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    Catwalk analysis: a novel approach to profiling age differences in gait
    (2016-03-23) Wong, Jessica; Vann, Phillip; Forster, Michael; Sumien, Nathalie; Mock, J.
    The Catwalk is a validated video-based gait analysis tool for quantitative analysis of both static and dynamic differences in rodent gait and weight-bearing. The Catwalk has been used to analyze disease states such as stroke, sciatic nerve crush and osteoarthritis, however age-related changes in rodent gait are not well characterized. Motor impairments have been associated with dysregulated cellular redox state, namely levels of reduced to oxidized glutathione. Glutamate-cysteine ligase modifier (gclm) is a key enzyme sub-unit in the production of glutathione (GSH), and knocking it out reduces GSH levels by 85%. The gclm -/- mice are a potential model of accelerated aging and should develop impairments earlier than wild-type mice. Our hypothesis was that gait measures would decrease with advanced age and that impairments would occur sooner in gclm -/- mice. Wild-type (wt) and gclm-/- male and female mice were tested at 4, 10 or 17 months of age (n = 8-11/group). All testing was done in pitch black and a camera below the illuminated transparent platform captured each paw print as animals walked across the platform. Paw prints were automatically labeled according to left/right or front/hind then manually checked. A criterion for a good run was set as less than 60% speed variation within the run and less than 10% speed variation between runs. Two to five runs per animal were used for two-way analyses of variance of the dependent measures collected using Genotype and Age as in between factors. The dependent measures were gait speed (SP), front and hind base of support (BoSf, BoSh), front and hind stride length (SLf, SLh), front and hind stride speed (SSf, SSh), front and hind step cycle (SCf, SCh), and front and hind duty cycle (DCf, DCh). Gait speed decreased with age in both genotypes. BoS decreased in front legs and increased in hind legs in both genotypes. Stride length was decreased with age, and more prominently in the gclm-/-, while stride speed decreased with age and was higher in gclm-/-. Step cycles increased with age in the wt but not in the gclm-/-. Duty cycles increased with age, especially in the gclm-/-. These preliminary data suggest that age leads to measurable changes in mouse gait and that GSH dysregulation had only minor effects on gait. In conclusion, Catwalk analysis is sufficiently sensitive to measure subtle age-related changes in gait across several age-ranges, and can be added to the current battery of behavioral tests.
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    Enhanced Excitability of Vasopressin Neurons in the Supraoptic Nucleus Following 48 hr. Water Deprivation
    (2016-03-23) Little, Joel; Knapp, Blayne; Cunningham, Tom; Farmer, George Jr.
    Purpose Arginine vasopressin (AVP) is a neurohypophyseal hormone released from the posterior pituitary by magnocellular neurosecretory cells (MNCs) located within the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus. AVP is involved in the regulation of body fluid homeostasis and influences blood pressure, plasma osmolality, and blood volume. The regulation of AVP release is critical to maintaining body fluid homeostasis and is dependent on the activity of MNCs. The balance of excitatory and inhibitory inputs are important elements of activity, however the mechanisms leading to changes in AVP release are not fully understood. Water deprivation (WD), a physiological challenge, was used to examine changes in excitatory neurotransmission in MNCs using patch-clamp electrophysiology. Methods Male Sprague-Dawley rats weighing 250 – 350 g received bilateral SON infusions of an adeno-associated virus (AAV) construct containing mouse AVP gene promoter and EGFP reporter. Two weeks following AAV infusions, rats were water deprived for 24 hr. or 48 hrs. Controls were allowed ad libitum access to water. Following water deprivation, coronal brain slices (300 µm) containing the SON were cut using standard procedures. Whole-cell patch clamp recordings were obtained from slices superfused with aCSF containing tetrodotoxin (TTX; 0.5 µM) and bicuculine methbromide (Bic; 10 µM). Baseline mEPSCs were recorded then NMDAR mediated components were pharmacologically isolated with 5 minutes drug application of AMPA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10uM). Parameters measured for mEPSCs were amplitude (pA), rise time (ms), decay time (ms), charge transfer, and mEPSC frequency. Results In EGFP labeled SON neurons (i.e. confirmed vasopressinergic neurons), water deprivation increased the frequency of mEPSCs in both the 24 hr. group (p Conclusion Although no changes in amplitude, rise time, decay time, or charge transfer were detected, there was an increase in the frequency of mEPSCs following both 24 hr. and 48 hr. WD. It remains unclear how water deprivation leads to enhanced mEPSC frequency in MNC of the SON. Future studies will investigate the mechanisms that underlie this water deprivation dependent plasticity.
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    Towards elucidating mechanisms of synaptic vesicle fusion: A computational study
    (2016-03-23) Zuo, Zhicheng; Liu, Jin; Bhatta, Ram
    Objective: Neurotransmission, a process by which neurotransmitters are released from synaptic vesicles to the synaptic cleft, is essential for the neuron communication. Each neurotransmitter can influence neurons in the brain and affect the behavior during the neurotransmission. The misfortune in the neurotransmitter release can cause several neurological complications such as depression, Parkinson’s disease, Alzheimer’s disease and autism. As a critical step in the neurotransmitter release process, synaptic vesicle fusion requires vesicle membrane and plasma membrane fusion, which in turn, is facilitated by the protein machinery including SNARE complex. However, how this protein machinery helps with synaptic vesicle fusion remains unclear because the highly dynamic feature of this machinery greatly challenges currently available experimental methods. The goal of this study is to build a computational model to simulate the synaptic vesicle fusion process. Methods: Chemistry at Harvard Molecular Mechanics (CHARMM) and Visual molecular dynamics (VMD) were used to generate SNARE/membrane complexes. The MD simulations were performed using Nanoscale Molecular Dynamics simulator on Texas Advanced Computing Center. CHARMM27 general force field was implemented to solve the equations of motion in 3D PBC. The Particle Mesh Ewald algorithm was used to calculate long-range Coulomb interactions. Results: We built a novel all-atom computational model including SNARE complex, vesicle membrane and plasma membrane. The bicelle model of these membranes was stabilized by DHPC present in 20 nm edge of each membrane. SNARE molecules were preferentially arranged in trigonal planar and pentagonal planar conformations between vesicle membrane and plasma membrane separated at 20 angstroms apart from one another. In each of the planar conformations, SNARE molecules are arranged is such a way that C-terminals are towards the center of membranes and N-terminals are towards the edge of membranes. The transmembrane region (TMR) helices of syntaxin-1 and synaptobrevin were stabilized deep inside the plasma and vesicle membrane, respectively. Conclusions: We successfully built a novel computational model at atomic level to simulate synaptic vesicle fusion process. The conformational dynamics of SNARE/membrane complex were investigated at the molecular level. Our efforts provided a novel tool and revealed new insights towards a clear understanding of the mechanism of neurotransmission.
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    Alcohol and HIV-1 differentially regulate Toll Like Receptor (TLRs) expression and signaling in Primary Human Astrocytes
    (2016-03-23) Ghorpade, Anuja PhD; Pandey, Richa
    About 69% of human immunodeficiency virus-1 (HIV-1)-positive individuals exhibit some form of HIV-associated neurocognitive disorders (HAND). Several studies have reported that HIV-1 virions, viral proteins and alcohol, individually have direct or indirect effects on HAND pathophysiology. Recently, we showed that alcohol activates astrocytes and regulates inflammation via cPLA2 in HAND. Toll-like receptors (TLRs) are a family of innate immune system receptors that respond to pathogen-derived and tissue damage-related ligands. TLR signaling in immune cells, astrocytes, microglia and neurons may play roles in the pathogenesis of multiple diseases. TLRs are a “missing” link in alcohol-mediated astrocytic response in context of HAND since TLR stimulation by alcohol in glial cells induces secretion of pro-inflammatory molecules. Thus, we explored the role of TLRs in alcohol-induced inflammation and cytotoxicity in primary human astrocytes with HAND. TLRs signaling gene array was performed to screen altered profiles for all 10 TLR family members and 74 downstream signaling molecules. Ingenuity pathway analysis (IPA) was performed to identify potential signaling nodes. Data suggested that HIV-1 and/or EtOH led to differential TLRs expression in astrocytes. We confirmed all 10 TLRs by real-time PCR in four independent astrocyte donors. Alcohol alone and with HIV-1, significantly upregulated TLR1, 2, 3, 4, 5 and 9 as compared to controls and HIV-1 alone. We propose that TLRs regulation plays an important role in astrocytes inflammation upon HIV-1 and EtOH exposure.
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    Functional performance and quality of life in transtibial amputees is influenced by the type of prosthesis
    (2016-03-23) Patterson, Rita; Godon, Steven; Ginzel, Elizabeth; Bugnariu, Nicoleta; Peters, Rebecca
    Purpose/Hypothesis: Lower limb amputees may receive a prosthetic limb based on their functional ability, known as a K-level, ranging from least functional, K0 to most functional K4. The purpose of this study was to evaluate effects of two types of prosthetic feet on balance, walking performance and quality of life in transtibial amputees classified as either K2 or K3. Number of subjects: Seven males and three female, aged 48 to 69 years old with transtibial amputations due to peripheral neuropathy or type II diabetes have completed the study; enrollment is ongoing. Materials/Methods: Study participants are transtibial amputees secondary to diabetes, trauma, or vascular disease currently ambulating with either a K2 or K3 prosthesis. Quality of life was established using SF 36 and the Reintegration to Normal Living Index. Clinical tests: Timed Up and Go, Short Physical Performance Battery, Dynamic Gait Index and Activity Specific Balance Confidence Scales were administered. The V-gait CAREN system measured standing balance sway, gait speed, walking kinematics and kinetics on level ground and on a 4.8 degree ramp. The Physiological Cost Index was calculated for level and ramp walking. Participants completed all test with their current prosthesis and then switched prosthesis types in order to determine immediate effects of switching. Participants were then randomized into groups for a 2-week trial period to evaluate prosthetic foot that are either at, above or below their current functional level. Results were analyzed with pared t-tests. Results:Baseline quality of life, balance, and gait measures for participants currently ambulating with a K2 vs k3 were significantly lower (p Conclusions: Preliminary results suggest that K3 prosthetic feet lead to greater quality of life and functional performance. A higher functioning prosthesis for lower functioning amputees may lead to less injuries and falls due to improved balance and coordination, and it may also improve cost effectiveness.
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    Treatment of Hypertensive Obese Zucker Rats with Metformin Enhances Phenylephrine-Induced Bradycardia and c-Fos Expression in the NTS
    (2016-03-23) Schreihofer, Ann PhD; Chaudhary, Parul
    With the progression of metabolic syndrome adult obese Zucker rats (OZR) develop impaired baroreflexes that coincide with a reduced ability to activate the nucleus tractus solitarius (NTS). The NTS, which receives baroreceptor afferent inputs, appears to respond less to acute rises in mean arterial pressure (MAP) as suggested by less phenylephrine (PE)-induced c-Fos expression compared to age-matched lean Zucker rats (LZR). In addition microinjection of glutamate into the NTS produces smaller reductions in splanchnic sympathetic nerve activity and MAP in adult OZR versus LZR. We have recently observed that prevention of hypertension improves baroreflexes in OZR, but they are still impaired compared to like-treated LZR. Because hyperglycemia impairs NTS function and baroreflexes in type I diabetic rats, we hypothesized that improvement of glycemia in OZR would also enhance baroreflexes and the ability to activate the NTS. OZR and LZR were treated with metformin (MET; 300 mg/kg/day in drinking water) for 3 weeks (began at 13 weeks of age) and compared to untreated age-matched rats. Blood samples were taken in conscious, unfasted rats at 9-10 am. Compared to untreated OZR, MET-treated OZR had lower blood glucose levels (179±19 vs. 120±8 mg/dl, P
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    The role of miRNA in regulating Progesterone's neuroprotective function in the ischemic brain
    (2016-03-23) Singh, Meharvan PhD; Su, Chang Phd, MD; Nguyen, Trinh
    Abstract: Stroke has been reported as the fourth leading cause of death for Americans and it is a leading cause of adult disability. The risk of ischemic stroke increases significantly with aging. Gender appears to play a profound role, with the incidence being higher in women. A large body of studies has suggested that women in postmenopausal state are at greater risk of ischemic stroke and are likely to experience much more severe impacts. A considerable amount of research has supported that progesterone (P4) is a potent neuroprotectant that may exert beneficial effects in various neurodegenerative diseases and stroke. Our laboratory has reported that Brain-derived neurotrophic factor (BDNF) is a critical mediator for P4 neuroprotective actions. BDNF has well-defined roles in synaptogenesis and neuronal survival. We recently reported that P4 enhances BDNF release from glia, but not from neurons, by acting via a novel membrane-associated progesterone receptor, Pgrmc1. Here, we identified a member of the Let-7 microRNA (miRNA) family as a potential negative regulator of Pgrmc1. Our data demonstrated an inverse association between the expression levels of Let-7 and the transcripts of Pgrmc1 and BDNF in post-ischemic mouse cortex. Literature supports the antagomir (synthetic inhibitor) of Let-7 miRNA significantly reduced infarct volume and improved neurological deficits in a rodent ischemic stroke model. When combined, these lines of evidence have strongly supported our hypothesis that in the stroked brain, Let-7 negatively regulates Pgrmc1 gene expression, which disrupts P4-induced BDNF release from glia and ultimately leads to the attenuation of P4’s positive effect on synaptogenesis. Methods: 1. Cell culture: Mouse primary cortical neurons and primary cortical astrocytes were derived from postnatal day 1 male pups. Primary astrocytes were maintained in DMEM with sodium pyruvate/10% FBS + 1% penicillin/streptomycin. Primary neurons were maintained in Neurobasal A media with 10% FBS + 1% penicillin/streptomycin. 2.Treatment of primary astrocytes: Primary astrocytes were transfected with either Let-7 mimics or inhibitors. qPCR was used to confirm the overexpression and knock down of Let-7, as well as expression of their potential targets. Western blotting was used the determine the protein levels of the miRNA potential targets. 3.Treatment of neuronal cultures with conditioned media (CM) from glia: Primary astrocytes were transfected with miRNA mimics/inhibitors. Then cultures were treated with vehicle control (0.1% DMSO ) or 10 nM P4 for 18 hrs. The CM was concentrated by Protein Concentrators with 9 KDa molecular-weight cutoff (Pierce) to remove P4. Concentrated CM was then applied to neuronal cultures. 4. Animal treatment: Female C57BL/6J mice were bilaterally ovariectomized at 4.5 months old to eliminate endogenous ovarian production of P4. One week afterward, P4 pellets or cholesterol pellets were implanted in these animal subcutaneously. 2 days after pellets implantation, ischemic stroke was induced in these mice using the middle cerebral arteries occlusion method. 24hrs after stroke induction, the antagomir of let-7or scrambled control were delivered to the penumbra via intracerebral ventricular (ICV) injection.
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    Inhibition of tyrosine hydroxylase in substantia nigra, but not striatum, reduces locomotor activity in an open-field.
    (2016-03-23) McInnis, Tamara; Salvatore, Michael F.
    Background: The risk of locomotor impairment increases substantially during aging. There are two primary sources of locomotor impairment; aging-related Parkinsonism and Parkinson’s disease (PD). One of the three cardinal symptoms of PD is bradykinesia, which is also observed in aging-related Parkinsonism. The neurobiological basis for the manifestation of bradykinesia in PD is associated with [greater than] 70% loss of dopamine (DA) in the striatum, the terminal field compartment of the nigrostriatal pathway. Coincident with bradykinesia onset in PD, others have reported 40-50% DA loss in the substantia nigra (SN), the somatodendritic compartment of this pathway. While this degree of DA loss occurs in SN in aging, DA loss in striatum has never been reported to reach 70%. We have previously shown in young male rats that DA tissue content can be specifically reduced in either DA compartment to levels previously reported in aging studies with direct delivery of the tyrosine hydroxylase (TH) inhibitor, α-methyl-p-tyrosine (AMPT) in either striatum or SN. Hypothesis: We tested the hypothesis that DA reduction in the SN alone would be sufficient to reduce locomotor activity. Methods: To ensure that locomotor function would be devoid of aging-related confounds to locomotor performance, 6 month old Brown-Norway Fischer 344 F1 rats were used. To selectively reduce DA tissue content in either striatum or SN, rats were first anesthetized to implant double guide cannula to target striatum (+1.0 AP, 2.5 ML, 5.0 DV) or the SN (-5.7 AP, 2.5 ML, 7.5 DV). Following recovery, rats were placed into an open-field for three hour sessions immediately following infusion of saline or AMPT on a different day. This was repeated 5 times to acquire locomotor activity following saline or AMPT infusion. Results: Infusion of a quantity of 1.4 nmole AMPT into the SN, but not striatum, produced a significant decrease in open-field locomotor activity out to 2 hours past infusion. Infusion of 14 nmole AMPT into striatum did not produce significant decreases in open-field locomotor activity. Conclusions: The inhibition of TH activity in the SN, but not striatum, reduces open-field locomotor activity in young rats. This inhibition has been previously reported to reduce DA in SN or striatum to levels comparable seen with aging. Therefore, TH function in the SN may be a molecular component in the ability to initiate locomotor activity.
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    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, Marshall
    Background: 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.
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    Astrocyte AEG-1 regulates ER stress responses in the context of HAND
    (2016-03-23) Ghorpade, Anuja PhD; Nooka, Shruthi
    Endoplasmic reticulum (ER) stress has recently been linked to neurological disorders, including HIV-associated neurocognitive disorders (HAND). We recently showed astrocyte elevated gene (AEG)-1, a multifunctional oncogene regulating astrocyte migration, proliferation and neuroinflammation. AEG-1 upregulation in Huntington’s disease model suggests its role in ER stress responses in aging and HAND. However, its involvement in ER stress responses during HIV-1 infection is not known. In this study, we investigated HIV-1 and anti-retroviral therapy (ART) drugs mediated ER stress i.e., unfolded protein response (UPR) pathway activation, and astrocyte AEG-1 expression, intracellular localization during ER stress. RT-PCR and western blot analysis revealed that HIV-1, IL-1β and ART drugs activated UPR pathway and autophagy in astrocytes. Moreover, astrocytes exposed to ER stress compounds upregulated AEG-1 expression. Confocal analysis and mPTP assay showed AEG-1 colocalization with calnexin and mitochondrial damage with ER stress. In addition, AEG-1 overexpression upregulated ER stress markers such as BiP, PERK, and CHOP that were further enhanced by IL-1β treatment. Immunocytochemical studies also showed increased autophagy markers i.e., LC3 and P62 in AEG-1 overexpressing astrocytes. In summary, our study highlights that HIV-infection and ART drugs induce ER stress in astrocytes that is further exacerbated by AEG-1. Therefore, elucidation of AEG-1 regulated UPR pathway could assist in targeting astrocyte-induced ER stress responses in HAND.
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    Neuropharmacological Responses are Observed in Mice After Systemic Treatment with a Prodrug of Thyrotropin-Releasing Hormone
    (2016-03-23) Bailey, Brooke; Nguyen, Vien; Prokai, Laszlo; Prokai-Tatrai, Katalin; Shurlknight, Kelly
    Short description: Thyrotrophin-releasing hormone (TRH) is a promising agent to treat various brain maladies. However, delivery of this peptide into the brain has been an obstacle. In this exploratory study, a TRH prodrug designed for brain-enhanced delivery was evaluated through the reversal of drug-induced narcosis (analeptic effect) and antidepressant-like effect. Purpose: To show the utility of ketamine/xylazine in a mouse model to evaluate analeptic effect evoked by TRH upon its brain-delivery via a novel prodrug approach, as well as to confirm the antidepressant-like effect of this neuroactive peptide when delivered into the brain. Methods: For the assessment of analeptic effect, CD1 mice were divided into groups of n = 8. Test compounds were dissolved in saline. Mice were treated with a single dose (10 µmol/kg) of prodrug, saline vehicle, TRH, respectively, through the tail vein by i.v. injections. Ten minutes after injection of drug, mice were injected i.p. with either a mixture of ketamine (100 mg/kg) and xylazine (10mg/kg), or sodium pentobarbital (60 mg/kg). Sleeping time was recorded starting from the loss of righting reflex until this reflex was regained. To evaluate antidepressant-like activity using the Porsolt’s swim test, test compounds were administered i.v. through the tail vein at the dose of 3 µmol/kg, in a separate study. For 6 min, the immobility time (the duration of motionless floating after the cessation of struggling and making only movements necessary to keep the head above the water) was recorded. Results: TRH also reduced sleeping times after ketamine/xylazine sedation. Administration of the TRH prodrug also manifested analeptic effect characteristic to the parent peptide. Like after TRH injection, immobility time in Porsolt’s swim test indicative of the peptide’s antidepressant-like activity also shortened after the administration of its prodrug when compared to saline control. Thus, the analeptic and antidepressant-like effects observed after systemic administration of the TRH prodrug has reflected its ability to penetrate the blood-brain barrier followed by the release of the parent peptide at the site of action. Delivery of TRH to the brain by specific prodrug approach would allow for reduction of TRH’s endocrine side effects and for a prolonged duration of action. Conclusions: Treatment by a TRH prodrug reduced the ketamine/xylazine- and sodium pentobarbital-induced sleeping time in mice, as well as shortened the immobility time in Porsolt’s swim test. These observations have indicated a successful delivery of the neuroactive peptide into the brain via its prodrug introduced here.
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    Increased duration of isoflurane exposure reduces cerebral infarct and improves neurological function in focal cerebral ischemia in rats
    (2016-03-23) Gaidhani, Nikhil
    Isoflurane is a widely used gas anesthetic for surgical procedure in rodents. Using a rodent model of transient ischemic stroke (transient midcerebral artery occlusion; tMCAO) our laboratory has demonstrated that longer exposure period to isoflurane anesthesia significantly reduce cerebral ischemic damage. The objective of this study was to demonstrate the correlation between the duration of isoflurane exposure and isoflurane-induced neuroprotection after tMCAO. Three groups were created based on total duration of isoflurane exposure during tMCAO and rats were randomly assigned to these groups; Isoflurane-20 min, Isoflurane-40 min, and Isoflurane-90 min. Focal cerebral ischemia was induced by a transient (90 min) middle cerebral artery occlusion in the rats from each group. Measurements of cerebral infarct volumes and neurological behavioral tests were performed 24hrs post-MCAO. We report that rats from Isoflurane-20 min group sustained significantly larger cerebral infarct than animals from Isoflurane-40 min and Isoflurane-90 min groups. Rats from Isoflurane-90 min groups showed near complete resistance to ischemic damage. Neurological function also significantly improved with increased duration of isoflurane exposure as evidenced by the results of Bederson and Cylinder tests. Our results clearly demonstrate the neuroprotective effect of isoflurane during ischemic stroke and provide strong evidence that increasing the duration of isoflurane exposure during the tMCAO procedure significantly reduce brain injury and improve neurological function after focal cerebral ischemic stroke.
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    Health Disparities and Novel Biomarkers for HIV-1 Disease Progression
    (2016-03-23) Ghorpade, Anuja; Nejtek, Vicki; Aryal, Subhash; Shenoy, Sangeeta
    Purpose: According to the World Health Organization, thirty five million people are living with a diagnosed HIV infection worldwide and approximately a million in the United States. The burden of illness in the United States falls disproportionately on ethnic/racial minorities. After the advent of antiretroviral therapy (ART) and greater life expectancy of infected individuals there is a significant increase in cognitive impairment or HIV-1 associated neurocognitive disorders (HAND-30-60%). Research indicates that pro-inflammatory proteins and other biomarkers may correlate with the level of neurological impairment. We propose biomarkers including sCD40L, correlate with the level of neurological impairment and these factors may vary in individuals living with HIV/AIDS dependent on their age, gender, racial/ethnic background and disease progression. Methods: HIV-1 seropositive Caucasian, African American and Hispanic men and women (20 in each category) older than 20 years were recruited from clinics in Dallas- Fort Worth metroplex area. The study visit included informed consent, drug screening, HIV-1 relevant medical history review, socio-demographic survey, neurocognitive assessment (CNS vital signs) and blood sampling. Patients will be followed up for a second visit a year later. Patient samples (Plasma, Cellular DNA, RNA and protein) were isolated for biomarker analysis. Database was compiled in socio-demographic, virological, medical history, neurocognitive, and inflammatory biomarkers parameters. Database was coded in categorical and numerical variables .The difference in protein levels between gender, cognitive status and race/ethnicity was analyzed by SPSS. Episomal 2-LTR circles were also tested and are important markers for ongoing viral replication and viral latency. Results: Preliminary screening of whole plasma samples from participants showed observable levels for sCD40L and other inflammatory biomarkers, which correlated with the socio-demographic and neurocognitive test data for all first visit subjects. Episomal 2-LTR circles were quantified. Conclusion: Pro-inflammatory biomarker levels can be accurately measured in patient plasma samples to correlate between different gender, racial/ethnic background and disease progression. HIV-1 cDNA levels can be a useful tool in monitoring therapy in HIV-1 infected individuals. We expect to obtain and verify trends in immune biomarkers in context of race/ethnic backgrounds.
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    Exploring GFRα-1 as a potential therapeutic target for preventing tyrosine hydroxylase loss in an experimental Parkinson’s Disease model
    (2016-03-23) Gajewski, Austin; Owens, Catherine; Pruett, Brandon; McDivitt, Lisa; Salvatore, Michael F.; Tan, Christopher
    Background: Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. Glial cell-line derived neurotrophic factor (GDNF) increases dopamine neuron function in vitro and in vivo in rodent models, making it a promising candidate for the treatment of PD. In fact, unilateral delivery of GDNF increased locomotor function bilaterally in two of three PD clinical trails. Lack of improvement in a subsequent clinical trial has halted further clinical work. Thus, evaluating downstream impact of GDNF may identify additional targets to improve locomotor impairment in the PD patient. Hypothesis: Striatal GDNF infusion increases expression of GFRα-1 and tyrosine hydroxylase (TH) phosphorylation in the substantia nigra (SN) of aged rats 4 weeks after GDNF delivery. Here we tested the hypothesis that GDNF may produce a bilateral increase in GFRα-1 expression in the striatum & SN, between one and four weeks after striatal delivery. We further hypothesized that the long-lasting effect of GDNF on dopamine and TH may be related to sustained expression of GFRα-1, and therefore determined if GFRα-1 could be protective against loss of TH and dopamine caused by 6-hydroxydopamine (6-OHDA) lesion. We used a rat model of PD to address this question. Methods: Part 1. To test whether unilateral GDNF could increase GFRα-1 bilaterally, GDNF (30 µg) was delivered unilaterally into the dorsal striatum of 24-month old Brown-Norway Fischer 344 F1 male rats. Striatal and SN tissue samples were bilaterally dissected at one day, one week, and four-week time intervals after GDNF infusion. Part 2. To determine if GFRα-1 alone could protect against 6-OHDA lesion, GFRα-1 was infused into either striatum or SN via guide cannula 6 days following lesion. Dopamine and TH expression were determined 4 days later. Results: Part 1. GDNF increased in GFR-α1 expression bilaterally in the striatum and SN four weeks following GDNF compared to 1 week expression levels. Part 2. GFR-α1 (1 ng) delivered into the SN produced a significant reduction in both dopamine and TH protein loss caused by 6-OHDA that was selective for the SN. However, GFR-α1 delivery into the striatum did not produce any reduction in either dopamine or TH loss. Conclusions: Increased GFR-α1 expression in the SN may be associated with the previously observed effects of GDNF upon locomotor function via expression of TH and increased dopamine content in the SN alone.
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    Examining Active Theater and Clinical Outcomes as Indicators of mild TBI in Post-Deployed Veterans vs. Civilians at No-Risk for mild TBI: A Longitudinal Evaluation
    (2016-03-23) Talari, Deepika M.P.H; O’Bryant, Sid Ph.D.; Nejtek, Vicki PhD
    Purpose: Of the ~300,000 veterans who experience mild TBI, 40% have ‘residual’ neuropsychological symptoms lasting 3-months or longer – a timeframe considerably longer than the gold standard for diagnosing mild TBI in the post-deployed condition. Failure to recognize residual symptoms as a consequence of mild TBI in veterans is common as these symptoms are often misattributed to posttraumatic stress disorder (PTSD). Determining risks for mild TBI retrospectively is understudied. Here, we examined active theater and clinical indices as retrospective risk indicators for mild TBI in post-deployed veterans compared to civilians. Hypotheses: Veterans at-risk for mild TBI will have poorer depression, anxiety, and quality of life outcomes than civilians with no risk. Risks for mild TBI in veterans will be influenced by military experiences and clinical indices. Methods: Longitudinal data from 182 veterans and 74 civilian clients (n=256) receiving cognitive behavioral therapy (CBT) at Recovery Resource Council from 2013-2015 were analyzed. Descriptive statistics, frequency distributions, ANOVA and regression modeling, were used to test the hypotheses using a 95% confidence level and an alpha level of 0.05 to determine statistical significance. Results: Depression, anxiety, and quality of life scores measured before, after 6- and 12- sessions of CBT were significantly worse in veterans compared to civilians. Active theater and clinical indices predicted risks for mild TBI. 74 civilians and 52 veterans had no-risk for mild TBI (n=126) and 130 veterans were at-risk for mild TBI. Veterans at-risk for mild TBI had significantly higher depression and anxiety scores and lower quality of life scores than the no-risk group measured before, after 6- and 12-sessions of CBT. Risks for mild TBI predicted PTSD severity, depression, anxiety, and quality of life scores at all three time points. Conclusion: Active theater and clinical indices identified veterans at-risk for mild TBI. Even with 12-sessions of CBT, veterans at-risk for mild TBI had poorer depression, anxiety, and quality of life scores than those with no-risk. These outcomes indicate that mild TBI presents with long-lasting psychological symptoms beyond a 3-months timeframe that do not fully resolve with CBT. Veterans returning from active theater should receive a thorough neuropsychological evaluation to differentiate mild TBI from PTSD in order to receive proper treatment.
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    Visuomotor Integration in Atypical Development
    (2016-03-23) Miller, Haylie; Patterson, Rita; Bugnariu, Nicoleta; Crocker, Kayla
    Visuomotor Integration in Atypical Development Purpose Children with Developmental Coordination Disorder (DCD) commonly present with impairments in both gross and fine motor functions, which could be attributed to abnormalities in visuomotor integration. These impairments negatively affect their ability to coordinate appropriate postural responses while interacting with the environment. It is unclear whether visuomotor deficits seen in DCD individuals occur in attaining visual input, integrating visual information with other sensory inputs, or implementing a motor response. The purpose of this study was to determine how individuals with DCD, compared to those of typical development and eventually to those with ASD, integrate visual information from the environment to maintain postural stability. Methods Twelve participants aged 8 to 11 years old, eight with DCD, two with ASD and two controls participated in this study. Enrollment is ongoing. This study utilized a 12-camera motion-capture system, a Computer Assisted Rehabilitation Environment Network (CAREN), ETG 2.0 eye tracking system, a 180° wrap-around screen, and computers for controlling and integrating all components. Participants completed one or more visuomotor tasks. In the Disc Match task, participants displace their center of pressure in medial lateral direction to maintain overlap with a disc moving on the screen from left to right at 8 different frequencies. In the Shooting Ducks task participants select, aim, and shoot 24 virtual moving ducks. Percentage of overlap at each frequency and time of execution per target were analyzed with t-tests. Results Percentage of overlap time between target stimulus and Center of Pressure (COP) representation during the Disc Match task revealed overall trend of decreased scores with increased frequency of stimulus frequency, with the highest average score for 0.2 Hz and lowest average score for 0.8 Hz. All participants completed the Shooting Ducks task within the allocated 2 minute trial, however difference in strategies used to select a target, track its movement across the visual field were identified between children with atypical development and controls, resulting in a longer time to complete task. Time hovering on a target until achieving accurate aim was inefficient for DCD and ASD participants respectively. Conclusions Preliminary results demonstrate support for the hypothesis that impaired postural responses in children with DCD and ASD are seen mostly when visuomotor integration is required to organize and execute the appropriate motor program.
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    Novel Androgen Receptor Variant 45 in Brain Tissue
    (2016-03-23) Contreras, Jo; Duong, Phong; Cunningham, Rebecca L.; Downs, Lincoln
    Purpose: A membrane associated androgen receptor has been implicated in the damaging effects of testosterone in neurodegenerative disorders, such as Parkinson’s disease. An androgen receptor variant (AR45) that has a C-terminus and a unique N-terminus has been found in peripheral tissues, such as the heart, skeletal muscle, uterus, and prostate, but has not been found in the brain. Recent findings have shown that a AR45 is present in the membrane fraction of a dopaminergic neuronal cell line (N27). Therefore, we wanted to determine if AR45 was present in cortical, hippocampal, and substantia nigral neurons that are lost during Alzheimer’s and Parkinson’s disease. Methods: In this study we used immunohistochemistry to determine the presence of AR45 presence in rat brain tissue. Tissue sections of 40 um were prepared using a cryostat. The sections were stained with primary antibodies specific for different androgen receptor sequences. The antibodies AR C-19 and AR N-20 were used to identify the C terminus and the N terminus, respectively. Results: Since AR-45 is lacking an N-terminus, we used N-20 antibody that binds to the N-terminus as a negative control. However, AR-45 does have a c-terminus, and thus we used the C-19 antibody as a marker for AR-45. C-19 immunofluorescence was present in the hippocampus, cortex, and substantia nigra. Interestingly, N-20 positive cells were not identified in the substantia nigra, indicating that only AR45 is present in these neurons. Conclusion: This is first study to show the presence of AR45-immunoreactive positive neurons in the brain. Now the receptor has been identified, more research is needed to determine the role of AR45 in neurodegenerative diseases.
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    Novel Risk Factors for TIA and Stroke in Young Adults
    (2016-03-23) Nejtek, Vicki M.S., Ph.D; Talari, Deepika M.B.B.S, M.P.H.; Lomax, Jerica
    Purpose: From 1993 to 2005 the incidence of stroke in older adults (ages 55-84) substantially decreased while in younger adults (ages 20-44) the incidence more than doubled. Traditional risk factors for stroke have been well documented in older adults such as hypertension, diabetes and obesity. Fewer studies explore transient ischemic attack (TIA)/stroke risks in young adults. Thus, it is clinically relevant to examine novel risk factors that may be unique to young adults. To better understand the increasing incidence for stroke in this population, we examined physical, psychological, and cognitive indices as novel risk factors for TIA and stroke in young adults. Hypothesis: Those identified as moderate/high risk for TIA/Stroke will have higher truncal body fat percent and waist-to-hip ratio (WHR) and will perform poorer on cognitive and stress tests compared to no/low risk. Methods: Men and women ages 18-45 of all race/ethnic backgrounds were eligible to participate in this prospective, cross-sectional, pilot study. Demographic data, WHR, body fat percent, personal and family history of chronic illness, TIA/stroke, etc. were collected. Assessments included the Perceived Stress Scale, Coping Self-efficacy Scale, and the Rey-Osterrieth Complex Test. Descriptive statistics, frequency distributions, independent sample t-tests, ANOVA, and regression modeling, were used as appropriate. All analyses were conducted using a 95% confidence level and an alpha level of 0.05 to determine statistical significance. Results: A total of 50 subjects participated in this study (n=50). Each subject was grouped as no/low risk (NL) or moderate/high risk (MH) based on their personal + family medical history + total body fat percent. The MH group had significantly higher BMI (p = 0.03), higher body fat percent (p = 0.001), and higher truncal body fat distribution (p=0.001). MH subjects had more difficulty with cognitive-based coping skills, higher perceived stress, and performed worse on the Rey-Osterrieth test than the NL group. Conclusion: These preliminary data suggest that those who have a moderate-to-high TIA/stroke risk profile have higher stress levels, less memory recall, and reduced cognitive-based coping skills. The results inform researchers about the need to further explore these novel risks factors in a larger controlled study. Future studies are needed to identify young adults who are at risk of TIA/stroke to reduce the incidence rate in this population.
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    Androgen Receptor-Independent Mechanisms for Dihydrotestosterone (DHT)-induced Protection in the C6 Glioma Model of Astrocytes
    (2016-03-23) Rybalchenko, Nataliya; Singh, Meharvan PhD; Kubelka, Nicholas
    Testosterone and dihydrotestosterone (DHT) exert protection through the activation of the intracellular androgen receptor (AR). However, studies suggest DHT may also exert protective effects by way of alternate mechanisms, including through prior conversion to 3beta-diol, a metabolite that can bind and activate estrogen receptors. Using the AR-deficient C6 glioma, a model of astrocytes, we found DHT was protective against iodoacetic acid (IAA) toxicity. The protective effects of DHT, as assessed by the Calcein-AM viability assay (which is a surrogate measure of cell number), were blocked by the co-application of the non-selective estrogen receptor antagonist, ICI-182,780. Using a complementary viability assay, the MTT assay, which is a surrogate for mitochondrial respiration/activity, we reproduced DHT protection and extended our results to find that 3beta-diol was also protective against IAA-induced reduction in mitochondrial activity. Interestingly, while the effects of 3beta-diol, the presumptive mediator of the effects of DHT, were blocked by ICI 182,780, they were not blocked by the estrogen receptor isoform-selective antagonists MPP (against ERa) and PHTPP (against ERb). Collectively, these data support our hypothesis that DHT is protective against cytotoxicity in a cell line devoid of the classical/intracellular androgen receptor, and that the metabolite of DHT, 3beta-diol, may be an important mediator of DHT’s effects in the central nervous system. Our results also suggest that the capacity to convert DHT to 3beta-diol may be relevant to the protective influence of androgens and estrogens in the postmenopausal women, a time when estrogen and progesterone levels decline significantly, but androgen levels persist.
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    Non-Feminizing Estrogens Do Not Exhibit Antidepressant-Like Activity
    (2016-03-23) Prokai, Laszlo; Prokai-Tatrai, Katalin; Nguyen, Vien
    Short description: In this exploratory lead compound evaluation, we aimed at addressing the utility of two non-feminizing estrogens, specifically 2-adamantyl-17β-estradiol (Ada-E2) and 2-adamantylestrone (Ada-E1), in a well-established animal model of depression-like behavior precipitated by estrogen deprivation. Purpose: To evaluate non-feminizing estrogens in fulfilling their overall premise for the treatment of climacteric symptoms. Methods: Mice were divided into six animals per treatment group. Test agents were dissolved either in corn oil vehicle or in 30% v/v aqueous 2-hydroxypropyl-β-cyclodextrin. The well-known antidepressant amitriptyline, as a reference standard, was used at 15 mg/kg dose, while the estrogen receptor (ER) antagonist fulvestrant was used at 4 mg/kg dose. The control groups received vehicle only. Test compounds in corn oil vehicle were administered subcutaneously (s.c.), while those in HPβCD were given intravenously (i.v.). Each group of animals was treated daily for five consecutive days injecting the test agents 100 µg/kg or 500 µg/kg doses on each day. Antidepressant-like activity were evaluated 30 min after the last injection using the Porsolt swim test (PST). The immobility time (in seconds, defined as the duration of floating motionless after the cessation of struggling and making only movements necessary to keep the head above the water) was recorded for 6 min simultaneously by a trained observer. Drug-likeness was evaluated via the online Osiris Property Explorer. Antioxidant potencies were determined experimentally by the ferric thiocyanate and thiobarbituric acid reactive substances methods. Results: Adding the bulky Ada to the already lipophilic E2 and E1 brought about further increase in the lipophilicity (logP) by [greater than] 2 log units. This increase was probably the most profound contributor to their unfavorable drug-likeness score. In agreement with our earlier quantitative structure–activity relationship study, our experimental assessment also supported that an increase in logP enhances antioxidant effect of estrogen-derived synthetic steroids and their analogs. However, while E2 and E1 did show significant reduction of immobility time in the mice PST, Ada-E2 and Ada-E1 failed to manifest activity in this paradigm (Immobility time is associated with depression-like behavior). Therefore, ERs play a pivotal role in triggering depression-like behavior in estrogen-deprived animals and non-feminizing estrogen offers no remedy for this symptom. Conclusions: Our lead evaluation has confirmed that both genomic and non-genomic mechanisms are required for broad-spectrum estrogen neuroprotection and treatment of menopausal symptoms. Therefore, non-feminizing estrogens such as Ada-E2 and Ada-E1 are not appropriate for the management of symptoms that manifest through ERs such as depression.