Browsing by Author "Forster, Michael J."
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Item Administration of 5-methoxyindole-2-carboxylic acid that potentially targets mitochondrial dihydrolipoamide dehydrogenase confers cerebral preconditioning against ischemic stroke injury(2018-03-14) Li, Rongrong; Li, Wenjun; Ren, Ming; Thangthaeng, Nopporn; Sumien, Nathalie; Liu, Ran; Yang, Shaohua; Simpkins, James; Forster, Michael J.; Yan, Liang-Jun; Wu, JinziPurpose: The purpose of this study was to investigate a possible role of mitochondrial dihydrolipoamide dehydrogenase (DLDH) as a chemical preconditioning target for neuroprotection against ischemic injury. Methods: We used 5-methoxyindole-2-carboxylic acid (MICA), a reportedly reversible DLDH inhibitor, as the preconditioning agent and administered MICA to rats mainly via dietary intake. Upon completion of 4 week's MICA treatment, rats underwent 1 h transient ischemia and 24 h reperfusion followed by tissue collection. Results: Our results show that MICA protected the brain against ischemic stroke injury as the infarction volume of the brain from the MICA-treated group was significantly smaller than that from the control group. Data were then collected without or with stroke surgery following MICA feeding. It was found that in the absence of stroke following MICA feeding, DLDH activity was lower in the MICA treated group than in the control group, and this decreased activity could be partly due to DLDH protein sulfenation. Moreover, DLDH inhibition by MICA was also found to upregulate the expression of NAD(P)H-ubiquinone oxidoreductase 1(NQO1) via the Nrf2 signaling pathway. In the presence of stroke following MICA feeding, decreased DLDH activity and increased Nrf2 signaling were also observed along with increased NQO1 activity, decreased oxidative stress, decreased cell death, and increased mitochondrial ATP output. We also found that MICA had a delayed preconditioning effect four weeks post MICA treatment. Conclusion: Our study indicates that administration of MICA confers chemical preconditioning and neuroprotection against ischemic stroke injury.Item Advancing the Role of Thyrotropin-releasing Hormone as a Central Nervous System Agent(2022-05) De La Cruz, Daniel L.; Prokai-Tatrai, Katalin; Prokai, Laszlo; Lacko, Andras G.; Forster, Michael J.Item Aging Confers an Increase in Sensitivity and Sensitization to Pain and Results in Shifts of Spinal NR1 expression(2009-05-01) Jenschke, Monica L; Ratka, Anna; Forster, Michael J.Purpose: Many elderly experience inadequate postoperative pain relief resulting in increased morbidity and mortality. Several experimental models of postoperative pain have been developed but none were adapted to study the effects of aging on the postoperative pain. Review of literature explored current knowledge of postoperative pain models and identified several models suitable for aging studies. A unique model of postoperative pain, the dorsal hairy skin incision model, was modified and adopted for aged rats. Using this model, we tested two hypotheses: a) aged rats will exhibit similar intensity but longer duration of postincision hyperalgesia compared to young rats and b) spinal cord NR1 expression will increase in response to nociceptive stimulation and that age-related differences in magnitude of NR1 expression will be evident. Methods: In study I, young (5-7 months old) and aged (22-23 months old) male Fischer 344 rats were exposed to nociceptive testing with von Frey filaments and the cutaneous trunci muscle reflex was measured. For each stimulation, a graded response of 0, 0.5, or 1, for no reflex, a small reflex, or vigorous reflex, respectively was recorded. After baseline testing, a 2 cm incision was made through the dorsal skin followed by skin closure and recovery. Subsequently, rats were tested at 3 hours, 6 hours, and on postoperative days (POD) 1, 3, 6, 10, and 14. In study II, young (4-6 months old) and aged (19-21 months old) male Fischer 344 rats were subjected to three sessions of mechanical nociceptive stimulus. After testing, spinal cords were harvested for western blot analysis of NR1 expression. Results: In study I, aged rats had greater baseline graded responses to nociceptive stimuli. After incision, young rats developed primary allodynia lasting until POD 3 and primary hyperalgesia until POD 8. Aged rats did not develop allodynia or primary hyperalgesia. Neither group developed secondary hyperalgesia. Aged rats demonstrated greater sensitivity to baseline nociceptive testing and greater maximal graded responses to repetitive testing sessions. In young rats, nociceptive stimulation resulted in a significant increase in NR1 expression. Increased NR1 expression in young tested rats positively correlated with an increase in graded response for one of 18 session/region/force categories tested. There was no increase in spinal cord NR1 expression in aged rats in response to nociceptive stimulation. Low NR1 expression in aged tested rats negatively correlated with an increase in graded response for 9 of 18 session/region/force categories tested. Conclusions: An experimental rat model to study effects of age on postoperative pain is presented. Age has a profound impact on the pre- and postoperative periods. Aged rats differ significantly from young rats in sensitivity and maximal graded response to acute incisional pain. Young rats exposed to mechanical punctate nociceptive stimuli experienced increased NR1 expression which positively correlated with an increase in graded response. In contrast, aged rats with decreased NR1 expression negatively correlated with an increased graded response. Lower sensitivity and maximal graded responses in the young rats reflect an intact endogenous modulatory pain pathway. Greater sensitivity and maximal graded responses in the aged rats reflect impairment of descending modulatory pain pathways.Item Blood Inflammatory Exosomes with Age Prime Microglia through Complement Signaling for Negative Stroke Outcomes(2020-05) Zhang, Hongxia; Jin, Kunlin; Forster, Michael J.; Yang, Shaohua; Shi, Xiangrong; Cunningham, J. ThomasThe systemic inflammatory milieu plays an important role in the age-related decline of functional integrity, but its contribution to age-related disease (e.g., stroke) remains largely unknown. Here, we found that activated complement molecules (C1q, C3a, C3b) in serum exosomes increased with age, whereas CD46, a C3b/C4b-inactivating factor, was higher in serum exosomes from young rats. These serum inflammatory exosomes passed the blood-brain barrier and primed the microglial response that led to exacerbation of synaptic loss and motor deficits after ischemic stroke via microglial C3a receptor (C3aR). When aged rats were exposed to serum exosomes from young rats, microglia-mediated synaptic loss was reduced and motor deficits after stroke were improved. Administration of C3aR inhibitor or microglial depletion attenuated synaptic loss associated with the treatment of serum exosome from aged rats, in parallel with improved post-stroke outcome. Our data suggest that peripheral circulating old exosomes act as inflammatory mediators and influence ischemic stroke outcome through a complement-microglia axis.Item CHARACTERIZATION OF COCAINE-CONDITIONED LOCOMOTOR RESPONSES BY MODULATION OF ENVIRONMENTAL CONTEXT AND NEURAL PLASTICITY-SIGNALING PATHWAYS(2014-03) Nguyen, Jacques D.; Forster, Michael J.Rodent models are commonly used for the study of substance abuse and addiction. The objective of this study was to characterize the cocaine-conditioned locomotor response, a behavioral phenomenon observed in mice, following an acute injection of cocaine, and to determine its mechanism of action for potential therapeutic targeting. Compounds known to modulate neural plasticity were evaluated for their ability to affect the acquisition and expression of the conditioned behavior. Purpose (a): In rodents, increase in locomotion is a hallmark effect of psychostimulant exposure and conditioning that is associated with activation of mesocorticolimbic dopamine signals mediating reinforcing/rewarding actions. The objective of this study was to characterize the cocaine-conditioned locomotor response following an acute injection of cocaine, specifically the modulating roles of environmental context and plasticity-associated signals. Methods (b): Cocaine (40mg/kg) was administered to different groups of Swiss-Webster, C57Bl/6, or DBA2 mice via intraperitoneal injection (i.p.), in either a locomotor activity testing apparatus or the home cage, 2 hours following an activity test under saline. Mice placed in the testing chambers were given 30 minutes to explore freely and locomotion was monitored using a Digiscan photocell apparatus. A conditioned effect of cocaine was inferred by an increase in horizontal activity counts relative to home cage cocaine controls during a test in the same apparatus on the following day. Compounds known to modulate neural plasticity-associated signaling cascades were evaluated for their ability to affect the acquisition and expression of cocaine-conditioned locomotor response, using a two-day protocol. Mice were administered haloperidol (0.05-1 mg/kg), dizocilpine (0.01-0.25mg/kg), nifedipine (0.1-10 mg/kg), cycloheximide (2.5-10mg/kg), or vehicle, prior to placement into the activity chambers on the test day for expression or prior to acquisition day. Results (c): Haloperidol (0.25-1 mg/kg) inhibited expression of the cocaine-conditioned locomotion, though failed to alter acquisition of the behavioral response. Dizocilpine (0.05-0.25 mg/kg) attenuated acquisition and exacerbated expression. Nifedipine had no effect on the conditioned locomotor response. Cycloheximide (2.5-10 mg/kg) attenuated acquisition of the conditioned response. Conclusions (d): These findings suggest that plasticity-dependent signaling pathways mediate associations of context following acute cocaine exposure and are necessary for the acquisition and expression of the cocaine-conditioned locomotor response.Item Dietary Curcumin And Caloric Restriction As Interventions For The Reversal Of Age Associated Functional Decline(2015-12-01) Sarker, Marjana R.; Forster, Michael J.; Franks, Susan; Sumien, NathalieAugmentation and exacerbation of oxidative stress and low-grade chronic systemic inflammation during mid-life has been proposed as modifiable causative factors for neurobehavioral decline reported with normal aging. Physiologically, the imbalance of pro-oxidants and endogenous antioxidants leads to an increase in tissue- damaging oxidative stress. Aging has also been associated with chronic systemic inflammation that can damage healthy tissues and diminish cognitive and motor capacity. The overall hypothesis of this project is that caloric restriction and dietary curcumin, via their strong anti-oxidant and anti-inflammatory properties; can delay the onset or ameliorate cognitive and motor decline in middle aged and aged mice respectively. Study 1: Fifteen month-old male C57BL/6 mice were tested as a model of sedentary mid-life obesity for the pilot study. They underwent dietary treatment for 12 weeks and were subjected to cognitive tests at the 8th week of treatment. Dietary treatments included regular chow fed ad libitum (AL), curcumin (1g/kg of diet) fed ad libitum (CURAL) and 30% to weight stable caloric restriction (CR). Mice were tested for spatial learning and cognitive flexibility testing. Blood was collected to measure inflammation and oxidative stress. Results from the pilot study indicated a significant weight loss and reduced adiposity in the CR group; whereas CURAL mice maintained stable weight throughout the treatment, consumed more food than the AL mice, and did not show a reduction of adipose tissue. However, both the CR and CURAL groups took fewer trials than AL to reach criterion during the reversal sessions of the active avoidance task, suggesting an improvement in cognitive flexibility. The AL mice had higher levels of CRP compared to CURAL and CR, and reduced glutathione as well as the GSH/GSSG ratio were increased during curcumin intake, suggesting a reducing shift in the redox state. Study 2: In the subsequent study, 15 and 20 month old female and male C57BL/6 mice were used as a normal aging model to study functional decline. This study included all of the dietary interventions from the pilot study and an additional combination diet of CR and curcumin (CURCR). Curcumin was added to the diet at 7g/kg of diet with mice under CURCR receiving 7.2g/kg of diet, adjusted to take difference in food intake into account. The mice underwent dietary treatments for 4 months, and cognitive and motor behavior tests were conducted at 8 weeks of treatment. Mice were tested on multiple tasks that are sensitive to age associated cognitive and motor dysfunction. Results from the second study indicated females to be more active than males. Mice under CR and CURCR performed better in the motor tests compared to their age matched controls, which included coordinated running, wire suspension and bridge walking. Cognitive flexibility was significantly better for middle-aged males under CR and CURAL compared to AL but not under CURCR, suggesting an antagonistic interaction. On the other hand, middle aged and aged female experimental groups did significantly better than AL. No interaction of CR and CUR was observed in aged males, with CURAL and CR yielding comparable benefits. None of the treatments had a significant effect on hippocampus- dependent rate of learning in middle age or the aged; however middle aged males under the CURCR intervention had poorer probe performance compared to their age matched controls. Data from both projects suggest that independent of weight loss; dietary curcumin and CR have positive effects on fronto-cortical functions in late middle age and senescence that could be linked to anti-inflammatory or antioxidant actions. These effects were similar across different behavioral tasks and were non-additive or antagonistic in a sex dependent manner, suggesting that they could involve the same or similar mechanisms including an influence of sex hormones. Therefore, curcumin intake may mimic the neurobehavioral outcomes of CR that could be age dependent, but the mechanism of action underlying the outcomes of the CR and curcumin combination treatments need to be further examined.Item Divergent Behavioral Phenotypes in Conditioned Place Preference(2017-12-01) Wagner, Alison N.; Forster, Michael J.; Gatch, Michael B.; Shetty, RituThe addictive properties of psychostimulants have been studied using a variety of animal models and behavioral paradigms. These studies consistently report individual variation in drug response that could reflect subgroups with different susceptibility to addiction. A place conditioning assay was used to assess the possibility that such divergent behavioral phenotypes explain variable outcomes in mice after conditioning with psychostimulants (cocaine, 3,4-methylenedioxypyrovalerone (MDPV), methamphetamine, and d-amphetamine). K-means clustering analysis partitioned individuals into groups (i.e. clusters) for analysis. The reliability of these phenotypes was supported with Pearson correlation analysis comparing adjacent time points, as well as Cronbach’s alpha and intraclass correlation coefficients for overall within-group relatedness at each time point. Furthermore, initial preference developing in a drug-naïve condition was reversed with drug conditioning, demonstrating that changes in salience were sufficient to reverse initial preference in some mice. By purposefully examining these behavioral phenotypes in place conditioning, we advance toward the development of pharmacological strategies for addiction and robust epigenetic outcomes.Item “Ecstasy” to Addiction: Mechanistic and Reinforcing Effects of Synthetic Cathinone Analogs of MDMA(2017-05-01) Dolan, Sean B.; Gatch, Michael B.; Huang, Ren-Qi; Forster, Michael J.Following widespread scheduling, many synthetic cathinone compounds have been diverted from “bath salts” to “Ecstasy” tablets or “Molly” powder formulations in addition to or in lieu of 3,4-methylenedioxymethamphetamine (MDMA). The current study aimed to assess the mechanism and reinforcing effects of three under-researched synthetic cathinone analogs of MDMA frequently used as adulterants in “Ecstasy” formulations: methylone, butylone, and pentylone. To assess the mechanism of these compounds in vitro, we utilized whole-cell patch clamp electrophysiology on HEK293 cells expressing the serotonin transporter (SERT). The abuse-related, in vivo mechanisms were determined using a drug discrimination assay with rats trained to discriminate methamphetamine, the hallucinogenic phenethylamine 2,5-dimethoxy-4-methylamphetamine (DOM), or MDMA from vehicle, and drugs that substituted were tested with the D1-like receptor antagonist SCH23390 to assess relative differences in dopaminergic signaling. The reinforcing effects were assessed in an intravenous self-administration assay using continuous and progressive ratio schedules of reinforcement. Methylone and butylone, like MDMA, produced inward currents at SERT, indicative of a substrate-like mechanism. Each test compound fully substituted for the discriminative stimulus effects of methamphetamine. MDMA, methylone, and butylone substituted partially for DOM, and methylone and butylone substituted fully for MDMA. Pentylone, conversely, substituted partially for MDMA, but failed to substitute for DOM. SCH23390 fully and dose-dependently attenuated methamphetamine-appropriate responding, with pentylone being least sensitive to these antagonistic effects, but failed to attenuate MDMA-like responding against MDMA, methylone, and butylone. Each test compound maintained robust self-administration under a continuous schedule of reinforcement, but pentylone was the most reinforcing test compound under a progressive ratio. These data indicate that methylone and butylone produce complex discriminative stimulus effects, similar to MDMA, that are mediated by both dopamine and serotonin, whereas pentylone is predominately dopaminergic. The underlying differences in relative dopaminergic and serotonergic mechanisms likely influence the relative abuse liability, with pentylone’s predominately dopaminergic mechanism conferring a greater reinforcing efficacy relative to the more serotonergic methylone and butylone. In conclusion, incorporation of these compounds into “Ecstasy” formulations, especially pentylone, may lead to compulsive, uncontrolled use of “Ecstasy”.Item Effect of Short-Term Alpha Lipoic Acid Supplementation on Age Related Cognitive and Motor Deficits(2001-08-01) Shetty, Ritu A.; Forster, Michael J.; Dillon, Glenn H.; Das, HridayRitu A. Shetty. Effect of short-term antioxidant supplementation on age related cognitive and motor deficits. Master of Science (Biomedical Sciences), August, 2001, 24 pp, 2 tables, 11 figures, 30 references. During aging there is an increase in oxidative damage and loss of brain function that may reflect and increased level of oxidative stress. Studies have suggested that the increased oxidative damage in old rodents can be reversed, relatively rapidly, by experimental interventions like caloric restrictions and antioxidants capable of lowering oxidative stress (Forster et.al., Joseph et.al., 1995). Based on those findings, it was hypothesized that age-related declines in cognitive and/or psychomotor function are the result of molecular damage associated with oxidative stress. The current study addressed the possibility a decreasing oxidative damage could be produced with the antioxidant alpha lipoic acid, in aged mice, leading to a reversal of age-related impairments of psychomotor and cognitive functions. C57BL/6 mice aged 6 or 23 months were gavaged daily with 100mg/kg alpha-lipoic acid or the vehicle (0.9% saline; 2% methylcellulose). After 3 weeks of treatment, the animals were subjected to a battery of behavioral tests for motor and cognitive functions. Following the behavioral tests the animals were sacrificed brains were dissected and frozen for analysis of carbonyl concentration. The results showed significant age-related deficits in spatial learning, accuracy for spatial memory, recent memory, and psychomotor performance. None of these age-related deficits was reversed following the treatment with alpha lipoic acid. There was no reduction in carbonyl concentration with alpha lipoic acid supplementation. Thus we concluded that alpha lipoic acid supplementation had neither beneficial nor detrimental effects in reducing oxidative damage in brain or in reversal the age-related decline in function.Item Effects of antioxidants on nicotine recognition in rats(2020-05) Galvez, Jonathan R.; Forster, Michael J.; Gatch, Michael B.; Ritu, Shetty A.According to the CDC, nicotine addiction accounts for over 7 million deaths a year worldwide, twice that of narcotics and alcohol combined (11). Most adults need 30 attempts to quit smoking for a year or longer, and this is likely an underestimate (1). The addictive properties of nicotine are thought to involve activation by acetylcholine of brain reward pathways of the Ventral tegmental area and nucleus accumbens (see Figure 1) (8). Drugs like bupropion and varenicline target monoamine and cholinergic nicotinic receptor mechanisms involved in these pathways and have been approved to treat nicotine addiction, although their success has been limited (19). Because of this, novel approaches of treating nicotine addiction are necessary. The goal of current studies will be to evaluate redox signals as a target for development of new interventional approaches to smoking cessation. A variety of approaches have been used to treat addiction. The nicotine patch is an example of a substitution approach for which the goal of treatment is to maintain a sustained low level of the addicting substance for the purpose of reducing craving for the drug (12). Varenicline and bupropion also represent examples of substitution-based approaches targeting a reduction of craving (18). A second approach which could be characterized as an antagonist approach involves preventing activation of neural systems involved in the addicting process (6). The following is a proposal to test blockers of redox signaling as potential treatment medications for addiction using the antagonist approach.In the current study, we will evaluate the hypothesis that redox signaling-related effects on neurotransmission participate in the subjective effects of nicotine using a drug discrimination paradigm. In this paradigm, rat subjects learn to recognize the effects of a drug and report its presence or absence using behavioral responses emitted to obtain food reward or avoid aversive stimuli (17). If the hypothesis is correct, then interference with redox signals should fully or partially block the nicotine discriminate stimulus effects. Vitamin C, vitamin E, apocynin, and FSNY-1 were chosen as potential antagonists for this study to evaluate multiple sources and targets of reactive oxygen species (ROS). Vitamin C is a cytosolic antioxidant, vitamin E is a membrane antioxidant, apocynin is a NADPH oxidase inhibitor, and FSNY-1 is thought to inhibit hydrogen peroxide and hydroxide radical. As positive controls, the proposed studies will evaluate the nonselective NN receptor antagonist mecamylamine for the ability to antagonize nicotine discrimination (see preliminary data in subsequent sections). The NN receptor antagonist hexamethonium will also be included as a negative control for mecamylamine, because it has same mechanism of action but does not cross the blood brain barrier. It is expected that mecamylamine, but not hexamethonium, will block the discriminative stimulus produced by nicotine.Item Elucidation of Mechanism and Molecular Determinants Important in Picrotoxin Action in the 5-Hydroxytryptamine Type 3 Receptor(2003-09-01) Das, Paromita; Basu, Alakananda; Forster, Michael J.; Luedtke, Robert R.Das, Paromita, Elucidation of mechanism and molecular determinants important in picrotoxin action in the 5-hydroxytryptamin type 3 receptor. Doctor of Philosophy (Pharmacology and Neuroscience), September 2003, pp. 192, 3 tables, 26 illustrations, 67 titles. The 5-HT3 receptor belongs to the superfamily of ligand-gated ion channels (LGIC), which mediate fast neurotransmission. Till date, only two subtypes of the receptor i.e. 5-HT3A and 5-HT3B have been investigated. The GABAA receptor antagonist picrotoxin inhibits other anion-selective members of the LGIC. Whether PTX inhibits the cation-selective 5-HT3 receptors was previously unknown. Thus, the primary goal of this study was to elucidate the mechanism of action of PTX and identify the amino acids involved in the action of PTX in 5-HT3 receptors. The overall hypothesis tested was that PTX inhibits the 5-HT3 receptor by interacting in the ion channel. PTX-mediated blockade of the 5-HT3A receptors was non-competitive and use-facilitated similar to GABAA receptors suggesting a conserved site of action of these ligands. The inhibitory effect of PTX was reduced drastically in heteromeric 5-HT3A/3B receptors, compared to homomeric 5-HT3A receptors. Picrotoxin should prove to be a useful probe for determining the presence of homomeric vs. heteromeric 5-HT3 receptors in native tissue and recombinant receptor preparations. In anion-selective ion channels, the 2’, 3’ and 6’resides in cytoplasmic aspect of TM2 are known to modulate PTX sensitivity. While mutation of 2’ and 3’ residues in 5-dramatic loss of sensitivity to PTX in 5-HT3A receptors. A converse mutation at 6’ residue in the 5-HT3B subunit caused gain of sensitivity to PTX, suggesting that 6’ is a key determinant of PTX sensitivity. A novel finding was the involvement of 7’ residue in increasing PTX sensitivity in 5-HT3A but not the 5-HT3B subunit. The lack of specific binding by radioligand [3H]EBOB in 5-HT3A receptors suggested that the site of action of convulsants may be different from that anion-selective receptors. The overall results suggest that PTX interacts from that in the anion-selective receptors. The overall results suggest that PTX interact in the ion channel in the 5-HT3 receptors but also underscores the complexity of its interaction with LGICs.Item Gait Analyses in Mice: Effects of Age and Glutathione Deficiency(International Society on Aging and Disease, 2018-08-01) Mock, J. Thomas; Knight, Sherilynn G.; Vann, Philip H.; Wong, Jessica M.; Davis, Delaney L.; Forster, Michael J.; Sumien, NathalieMinor changes (~0.1 m/s) in human gait speed are predictive of various measures of decline and can be used to identify at-risk individuals prior to further decline. These associations are possible due to an abundance of human clinical research. However, age-related gait changes are not well defined in rodents, even though rodents are used as the primary pre-clinical model for many disease states as well as aging research. Our study investigated the usefulness of a novel automated system, the CatWalk XT, to measure age-related differences in gait. Furthermore, age-related functional declines have been associated with decreases in the reduced to oxidized glutathione ratio leading to a pro-oxidizing cellular shift. Therefore the secondary aim of this study was to determine whether chronic glutathione deficiency led to exacerbated age-associated impairments. Groups of male and female wild-type (gclm(+/+)) and knock-out (gclm(-/-)) mice aged 4, 10 and 17 months were tested on the CatWalk and gait measurements recorded. Similar age-related declines in all measures of gait were observed in both males and females, and chronic glutathione depletion was associated with some delays in age-related declines, which were further exacerbated. In conclusion, the CatWalk is a useful tool to assess gait changes with age, and further studies will be required to identify the potential compensating mechanisms underlying the effects observed with the chronic glutathione depletion.Item Impact Of Culture Conditions on Primary Astrocyte Phenotype(2019-05) Prah, Jude K.; Yang, Shaohua; Singh, Meharvan; Forster, Michael J.; Yan, Liang-Jun; Fudala, RafalAlthough previously thought to be passive support cells in the central nervous system (CNS), recent findings introduced critical contributions of astrocytes to numerous CNS functions like energy metabolism, ion and water homeostasis, blood brain barrier formation and neurotransmission. Their dysfunction has been implicated in the initiation and progression of specific CNS pathologies with astrocyte now given serious attention as cellular target for neuroprotection and treatment of neurological disorders. In spite of the aforementioned advances, our understanding of the mechanisms and pathways regulating astrocytic function, dysfunction and astrogliosis is still rudimentary. This is as a result of the complex interwoven nature of different cells in the CNS. Because of the complexities of the brain structure and function in vivo, methods of in vitro primary culture that overcome the influence of complex brain environment provide critical tools for understanding brain cell function at the cellular and molecular levels. The current primary astrocytes cultures are mostly maintained in serum-containing hyperglycemic medium which is non-physiological and produces astrocyte with a reactive, morphological and functional phenotype different from in vivo quiescent astrocytes. The first study presented in the dissertation delineates a serum free astrocyte culture condition that maintains primary astrocytes in a quiescent state. Results showed that primary astrocytes isolated from the cerebral cortex of postnatal day 1 C57BL6 mice and cultured in an astrocyte base medium supplemented with fibroblast growth factor (FGF2) and epidermal growth factor (EGF) (ABM- FGF2-EGF) have higher process bearing morphologies similar to in vivo astrocytes and different from the flat polygonal fibroblast like morphologies exhibited by astrocytes cultured under the traditional FBS condition developed by McCarthy and de Vellis (1980) (MD-10% FBS). Additionally astrocytes cultured in ABM-FGF2-EGF had enhanced glycolytic metabolism, higher glycogen content, lower GFAP and vimentin, increased glutamine synthase and glutamate transporter mRNA levels compared to astrocytes in the MD-10% FBS condition. These findings strongly indicates that astrocytes cultured in ABM-FGF2-EGF medium compared to the usual FBS medium promote quiescent and biosynthetic phenotype similar to in vivo astrocytes. This media provides a novel method for studying astrocytes function in vitro under physiological and pathological condition. Hyperglycemia could increase neuronal glucose level which leads to neuronal damage in a phenomenal referred to as glucose neurotoxicity. On the other hand the impact of hyperglycemia on astrocytes has been less explored although astrocytes are critical for glucose uptake and metabolism and many primary astrocyte cultures are maintained in high glucose conditions. In the second part of this dissertation we investigated the impact of hyperglycemia on astrocyte phenotype and function. Our studies demonstrated that hyperglycemic levels (25 mM) induce cell cycle arrest, ROS production, cytokine expression and inhibited astrocyte proliferation. High glucose enhanced glycolysis and increased metabolic potentials of astrocyte. In addition high glucose activated AMP-activated kinase (AMPK) signaling pathways and induces reactive astrocyte phenotype. In conclusion both studies presented a unique perspective of how culture conditions influence astrocyte phenotype and experimental outcome. Our study also provided a mechanism which may underline the role of astrocytes in hyperglycemia induced neurological complications.Item LLT1-Mediated Immunotherapy for Hepatocellular Carcinoma(2022-08) Allison, Michaela M.; Mathew, Stephen O.; Chaudhary, Pankaj; Mathew, Porunelloor A.; Forster, Michael J.Item Locomotor and discriminative stimulus effects of three benzofuran compounds in comparison to abused psychostimulants(Elsevier B.V., 2023-08-21) Hill, Rebecca D.; Shetty, Ritu A.; Sumien, Nathalie; Forster, Michael J.; Gatch, Michael B.AIMS: Benzofurans are used recreationally, due their ability to cause psychostimulant and/or entactogenic effects, but unfortunately produce substantial adverse effects, including death. Three benzofurans 5-(2-aminopropyl)-2,3-dihydrobenzofuran (5-APDB), 5-(2-aminopropyl)-2,3-dihydrobenzofuran (5-MAPB) and 6-(2-aminopropyl) benzofuran (6-APB) were tested to determine their behavioral effects in comparison with 2,3-methylenedioxymethamphetamine (MDMA), cocaine, and methamphetamine. METHODS: Locomotor activity was tested in groups of 8 male Swiss-Webster mice in an open-field task to screen for locomotor stimulant or depressant effects and to identify behaviorally active doses and times of peak effect. Discriminative stimulus effects were tested in groups of 6 male Sprague-Dawley rats trained to discriminate MDMA (1.5 mg/kg), cocaine (10 mg/kg), or methamphetamine (1 mg/kg) from saline using a FR 10 for food in a two-lever operant task. RESULTS: In the locomotor activity test, MDMA (ED(50) = 8.34 mg/kg) produced peak stimulant effects 60 to 80 min following injection. 5-MAPB (ED(50) = 0.92 mg/kg) produced modest stimulant effects 50 to 80 min after injection, whereas 6-APB (ED(50) = 1.96 mg/kg) produced a robust stimulant effect 20 to 50 min after injection. 5-APDB produced an early depressant phase (ED(50) = 3.38 mg/kg) followed by a modest stimulant phase (ED(50) = 2.57 mg/kg) 20 to 50 min after injection. In the drug discrimination tests, 5-APDB (ED(50) = 1.02 mg/kg), 5-MAPB (ED(50) = 1.00 mg/kg) and 6-APB (ED(50) = 0.32 mg/kg) fully substituted in MDMA-trained rats, whereas only 5-MAPB fully substituted for cocaine, and no compounds fully substituted for methamphetamine. CONCLUSIONS: The synthetic benzofuran compound 5-APDB and 5-MAPB produced weak locomotor effects, whereas 6-APB produced robust locomotor stimulant effects. All compounds were more potent than MDMA. All three compounds fully substituted in MDMA-trained rats suggesting similar subjective effects. Taken together, these results suggest that these benzofuran compounds may have abuse liability as substitutes for MDMA.Item Novel pharmacotherapy: NNI-362, an allosteric p70S6 kinase stimulator, reverses cognitive and neural regenerative deficits in models of aging and disease(BioMed Central Ltd., 2021-01-13) Sumien, Nathalie; Wells, Matthew S.; Sidhu, Akram; Wong, Jessica M.; Forster, Michael J.; Zheng, Qiao-Xi; Kelleher-Andersson, Judith A.Aging is known to slow the neurogenic capacity of the hippocampus, one of only two mammalian adult neurogenic niches. The reduction of adult-born neurons with age may initiate cognitive decline progression which is exacerbated in chronic neurodegenerative disorders, e.g., Alzheimer's disease (AD). With physiologic neurogenesis diminished, but still viable in aging, non-invasive therapeutic modulation of this neuron regeneration process remains possible. The discovery of truly novel neuron regenerative therapies could be identified through phenotypic screening of small molecules that promote adult-born neurons from human neural progenitor cells (hNPCs). By identifying neuron-generating therapeutics and potentially novel mechanism of actions, therapeutic benefit could be confirmed through in vivo proof-of-concept studies. The key aging and longevity mTOR/p70S6 kinase axis, a commonly targeted pathway, is substrate for potential selective kinase modulators to promote new hippocampal neurons from NPCs. The highly regulated downstream substrate of mTOR, p70S6 kinase, directly controls pleiotropic cellular activities, including translation and cell growth. Stimulating this kinase, selectively in an adult neurogenic niche, should promote NPC proliferation, and cell growth and survival in the hippocampus. Studies of kinase profiling and immunocytochemistry of human progenitor neurogenesis suggest that the novel small molecule NNI-362 stimulates p70S6 kinase phosphorylation, which, in turn, promotes proliferation and differentiation of NPCs to neurons. NNI-362 promoted the associative reversal of age- and disease-related cognitive deficits in aged mice and Down syndrome-modeled mice. This oral, allosteric modulator may ultimately be beneficial for age-related neurodegenerative disorders involving hippocampal-dependent cognitive impairment, specifically AD, by promoting endogenous hippocampal regeneration.Item Pancreatic mitochondrial complex I exhibits aberrant hyperactivity in diabetes(Elsevier Inc., 2017-07-19) Wu, Jinzi; Luo, Xiaoting; Thangthaeng, Nopporn; Sumien, Nathalie; Chen, Zhenglan; Rutledge, Margaret A.; Jing, Siqun; Forster, Michael J.; Yan, Liang-JunIt is well established that NADH/NAD(+) redox balance is heavily perturbed in diabetes, and the NADH/NAD(+) redox imbalance is a major source of oxidative stress in diabetic tissues. In mitochondria, complex I is the only site for NADH oxidation and NAD(+) regeneration and is also a major site for production of mitochondrial reactive oxygen species (ROS). Yet how complex I responds to the NADH/NAD(+) redox imbalance and any potential consequences of such response in diabetic pancreas have not been investigated. We report here that pancreatic mitochondrial complex I showed aberrant hyperactivity in either type 1 or type 2 diabetes. Further studies focusing on streptozotocin (STZ)-induced diabetes indicate that complex I hyperactivity could be attenuated by metformin. Moreover, complex I hyperactivity was accompanied by increased activities of complexes II to IV, but not complex V, suggesting that overflow of NADH via complex I in diabetes could be diverted to ROS production. Indeed in diabetic pancreas, ROS production and oxidative stress increased and mitochondrial ATP production decreased, which can be attributed to impaired pancreatic mitochondrial membrane potential that is responsible for increased cell death. Additionally, cellular defense systems such as glucose 6-phosphate dehydrogenase, sirtuin 3, and NQO1 were found to be compromised in diabetic pancreas. Our findings point to the direction that complex I aberrant hyperactivity in pancreas could be a major source of oxidative stress and beta cell failure in diabetes. Therefore, inhibiting pancreatic complex I hyperactivity and attenuating its ROS production by various means in diabetes might serve as a promising approach for anti-diabetic therapies.Item The Effect of Late-Life Antioxidant Supplementaion on Brain Function(2007-10-01) Shetty, Ritu A.; Forster, Michael J.; Sumien, Nathalie; Singh, MeharvanShetty, Ritu A., The effect of late-life antioxidant supplementation on brain function. Doctor of Philolosophy (Biomedical Sciences), October, 2007, 229 pp., 5 tables, 18 figures, bibliography, 284 titles. Purpose: Aging is associated with mild to moderate loss in brain function over time. These functional losses are thought to involve reversible changes disrupting important cellular signaling processes. One of the theories that proposes to explain the reversible losses of function is the ‘oxidative stress’ hypothesis of aging. According to the oxidative stress hypothesis, there is an inherent cellular imbalance between production of oxidants and antioxidative defenses that increases with age and that leads to an increase in oxidative damage to macromolecules that are involved in crucial cell functions. Previous studies have established a link between these cellular changes associated with aging and the impairments in cognitive and psychomotor function. Further it has also been suggested that dietary interventions can modulate the level of oxidative stress, reducing oxidative damage and perhaps even ameliorate age-related dysfunction. Most interventions have been implemented relatively early in life and maintained until old age. However, the current studies were based on the rationale that interventions initiated in late-life could potentially lower oxidative damage and thereby alter cellular components responsible for functional impairments. Methods: In study I, separate groups of young (4 months) and old mice male C57BL/6 (18 months) were fed a control diet or a diet supplemented with low (105 mg/kg/day) or high (368 mg/kg/day) concentrations of CoQ10 for a period of 15 weeks. After 6 weeks on the diets, the mice were subjected to a battery of age-sensitive behavioral tests. In study II, separate groups of male C57BL/6 young mice aged 3-4 months and old mice 17-18 months (total of n=124) were fed ad libitum either a control diet (cyclodextrin in base diet), or the same diet supplemented with D- α-tocopheryl acetate (Toc) (200 mg/kg body wt/day), or with CoQ10 (148 mg/kg body wt/day) or a diet containing a combination of CoQ and Toc (200 mg/kg body wt/day + 148 mg/kg body wt/day) for a period of 13-14 weeks. In both studies mice were subjected to a battery of behavioral tests that required utilization of various component of memory and learning and sensorimotor reflexes. Results: In study I, low CoQ10 failed to improve cognitive and psychomotor function in old mice. However, the high CoQ10 marginally helped the old mice to navigate in the swim maze task with greater efficiency than control mice but did not affect their performance in probe trials. Conversely, the high CoQ10 diet selectively impaired the spatial performance in young mice in probe trials. The results from study I indicated that intake of CoQ10 initiated in late-life had minimal beneficial effects on behavior function. In study II, an age-associated decline of behavioral functioning was observed; however CoQ10 treatment failed to improve the performance of mice in any of the age-sensitive tests. Moreover, young mice supplemented with a high CoQ diet performed poorly in the probe trial in a swim maze task, suggesting a possible deleterious effect. The results from study II indicated that there was a significant improvement in performance of old mice in the coordinated running and the learning ability in discriminated avoidance task when supplemented with Toc or with a combination of CoQ10 and Toc. Conclusions: In conclusion, these studies suggest that benefits of single antioxidant supplementation when initiated late in life are limited; however dietary supplementation with a combination of antioxidants has a greater impact in reversing age-related decline in behavioral function.Item The Effects of Lifelong Glutathione Deficiency on Functional Decline and Redox Signaling(2018-08) Mock, J. Thomas; Sumien, Nathalie; Forster, Michael J.; Salvatore, Michael; Yang, Shaohua; Zode, Gulab S.Purpose: A recent paradigm shift has implicated redox state as a potential key determinant underlying the aging process. Specifically, a pro-oxidizing shift in the ratio of reduced to oxidized glutathione (key substrate in redox status) is hypothesized to disrupt cellular signaling leading to functional impairments and mortality. Chronic glutathione deficiency is achieved by global knockout of glutamate-cysteine ligase modifier (GCLM), an enzyme subunit at the rate-limiting step in glutathione synthesis. Glutathione levels in GCLM-/- mice are 10-30% of those in GCLM+/+ mice. Our hypothesis stated that diminished glutathione synthesis would be sufficient to produce an accelerated, aging-like pattern effect on function, a shortened lifespan, and negative alterations in redox state. Methods: We characterized GCLM+/+ and GCLM-/- male and female mice with a functional battery (n = 15-23 / sex / age / genotype) measuring motor, cognitive and affective function. We also measured redox state, inflammation, metabolism and autophagy markers in central and peripheral tissues (n = 3-6 / group) at 5, 10, or 20 months of age. Lastly, survivorship and body weights were recorded for all animals (n = 455). Results: Overall, age-related declines in function were observed in all functional tests. In young and adult mice glutathione deficiency did not negatively affect function, rather it decreased anxiety-related behavior, improved coordinated running performance in young females and adult males, and delayed general motor decline in both sexes. In old mice, glutathione deficiency improved balance in males and worsened age-related motordecline in females, yet it had no negative effects on cognition in either sex. Lifespan was also extended in male and female GCLM-/- mice (median and maximum). Lastly, GCLM-/- had reduced liver redox state throughout life but only at 5 months in the young, and had increased inflammatory markers in old mice. Discussion: These data imply that (i) motor and cognitive domains appear to be differentially affected by glutathione deficiency and led to benefits in young/adult GCLM-/- mice, (ii) functional and biochemical outcomes were sexually dimorphic, (iii) glutathione deficiency did not decrease lifespan, but rather extended lifespan, and (iv) redox state was impaired in GCLM-/- mice across the lifespan peripherally, but primarily only at 5 months in central tissues. These data do not support the redox stress hypothesis of aging and require further investigation of the beneficial outcomes associated with chronic glutathione deficiency.Item The Influence of CNS stimulants, opioid antagonists and an NMDA antagonist on the reinforcing effect of cocaine using a progressive-ratio schedule(1996-07-01) Li, Donghang; Forster, Michael J.; Martin, Michael; Luedtke, Robert R.It has been hypothesized that there is a common dopaminergic pathway mediating the reward properties of abused drugs, and that dopamine is involved in tolerance to the reinforcing effect of cocaine. The progressive-ratio (PR) schedule can be used to test both potentiation and reduction of the reinforcing effects of cocaine by other factors. Under the PR schedule, an increasing number of responses is required to obtain each subsequent cocaine injection, and failure to complete the required number of responses within 1 h of the previous cocaine injection terminates the session. The number of total reinforcers obtained during a session is defined as “the breaking point” and was used as the primary dependent measure. Fisher F344 male rats acquired the self-administration task under the PR schedule within forty sessions and showed a stable daily acquisition baseline. The breaking point and inter-reinforcer time (ISRT) were positively correlated within each ratio. A motor-incapacitating side effect of a pretreatment can be determined by a change in the relationship between the ISRT and the breaking point. d-Amphetamine pretreatment (0.32-3.2 mg/kg, i.p., 30 min) potentiates the reinforcing effect of cocaine as demonstrated by a higher breaking point of self-administration without changing the ISRT. Morphine pretreatment (0.32-3.2 mg/kg, i.p., 30 min failed to change the breaking point of cocaine self-administration but it did increase the ISRT. These results support an additive reinforcing effect for amphetamines and cocaine, but do not support an additive reinforcing effect of morphine and cocaine. The reinforcing effect of cocaine was reduced by pretreatment with ketamine (0.032-0.32 mg/kg, i.p., 20 min) as indicated by a reduction in the breaking point. In a concurrent experiment, animals were trained to self-administer cocaine under a fixed ratio 2 schedule (FR2). Ketamine pretreatment did not modify the ISRT in FR2 trained animals except at the highest dose (0.32 mg/kg, i.p., 20 min), where significant motor incoordination was observed. Both chronic treatment with cocaine (20 mg/kg/ 8hr x 7 days, iv) or amphetamine (3.2 mg/kg /12 hr x 7 days, i.p.) resulted in a reduction in breaking point at any given dose, providing direct evidence of tolerance and cross-tolerance to the reinforcing effects of cocaine. Chronic treatment with ketamine (0.32 mg/kg/8hr x 7 days, i.v.) failed to modify either the breaking point under a PR schedule of reinforcement or the ISRT under a FR2 schedule of reinforcement. Co-administration of ketamine (0.32 mg/kg/8hr x 7 days, i.v.) with chronic cocaine (20 mg/kg/8hr x 7days, i.v.) failed to prevent tolerance to the reinforcing effect of cocaine as indicated by either the breaking point under a PR schedule of reinforcement of the ISRT under an FR2 schedule of reinforcement. These data indicate that the breaking point in the PR schedule is more sensitive to changes in the dopamine reward system, whereas changes in rate of response are not consistently related to the changes in the dopamine reward system. These data support the use of PR schedule as a better method than FR schedule for determining reward properties of drugs of abuse with fewer complications due to the central nervous system inhibitory effects of some drugs of abuse.