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    Chronic Intermittent Hypoxia Alters the Chloride Gradient in Median Preoptic Nucleus (MnPO) Neurons of Rats
    (2021) Farmer, George E.; Little, Joel; Bachelor, Martha E.; Rybalchenko, Nataliya; Cunningham, Joseph
    Rats exposed to chronic intermittent hypoxia (CIH), an animal model simulating the hypoxemia associated with obstructive sleep apnea, exhibit persistent elevations in blood pressure during normoxic periods. In MnPO neurons, angiotensin II type 1 receptor function mediates reductions in GABAa inhibition that become excitatory following CIH. Here, we use the ratiometric Cl- sensor, ClopHensorN, to monitor the chloride flux of MnPO neurons in normoxic (Norm) and CIH treated rats following GABAa activation. Using isoflurane anesthesia, male Sprague-Dawley rats (250-350g) received microinfusions of AAV9-Cre in PVN and DIO-ClopHensorN in MnPO. After recovery, rats underwent 7 consecutive days of CIH (6 min cycles of 3 min 21% O2, 3 min 10% O2 repeated 10x/hr for 8 hours) or Normoxia. For ClopHensorN imaging, rats were anesthetized with isoflurane and coronal slices containing MnPO were cut using standard in vitro slice procedures. Images were captured every 3 sec. Cl- flux was determined from ratiometric responses to 10 s focal application of muscimol (100 uM). Twelve rats (6 Norm, 6 CIH) were used for ClopHensorN studies. In MnPO CIH neurons, 20.1% showed decreased fluorescent ratios while 0.3% showed increased ratios indicative of Cl- efflux. In MnPO Norm neurons, 41.9% showed a muscimol dependent decrease in fluorescent ratio with 0 cells showing an increase. The magnitude of muscimol dependent decreases in fluorescent ratios were reduced in CIH treated rats suggesting reduced GABAa inhibition. Results demonstrate CIH alters Cl- flux of PVN projecting MnPO. These changes may contribute to hypertension associated with CIH.
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    De-tethering of MRI demonstrated tethered cord
    (2021) Howells, Madeleine; Hamby, Tyler; Donahue, David; Honeycutt, John
    Recognition of tethered cord syndrome (TCS) in children is important because as the child grows, the spinal cord is stretched; and for patients with TCS, this leads to ischemia and subsequent neurological deficits, including bowel or bladder dysfunction, back pain, or lower extremity weakness. Radiologically, the presence of a fatty filum, thickened filum, or conus medullaris located caudal to L2 is considered a marker for tethering. The objective of this study is to ascertain how symptomatic tethering is defined in our institution and assess the rationale for surgical detethering in asymptomatic patients. Charts for patients who underwent detethering surgery between 4/1/2015 and 3/31/2019 at our institution were examined. Univariate logistic regression analyses were used to determine whether demographic and diagnostic factors predicted symptoms at presentation with TCS. There were 289 patients included. The modal patient was < 5 years of age, male, had a fatty filum and thickened filum, and did not have a low-lying conus. Patients < 5 years of age were less likely to have urological, gastrointestinal, and neurological or orthopedic symptoms; males were less likely to have urological symptoms; those with fatty fila were more likely to have gastrointestinal and neurological or orthopedic symptoms; patients with thickened fila were less likely to have urological symptoms; patients with low lying coni were less likely to have urological and gastrointestinal symptoms. Timely diagnosis and treatment of patients experiencing signs and symptoms related to tethering offers the possibility of resolution of deficits related to tethering, or at least prevents their progression.
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    (2021) Nguyen, Vien; De La Cruz, Daniel; Prokai-Tatrai, Katalin; Prokai, Laszlo
    Decreased acetylcholine [ACh, (CH3)3N+CH2CH2OCOCH3] levels have been associated with several diseases of the central nervous system (CNS) such as Alzheimer's disease; therefore, it has been considered a potential biomarker. However, quantification of ACh in biological samples even in cerebrospinal fluids obtained through proximity sampling is difficult due to its often extremely low concentration owing to rapid enzyme-catalyzed hydrolysis to choline. Most limitations of previously used liquid chromatography-based (LC) assays relying on electrochemical detection have been addressed by coupling LC with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). However, numerous applications have remained challenging because of inadequate detection sensitivity achievable with most commercial instruments and columns. We improved the method using atmospheric pressure spray ionization (APSI) as an alternative to ESI and ultrahigh-performance liquid chromatography (UPLC) resulting in an about 100-fold increase in detection sensitivity over the previous LC–ESI-MS/MS assay. The presentation will feature application of the method to the sensitive determination of ACh in rodent brain microdialysates, which indicates fit for purpose to use in translational research focusing on this important neurotransmitter. The developed assay also has been rigorously validated to show that it would meet regulatory requirements for application to assess ACh as a potential clinical biomarker of CNS diseases.
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    Long-term cognitive and neurodegenerative effects of repetitive mild traumatic brain injury
    (2021) Abad-Jacobi, Christopher
    1.6 – 3.8 million sports-related traumatic brain injuries (TBI) occur every year in the U.S. Recent retrospective studies suggest that repetitive mild TBI (rmTBI) is associated with the earlier onset of neurodegenerative diseases. Mild TBI can be hard to detect, and there are currently no widely accepted biomarkers that could aid in its diagnosis. Further, there is currently no standard pharmacological treatment for TBI. Our previous work demonstrated neurological deficits 1 week following 20-25 rmTBI in young male mice. We hypothesized that these deficits would persist up to 5 weeks following injury and that pretreatment with an agonist of the Sigma-1 receptor (PRE-084) could reduce these deficits, as has been demonstrated in other neurodegenerative models. Eight-week-old male C57BK6 mice were divided into sham injury, rmTBI, and rmTBI+PRE084 groups (n=14/gp). Mice were anesthetized and administered either PRE084 (1mg/kg sc) or vehicle immediately before experiencing closed head-injury with rotational acceleration via a 65g weight drop 5 days a week for 5 weeks. Five weeks after the final injury mice were assessed for neurological deficits. Injured mice demonstrated significant (P< 0.05) deficits in motor and vestibular-motor performance (Rotarod, balance beam) and cognitive performance in the Morris water maze. Treatment with PRE-084 did not ameliorate deficits. These data suggest there are chronic deficits for at least 5 weeks after rmTBI and that sigma-1 activation does not inhibit rmTBI deficits.
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    A 3-Year-Old Boy with Seizures
    (2021) Tatineni, Chandana; Nguyen, Alexander; Luke, Rebecca
    Background: First described in 2007, Anti-NMDA Receptor Encephalitis is an autoimmune disease caused by immunoreactivity against the NR1/NR2 subunit of the NMDA receptor in the central nervous system. According to the California Encephalitis Project, Anti-NMDA was a more common presentation of encephalitis than viral causes. Case Presentation: A 3-year-old presents to the ER with a possible seizure and recent history of decreased language output and abnormal body stiffness with flexed arms and shaking movements that is prominent on the left side. His initial ER presentation shows signs of fever with unremarkable physical exam, labs and imaging. After no overnight seizures, he was discharged home. A week later, he developed mood lability and other behavioral changes. His second ER presentation shows lymphocytic pleocytosis and a brain MRI shows signs of cerebral inflammatory processes with bilateral non-specific hyperintense lesions. He was started on steroids and IVIG based on the suspicion of Anti-NMDA Receptor Encephalitis. On the 8th day, serum and CSF assays came back positive for the anti-NMDA Receptor antibodies. He continued to receive seizure medications to control his symptoms and was discharged 3 months later to in-patient rehab. Conclusion: This case presents a unique presentation of Anti-NMDA Receptor Encephalitis that was found in a young male instead of the typical tumor associated disease in a young female. This case study emphasizes the need to test for anti-NMDA Receptor antibodies when evaluating young patients with signs of encephalitis.
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    Restoration of HIV infected astrocytes functions by utilizing CRIPSR-Cas9
    (2021) Leng, Boyan; Borgmann, Kathleen
    Purpose: HIV enters the brain, resides in neuroglia and invades immune cells. Antiretroviral therapy has been unsuccessful at reducing the proportion of HIV+ patients developing neurocognitive disorders, highlighting the necessity of eradicating HIV from the brain. As one of the most abundant neural cells, astrocytes provide physical and metabolic support to neurons and remain a significant HIV reservoir capable of reconstituting peripheral HIV infection in humanized mouse models. Current strategies aiming to eradicate the HIV genome from the brain have proved to be detrimental to astrocyte function. We hypothesize that successful HIV excision will improve astrocyte function and restore a neuro-supportive phenotype. Methods: Primary human astrocytes were infected with a dual red/green reporter HIV construct (R/G-HIV) to investigate the function of HIV+ astrocytes. CRISPR/Cas9/gRNAs were designed based on previous studies and evaluated for off target effects. The constructs will be cloned into a glial-targeting adenoviral vector to improve transfection efficiency and permit in vivo studies. Results: Pseudotyped HIV successfully integrated into the astrocyte genome and led to expression of construct reporters and HIV proteins. HIV+ astrocytes showed increased expression of proinflammatory mediators, reduced glutamate clearance and proliferation as compared to controls. The CRISPR/Cas9/gRNA construct will induce cuts in the 5' and 3' long terminal repeats to excise HIV. Conclusion: Successful completion of this study will identify HIV excision as an important tool capable of restoring the neuro-supportive phenotype of HIV(-) astrocytes, which could provide a worthy therapeutic strategy against HIV-associated neurocognitive disorders.
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    Effect of dietary genistein on functional recovery and chronic post-stroke inflammation in ovariectomized middle-aged rats
    (2021) Oppong-Gyebi, Anthony; Metzger, Daniel; Vann, Philip; Sumien, Nathalie; Schreihofer, Derek
    PURPOSE: Increasing age increases stroke risk in women after menopause. A drop in circulating estrogens after menopause has been described as a key reason for this age-related risk, considering that estrogen has shown neuroprotection preclinically. However, using estrogen therapy for chronic prevention of cardiovascular diseases is limited by inconsistent beneficial and detrimental outcomes. For this, other agents are investigated as alternatives to protect women against changes that come with aging and low estrogen concentrations. In this study, we hypothesized that genistein, a neuroprotective plant-derived estrogen will confer neuroprotection following hypogonadism and experimental stroke. METHOD: We used ovariectomized proven retired breeder Sprague-Dawley rats (aged ~9months old), categorized into two hypogonadal time points (2weeks=short-term deprivation(STD) and 12 weeks=long-term deprivation(LTD)) and treated with isoflavone-free diet, genistein diet(GEN) or 17-β estradiol(E2) implant. Animals were subjected to intraluminal middle cerebral artery occlusion or sham surgery followed by motor and cognitive behavioral tests and biochemical analyses for chronic post-stroke inflammation. RESULTS: Sham-operated animals showed locomotor symmetry after both STD and LTD. Both GEN and E2 improved locomotor symmetry after LTD. GEN but not E2 improved cognitive flexibility after STD. Both GEN and E2 reduced activated calcium-binding adaptor molecule 1(Iba1) after STD. GEN but not E2 increased transforming growth factor-β1 and growth-associated protein at the contralateral hemisphere of stroke after STD. CONCLUSION: Dietary Genistein may improve locomotor function in the acute phase of stroke following LTD, improve aspects of cognition and reduce inflammation after STD.
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    Long-term effects of late gestational maternal hypoxic stress on neurodegeneration: Sex and age differences
    (2021) Wilson, Elizabeth; Mabry, Steve; Rybalchenko, Nataliya; Engelland, Rachel; Fadeyibi, Oluwadarasimi; Osikoya, Oluwatobiloba; Cushen, Spencer; Goulopoulou, Styliani; Cunningham, Rebecca
    Introduction: In utero insults can lead to onset of neurodegenerative diseases, such as Parkinson's disease (PD). In utero hypoxic insults are associated with maternal sleep apnea or preeclampsia. It is unknown whether late gestational maternal hypoxic insults have long-term effects on brain regions associated with PD, such as the nigrostriatal pathway. We hypothesized that late gestational maternal hypoxia will result in sustained nigrostriatal impairment in male and female progeny. Methods: Timed pregnant Long-Evans rats were exposed to five days (gestational days: 15-20) of chronic intermittent hypoxia (CIH) or room air (normoxia 21% O2) for 8 hours during their sleep phase. Each CIH cycle was 6 min of alternating 3 min hypoxia (10% O2) and normoxia (21% O2) totaling 10 CIH cycles/hour. Gestational age and biometrics were recorded 12-16 hours after birth. At postnatal day, PND 28, progeny were pair-housed with a conspecific of the same sex and similar weight. We focused on PD associated oxidative stress and behavioral impairments in the nigrostriatal pathway. Gross motor (open field), fine motor (ultrasonic vocalizations), and cognition (spatial memory) were examined during puberty and young adulthood. Results: Maternal CIH had no effect on gestational age, progeny biometrics, or progeny circulating oxidative stress. Gross motor and cognitive functions were unaffected by maternal CIH. However, a sustained fine motor impairment was observed in both male and female progeny. Conclusion: Maternal hypoxia during late gestation induced sustained nigrostriatal pathway impairment, which may increase the risk for neurodegeneration.
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    Long-term effects of prenatal chronic intermittent hypoxia insult on the substantia nigra
    (2021) Engelland, Rachel; Fadeyibi, Oluwadarasimi; Rybalchenko, Nataliya; Wilson, Elizabeth; Mabry, Steve; Osikoya, Oluwatobiloba; Cushen, Spencer; Goulopoulou, Styliani; Cunningham, Rebecca
    Purpose: Prenatal chronic intermittent hypoxia (CIH) was employed to evaluate the effects of hypoxic insults on the substantia nigra (SN), which is impacted by Parkinson's disease (PD). SN loss during PD is linked with oxidative stress (OS) and apoptosis. We hypothesized that exposure to late gestational maternal hypoxia would result in an increase in increased OS, but not apoptosis, in the SN of adult male and female progeny. Methods: During gestational days 15-20, pregnant Long-Evans rats were exposed to CIH or room air (normoxia) for 8 hours. CIH consisted of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2). Animals were sacrificed at puberty (PND 44) or adulthood (PND 66). SN micropunches were obtained. OS was quantified by measuring calpain cleavage of spectrin. Results: OS (calpain cleavage of spectrin) was increased in the SN of adult male and female rats exposed to prenatal CIH compared to control (F1,17 = 3.606; p = 0.075). No effects on OS were observed in pubertal rats. Apoptosis (caspase-3 cleavage of spectrin) was not observed in any of the groups. Conclusions: These data suggest that prenatal CIH programming has a long-lasting impact on the SN of adult progeny, which may increase the susceptibility of SN to damage and PD risk. Although no sex differences were observed in this pilot study, we may see a sex effect upon increasing animal number, especially in male rats. This is consistent with the higher incidence of PD in men than in women.
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    Long-term effects of late gestational maternal hypoxic stress on mood disorders: Sex and age differences
    (2021) Mabry, Steve; Wilson, Elizabeth; Rybalchenko, Nataliya; Engelland, Rachel; Fadeyibi, Oluwadarasimi; Osikoya, Oluwatobiloba; Cushen, Spencer; Goulopoulou, Styliani; Cunningham, Rebecca
    PURPOSE: In utero insults have been linked with increased fear and anxiety in progeny. In utero hypoxic stress is also associated with multiple gestational complications. We hypothesized that exposure to maternal hypoxia during late gestation will have a long-term impact on anxiety in progeny. METHODS: Pregnant female Long-Evans rats were exposed to five days (gestational days: 15-20) of chronic intermittent hypoxia (CIH) or room air (normoxia: 21% O2) for 8 hours during their sleep phase. Each CIH cycle was 6 min of 3 min hypoxia (10% O2) and 3 min normoxia for a total of 10 CIH cycles/hour. At weaning (PND 28), progeny was pair-housed with a conspecific of same sex and similar weight. To examine anxiety disorders, we quantified anxiety-related behaviors (time spent in center of open field arena, marble burying test, social and anti-social behaviors with conspecifics) along with quantifying food intake and circulating sex hormone levels during puberty (postnatal day, PND 40-45) and young adulthood (PND 60-65) in male and female progeny. RESULTS: Maternal CIH did not impact circulating sex hormones or food intake, regardless of sex or age of progeny. However, maternal CIH increased anxiety related behaviors in pubertal females but were not observed in young adulthood. Maternal CIH did not impact male progeny, regardless of age. CONCLUSIONS: Maternal CIH during gestation resulted in increased anxiety related behaviors in pubertal female progeny. Maternal hypoxia during late gestation may temporarily increase the risk for anxiety disorders in pubertal females.
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    Discovery of Small Molecule Slack Inhibitors for the Treatment of MMPSI: SAR Development in the Western and Central Region of Hit Compound VU0606170
    (2021) Mishra, Nigam; Qunies, Alshaima'a; Spitznagel, Brittany; Acuna, Valerie; Acebo, Jonathan; Weaver, C.; Emmitte, Kyle
    Introduction: Malignant Migrating Partial Seizures of Infancy (MMPSI) is a severe and pharmacoresistant form of epilepsy. Slack channels are sodium-activated potassium channels regulating essential electrical activity in CNS. Gain-of-function mutations in Slack channels have been linked to MMPSI. Objective: To develop small molecule selective Slack inhibitors employing a library synthesis based iterative hit optimization approach to discover leads for development into MMPSI therapeutics. Methods: Classical and state-of-the-art synthetic chemistry techniques including microwave assisted organic synthesis and flow chemistry were employed. Purification was by automated liquid chromatography. Bruker Fourier 300HD and Agilent 6230 time-of-flight LC/MS were utilized to obtain NMR and HRMS, respectively. Inhibitory activity of Slack was measured utilizing a Thallium flux assay in HEK293 cells stably expressing either WT or Slack mutants. Results: SAR studies around hit compound VU0606170 identified a chiral-methyl analog in the piperizine core as optimal for potency. Other new core analogs were less potent than their piperazine counterparts. Several western urea and amide analogs were prepared, and a few moderately potent compounds were identified. Replacement of sulfamide linkers with a sulfonamide gave encouraging results. Lastly, in-vitro DMPK studies with selected compounds revealed high clearance, high protein binding, and good permeability. Conclusion: SAR was identified for Slack activity, mutant selectivity, and DMPK properties around the western and central region of VU0606170. At present, synthesis of analogs that combine optimal functional groups from the entire chemotype are underway with a goal of improving potency and DMPK properties.
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    AT1R sniffer cells detects spontaneous and evoke release of AngII in the AP-NTS pathway
    (2021) Gusson Shimoura Almeida Lima, Caroline; Paundralingga, Obed; Farmer, George; Cunningham, Joseph
    Although angiontesin II (AngII) has multiple actions in the brain, the existence of a brain RAS is still controversial. Our previous studies have used angiotensin sensitive sniffer cells to test whether angiotensin peptides are released from subfornical organ projections to the median preoptic nucleus. In these studies, we examined another pathway involving the area postrema (AP) and nucleus of tractus solitarius (NTS). The AP is angiotensin sensitive and projects to the NTS, so the purpose of this study was to test for the release of angiotensin peptides in the NTS after stimulation of AP. Sniffer cells were produced by transfecting Chinese Hamster Ovary cells with commercially available plasmids for the angiotensin 1a receptor (Origene Tech.) and R-GECO (Addgene #32462). These sniffer cells are sensitive to AngII and III but not angiotensin 1-7, bradykinin, or neurotransmitters such as glutamate or acetylcholine. Sniffer cells were placed on coronal brainstem slices containing both AP and NTS from adult male Sprague – Dawley rats. Changes in fluorescent intensity of sniffer cells in the NTS was determined following electric stimulation of the AP (100Hz, 10ms, 1mA). Electrical stimulation increased fluorescence intensity 134 ± 11%, n=13 of sniffer cells on the NTS with a mean response latency of 4 ± 0.7sec, n=13. Some cells demonstrated spontaneous changes in fluorescence intensity 2±0.1, n=28 that were not observed in cells located outside of the NTS. The results indicate that sniffer cells placed on the NTS demonstrated evidence of spontaneous and evoked release of angiotensin peptides.
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    Neuroprotective Effects of Exercise on Parkinson's: The Role of BDNF in 6-OHDA Lesioned Rats
    (2021) Parry, Caleb; Salvatore, Michael; Kasanga, Ella
    Purpose: Parkinson's disease (PD) is a neurodegenerative disorder of the nigrostriatal pathway that largely affects elderly individuals and leads to steady motor decline. Exercise may slow motor decline in PD patients if it is tolerable and consistent. Thus, it is important to define physiologic mechanisms by which exercise affects PD to develop similar therapies. Mechanisms may include growth factors like brain derived neurotrophic factor (BDNF). This project aims to evaluate BDNF in a tolerable exercise regimen after 6-hydroxydopamine (6-ODHA) lesion. Methods: Sprague Dawley rats were conditioned to exercise. Nigrostriatal pathways were lesioned with 6-OHDA to model PD in one group. After motor impairment was measured, exercise was initiated and occurred 3 days a week for 3 weeks with 1 day of rest between each day of exercise. Exercise consisted of a warm-up phase (8m/min for 5 min) and training phase (10 m/min for 35 min). Striatal tissue was analyzed for BDNF expression using anti-BDNF antibody and western blot. Results: Exercised 6-OHDA lesioned rats showed motor recovery compared to non-exercised rats. Anti-BDNF antibody showed bands indicating mature and pro forms of BDNF. Bands were analyzed to compare BDNF expression between exercise and non-exercise groups. Conclusion: Understanding the CNS mechanisms of exercise and its impact on PD could reveal novel therapeutic strategies that target specific proteins. This study demonstrates a tolerable training regimen that improves motor deficit and a means to study the possible role of BDNF expression in motor improvement.
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    Exercise is a Protective Factor for Motor and Cognitive Function in Early-Stage Parkinson's Disease
    (2021) Soto, Maria
    Parkinson's disease (PD) is a neurodegenerative disease with no known cure. Previous research has identified aerobic exercise as a non-pharmacological therapeutic option to potentially reverse or slow the progression of motor decline. However, the impact of regularly maintaining aerobic activity on domains of cognition remains poorly understood. Here, the primary goal was to identify which cognitive domains may be more responsive to aerobic exercise to better understand its therapeutic benefits on motor and cognitive function in early-stage PD. Independent groups included exercising and non-exercising early-stage PD patients compared to matched non-Parkinson's healthy controls (NPHC). Neurocognitive testing revealed that PD exercise subjects made significantly fewer errors on Trail-Making Test (TMT) B (p < .05; cognitive flexibility, attention) than the non-exercise group and performed comparably to NPHC. The longer number of years PD subjects regularly exercised the better their cognitive flexibility, attention (TMT A and B; p < .001) and visuospatial memory (CLOX2, p < .05). Several significant relationships emerged between motor (Timed Up and Go, Gait speed, and 6-minute Walking Test) and cognitive flexibility, attention (TMT-A, p=0.001; TMT-B, p=0.000), and verbal fluency (FAS, p=0.009; Animal Naming, p=0.002). In summary, PD subjects maintaining a regular aerobic regimen of activity show better motor function, cognitive flexibility, attention, verbal and visuospatial memory than their sedentary counterparts. Future directions include translating these findings using a genetic PD rat model to further elucidate the neural mechanisms underlying exercise-related cognitive and motor performance.