these observations are underway.
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    NADPH oxidase (NOX1) mediates testosterone-induced neurodegeneration
    (2018-03-14) Duong, Phong; Cunningham, Rebecca; Tenkorang, Mavis
    Background: Parkinson’s disease (PD) has been recorded as the second most common neurological disease. Oxidative stress (OS) plays a key role in the pathogenesis of PD. Several studies have established that Parkinson’s disease (PD) is sex biased, affecting more men than women. Testosterone, a primary male sex hormone and a known oxidative stressor, has been implicated in PD. Previous studies in our lab have shown that testosterone via a non-genomic mechanism exacerbates OS damage in dopaminergic neurons. However, the mechanism by which testosterone increases OS is unknown. We found that testosterone acts through a membrane associated androgen receptor (mAR) variant – AR45 leading to the activation of proinflammatory mediators; NF-κB and COX2. NADPH Oxidase 1 (NOX 1) is a major OS generator in cells, hence a potential contributor to the pathogenesis of neurological diseases. It is possible that NOX 1 complexes with the mAR to mediate this destructive process. Purpose: The primary objective of this study is to determine the underlying mechanism by which testosterone increases OS in dopaminergic neurons. We therefore hypothesize that in dopaminergic cells, testosterone increases oxidative stress by activating NOX 1. Ultimately, our goal is to identify pathways regulated by testosterone in dopaminergic neurons in order to provide effective pharmacological targets to enhance the treatment of PD. Methods: We used a dopaminergic cell line (N27 cells). For an oxidative stressor, we used tert-butyl-hydrogen peroxide (H2O2) to induce 20% cell loss prior to testosterone (100nm) administration. NOX 1 inhibitor, Apocynin was administered before H2O2 exposure. To examine membrane associated androgen receptor and not the classical androgen receptor, we used cell impermeable DHT-BSA (500 nM) to confirm that NOX 1’s effect is through a non-genomic mechanism. Cell viability and OS were quantified using the MTT and Reduced Thiols assays respectively. To determine if NOX 1 interacts with a mAR, we immunoprecipitated the mAR and probed for NOX 1. Results: Apocynin alone had no effect on cell viability and OS. Further, Apocynin alone, did not alter H2O2-induced cell loss, indicating that H2O2 increases OS via a non-NOX 1 mechanism. However, Apocynin blocked testosterone’s induced cell loss and OS generation suggesting that NOX 1 mediates testosterone’s damaging effects in an OS environment. Inhibition of NOX 1 also blocked DHT-BSA’s damaging effects on cell viability in an OS environment. NOX 1 protein also complexes with the mAR. Conclusion: Testosterone-induced cell loss is mediated by a NOX1/mAR complex, indicating that NOX 1 is involved in testosterone-induced OS generation. The findings of these experiments provide a better insight into testosterone’s role in neurodegeneration and its underlying mechanism.
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    Life on the Catwalk – Age and redox state effects on mouse gait
    (2018-03-14) Knight, Sherilynn; Vann, Phillip; Wong, Jessica; Davis, Delaney; Forster, Michael; Sumien, Nathalie; Mock, J.
    1. Purpose Human clinical research has determined that changes in gait speed can be predictive of other impairments and can help identify at-risk individuals for further declines. However, age-related gait changes are not well defined in rodents, even though they are used as the primary pre-clinical model for a multitudes of diseases and for aging research. The purpose of our study was to measure age-related differences in gait, using an automated system, the CatWalk™ XT. Furthermore, age-related functional declines have been associated with a decrease in the reduced to oxidized glutathione ratio leading to a pro-oxidizing cellular shift. Using a model of chronic glutathione deficiency, we sought to determine whether redox state was a key factor in age-related gait impairments. Our hypothesis was that gait declines observed with aging would be exacerbated in glutathione-depleted mice. 2. Methods Groups of wild-type (gclm+/+) and knock-out (gclm-/-) mice aged 4, 10 and 17 months were tested on the CatWalk and gait measurements were recorded. Resulting dependent measures including gait speed, front and hind base of support, front and hind stride length, front and hind stride speed, front and hind step cycle, and front and hind duty cycle were analyzed using two-way analyses of variance with Age and Genotype as between-groups factors, followed by pairwise comparisons. 3. Results Age-related declines were observed in all aforementioned measures of gait, and chronic glutathione depletion was unexpectedly associated with delays in age-related declines for some of the measures. 4. Conclusions The CatWalk is a useful and sensitive tool to assess gait changes with age in rodents, and further studies will be required to identify the potential compensating mechanisms underlying the effects observed with the chronic glutathione depletion. Research Area Neuroscience Presentation Type Oral presentation
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    Histone deacetylase signaling plays a critical role in age-related decrease in adult neurogenesis
    (2018-03-14) Yang, Chenqi; Jin, Kunlin; Zhang, Hongxia
    Aging is associated with a striking increase in the incidence of stroke and neurodegenerative diseases, both of which are major causes of disability among those age 70 years and older in the United States. Adult neural stem/progenitor cells (NSCs) hold great promise for brain repair due to their unique location within the central nervous system, which continues through the life span. However, neurogenesis is significantly declined with aging. Purpose: the underlying mechanisms remain largely unexplored. Methods: HDAC expression in the young adult and aged brain was determined by immunohistochemistry. And then the young adult and old rats are administrated with histone deacetylases (HDACs) inhibitor by intraperitoneally and then immunohistochemistry test was performed. Results: First, we found that HDACs I and II were expressed in the NSCs in the subventricular zone (SVZ) and the subgranular zone (SGZ) of hippocampus in normal adult rat brain using immunohistochemistry. Second, the expression level of HDACs in the SVZ and SGZ was significantly altered in aged brain compared with young adult brain. In addition, the number of NSCs in aged brain was significantly increased after administration of HDAC inhibitor. Conclusion: Our data suggest that HDAC signaling may be an important factor in determining the neurogenesis in aged brain.
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    Development and Use of “Sniffer Cells” to Detect the Presence of Neuropeptides
    (2018-03-14) Amune, Anna; Bachelor, Martha; Yuan, Joseph; Cunningham, Tom; Farmer, George Jr.
    Purpose: The brain is sensitive to Angiotensin (Ang) II and expresses the enzymes necessary for synthesis. However, neuropeptide release has historically been difficult to study and it is currently unclear if Ang II is utilized as a neurotransmitter within the brain. To address this question, our laboratory has adopted a relatively new approach to study the brain renin-angiotensin system – sniffer cells. Methods: To make the sniffer cells, Chinese Hamster Ovary (CHO) cells were transfected with plasmids to express angiotensin type 1a (AT1a) receptors and a genetically encoded fluorescent Ca2+ sensor (GCaMP or R-GECO) to address the mechanisms of brain angiotensin II release. Sniffer cells were plated on glass cover slips and continually perfused with aCSF. Calcium imaging was performed at 2 sec intervals using excitation/emission wavelengths of 488/525 nm (GCaMP) or 589/615 nm (R-GECO) and fluorescent intensity was measured in response to bath application of neuropeptides. Sniffer cells were also placed on the median preoptic nucleus (MnPO) in in vitro brain slices (produced using standard slice procedures) from male Sprague-Dawley rats (250-300 g). Fluorescent intensity was measured at 1 sec intervals in response to electrical and optogenetic stimulation of the subfornical organ (SFO). Results: The sniffer cells exhibit an increases in fluorescence in response to exogenously applied Ang II that is blocked by the AT1aR antagonist Losartan. The observed increases in fluourescence was specific to AT1aR activation as exogenous application of other common neurotransmitters (Glutamate, Glycine, GABA, NE, ACh) failed to increase sniffer cell. The intensity of sniffer cell responses to Ang II and Ang III were dose dependent with the sniffer cells being more sensitive to Ang III. Using these sniffer cells we were able to detect spontaneous release of Ang II in the MnPO (n = 63). The spontaneous release was activity dependent (i.e. blocked by TTX, n = 10). We were also able to evoke release of Ang II onto sniffer cells via both electrical stimulation (n = 5) and optogenetic stimulation (n = 23) of the SFO. Conclusion: Using sniffer cells we were able to detect release of Ang II from in vitro brain slices. Future studies using these sniffer cells will 1) verify the existence of the brain-renin-angiotensin system, 2) characterize the phenotype of Angiotensin II releasing neurons, and 3) investigate potential changes in brain Angiotensin II release in sleep apnea. Sniffer cells are a useful tool for the detection and quantification of neuropeptide release in in vitro neuronal slice preparation and may offer utility in other applications.
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    Neurocognitive decline and dysregulation of Astrocyte-TIMP-1 in a Tat-transgenic mouse model
    (2018-03-15) Sumien, Nathalie; Ghorpade, Anuja; Joshi, Chaitanya
    Purpose: Despite antiretroviral therapy, HIV-associated neurocognitive disorders (HAND) persist in 60-70% of patients. In the brain, HIV-1 non-productively infects astrocytes, which produce and release HIV-1 proteins such as transactivator of transcription (Tat). Tat induces neuronal death and inflammation by direct and indirect mechanisms. During HAND, elevated matrix metalloproteinases (MMPs) aid ECM breakdown facilitating disease progression; whereas, tissue inhibitors of MMPs (TIMPs) impede their activity. Astrocyte TIMP-1 is an inducible protein and its neuroprotective effects have been shown. Astrocyte TIMP-1 expression increases with acute neuroinflammation in vitro, but its levels are reduced during chronic inflammatory brain diseases; indicative of a concomitant loss of TIMP-1 mediated neuroprotection. However, little is known about Tat regulation of astrocyte TIMP-1 expression. We hypothesize that HIV-1 Tat downregulates astrocyte TIMP-1 and induces inflammatory changes that contribute to neurocognitive decline. Methods: A doxycycline-inducible, glial fibrillary acidic protein (GFAP) promoter-restricted HIV-1 Tat mouse model (GT-Tg) was used to investigate astrocyte associated disease mechanisms. Neurocognitive decline was assessed using a battery of behavior tests in GT-Tg and wild-type (WT) mice. Subsequently, mouse brains were harvested to evaluate gene and protein expression. Results: GT-Tg mice had higher anxiety and lower initiation latency in elevated plus maze and locomotor activity tests, respectively. While GT-Tg mice swam faster in Morris water maze, latency and pathlength were comparable to WT. Discriminated reversal test and novel object recognition did not differ significantly between GT-Tg and WT mice. Although TIMP-1 gene expression was elevated in GT-Tg verses WT mice, it negatively correlated with Tat expression consistent with human astrocytes chronic responses. Gene and protein expression for other inflammatory biomarkers and GFAP were evaluated, and correlated with Tat expression. Conclusions: Collectively, our data from GT-Tg mouse model confirmed that TIMP-1 dysregulation is associated with neurocognitive decline in the context of HAND suggesting replenishing TIMP-1 levels could be used as a novel therapeutic option.
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    Cocaine-induced stroke susceptibility: motor and cognitive outcomes
    (2018-03-14) Taylor, Cynthia; Li, Wenjun; Forster, Michael; Yang, Shaohua; Sumien, Nathalie; Vann, Philip
    Title: Cocaine-induced stroke susceptibility: motor and cognitive outcomes Presenters: Philip Vann, Cynthia Taylor, Wenjun Li, Michael J. Forster, Shaohua Yang, Nathalie Sumien Purpose: Epidemiological findings suggest that the number of young individuals suffering from stroke seems to be increasing, and one of the most common cause for such an increase is the use of illicit drugs. Prior work in our laboratory suggested that life-long cocaine intake impaired cognitive function and that short-term intake induces brain changes conferring vulnerability. In this study, we tested the hypothesis that repeated cocaine use will induce brain vulnerability to ischemic stroke. Methods: Fifty seven young male Sprague-Dawley rats (3 months) were injected i.p. with cocaine (10mg/kg) or saline (3 times/wk) for 4 weeks. From each treatment group, half of the rats received an ischemic stroke (transient Middle Cerebral Artery Occlusion) and the other half a sham surgery. After a one month recovery period, the rats were subjected to a behavioral battery of tests measuring balance, motor function, spatial learning and long term memory (locomotor activity, bridge walking, rotorod, and Morris water maze). Once behavioral testing was finalized the rats were euthanized and brain regions were collected for further biochemical analyses. Data were analyzed using 2- or 3-way ANOVAs followed by pairwise comparisons. Results: The stroke surgery resulted in decreased body weights and increased overall activity (total distance travelled and horizontal activity). Maximum performance on the rotorod was lower for the stroked rats than for the shams, and treatment with cocaine did not affect the outcome. However, during training the cocaine-treated rats had higher latencies than the controls. On the bridge walking test, the stroke surgery did not seem to affect performance, however the cocaine-treated stroke rats performed the worst. The stroked rats took longer path length and latencies to reach the platform, and cocaine seem to exacerbate the impairment, more specifically at the end of training and during retention. Conclusions: While preliminary, these results suggest that cocaine-treated rats were more vulnerable to stroke than the saline-treated ones but exhibiting exacerbated impairments on balance and spatial learning and memory. Studies to identify the underlying mechanisms of this vulnerability are underway. IACUC 2016-0022 Intramural grant UNTHSC RI10014
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    Positive allosteric modulation of alpha7 nicotinic acetylcholine receptors as a novel approach to treatment of ischemic stroke
    (2018-03-14) Gaidhani, Nikhil
    Ischemic stroke is a leading cause of disability and death worldwide. Despite substantial investments in developing anti-stroke medicines, clinically effective pharmacological treatments remain inadequate. Clinical utility of tissue plasminogen activator (tPA, Alteplase), the only FDA-approved drug treatment is limited (72 h) intravenous (i.v.) or subcutaneous (s.c.) administration of PNU significantly reduced brain injury and neurological deficits after MCAO. The therapeutic efficacy of PNU after stroke may arise from activation of multiple converging α7-dependent therapeutic pathways including direct cytoprotection and central/peripheral anti-inflammatory mechanisms and may hold significant translational potential. Our results may become a starting point for developing clinically efficacious therapies utilizing α7 agents and may enable health-care providers to overcome limitations linked to the lack of effective treatments after stroke
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    Using human pluripotent stem cell-based models to understand a congenital deglycosylation disorder-induced abnormality in cerebral development
    (2018-03-14) Salick, Max; Zolekar, Ashwini; Shakhbazau, Antos; Ho, Yin; Hoffmann, Peter; Kayakas, Ajamete; Wang, Yu-chieh; Lin, Victor
    Background: Human pluripotent stem cells (hPSCs) provide significant promise for regenerative medicine and in addressing questions relevant to the disease physiopathology. Therefore, the generation of hPSCs using somatic cells obtained by noninvasive approaches would be ideal for securing patient samples and further implement the notion of personalized insight into disease and treatment. Neurodysfunction caused by a congenital disorder of deglycosylation, NGLY1 deficiency, was recently identified. How NGLY1 deficiency disturbs normal cerebral development and causes neurological abnormalities in the pediatric population is unknown. Purpose: Our desire is to establish a hPSC-based disease model of NGLY1 deficiency and study the pathogenic neurogenesis associated with NGLY1 loss and its influences on early development of the human brain. Method: Urinary epithelial cells from urine specimens of normal individuals and patient skin biopsy-derived fibroblasts were collected and reprogrammed to generate transgene-free human induced pluripotent stem cells (hiPSCs). Through gene editing, NGLY1-deficient hPSCs were obtained from the normal hPSCs which included human embryonic stem cells (hESCs) and hiPSCs. The NGLY1-deficient hPSCs and their normal counterparts were differentiated into neural cells using 2D- and 3D-differentiation protocols optimized by our group. Systems biology and 3D-imaging techniques were employed to characterize the cellular and molecular features of NGLY1-deficient neurodevelopment. Results: Urine-derived hiPSCs were pluripotent and capable of forming cerebral organoids that contain multiple types of neural cells in a self-organized and layered 3D structure closely mimicking the early human forebrain development. Like normal hPSCs, NGLY1-deficient hPSCs also form cerebral organoids. Unique gene expression patterns in NGLY1-deficient samples at different stages of development were identified by transcriptomic analysis. Structural abnormalities were also observed in NGLY1-deficient organoids compared with normal organoids. Conclusions: We have streamlined the production of cerebral organoids using hiPSCs reprogrammed from non-invasively collected cells via urine specimens and NGLY1-deficient hPSCs. Additionally, we demonstrate our ability to recapitulate NGLY1 deficiency down to the molecular level, and to continually uncover insights into a pathologically enigmatic disease. In doing so, we clearly exemplify a beginning to end approach that can be used to study other neurological diseases, as well as, assay their potential therapies in an all human-based and personalizable system.
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    Connexin 43 as a Mediator of Androgen and Estrogen-induced Protection against Oxidative Stress in Astrocytes
    (2018-03-14) Rybalchenko, Nataliya; Singh, Meharvan; Kubelka, Nicholas
    Purpose: Connexin 43 (Cx43) is a transmembrane protein highly expressed in astrocytes, whose expression and/or channel permeability can impact cell viability in the presence of oxidative stress. Androgens and estrogens are also known to protect against oxidative stress. However, it remains unknown whether these hormones alter Cx43 channel expression or permeability as a mediator of their protective effects. Here, we propose that the androgen, dihydrotestosterone (DHT) and the estrogens, E2 and 3betadiol (the latter being a metabolite of DHT), protect against oxidative stress in astrocytes through the regulation of Cx43 expression and/or permeability. Hypothesis: Reducing the permeability of Cx43 gap junctions or Cx43 hemichannels enhances the protective efficacy of E2, DHT, or 3betadiol against oxidative stress in cerebral cortical astrocytes. Methods: Using cortical astrocytes from neonatal female C57/Bl6 mice and the astrocytic C6 glioma cell line, we assessed changes in Cx43 mRNA expression, Cx43 permeability, and cell viability following treatment with 100 nM E2, DHT, or the DHT metabolite, 5-alpha-androstane-3-beta,17-beta-diol (3betadiol), and compared it to the effect of the vehicle (DMSO) control, in the presence or absence of the metabolic and oxidative stressor, iodoacetic acid. Changes in Cx43 mRNA expression relative to GAPDH were assessed by RTPCR; The MTT assay was used to assess cell viability under conditions of metabolic/oxidative stress, with or without the pharmacological manipulation of Cx43 channels; changes in Cx43 hemichannel permeability were assessed using the Ethidium Bromide dye uptake assay. Results & Conclusions: While only E2 transiently increased astrocyte Cx43 mRNA expression, E2, DHT, 3betadiol each blocked IAA induced increase in Cx43 hemichannel permeability to an extent similar to the hemichannel antagonist Boldine. Given that Boldine-induced hemichannel blockade significantly protected cells from oxidative stress, hemichannel blockade may also be a component of these steroids’ protective mechanisms. These data indicate that enhanced Cx43 hemichannel permeability not only inhibits the protective effects of E2 or DHT, but that the protective effects of E2 and DHT may be mediated, at least in part, by the regulation of Cx43 hemichannel permeability.