Browsing by Subject "Chemical Actions and Uses"
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Item 17 Beta-Estradiol, Integrins, and Synaptic Proteins(2009-05-01) Chandra, Manjari; Simpkins, James W.Item Analyzing the Scientific Debate of Coxibs and The Ethics Impact of Vioxx's Withdrawal on Drug Regulation and an Ongoing Phase III Clinical Trial with a New Cox-2 Inhibitor(2005-11-01) Fu, Jingwei; Gwirtz, Patricia A.; Rubin, Bernard R.; Jiminez-Williams, CynthiaOn September 30, 2004, Merck & Co. Inc announced voluntary withdrawal of its $25 billion blockbuster drug Vioxx from the market, five and a half years after Voixx received FDA approval. This is the largest prescription drug withdrawal in history. Merck made the decision based on the results of APPROVe (Adenomaous Polyp Prevention on Vioxx) clinical trial, which showed that Voixx had an increased risk of myocardial infarction and cerebral vascular stoke compared with placebo. The repercussions of Merck’s action were tremendous from both a financial aspect and an ethical aspect. The recalling of Vioxx has become an important public health issue and has placed drug regulation agencies in controversy. In April, 2005, Pfizer agreed to voluntarily suspend sales and marketing of its COX-2 inhibitor, Bextra in the United States as requested by the FDA. Vioxx and Bextra withdrawal has left a huge impact on the pharmaceutical industry. Debates are ongoing in the scientific community regarding the use of Cox-2 inhibitors and have caused much confusion in the medical community and in those who use these drugs for pain control for osteo- and rheumatoid arthritis disease. The goal of this report is to analyze the impact of Vioxx withdrawal and comment on how to apply this incident in guiding the industry with regards to drug development, drug regulation, and clinical practice in order to ensure the effectiveness in the drug development and safe usage of new pharmaceutical agents.Item Antioxidants, Exercise, APOE Genotype and Brain Function(2014-12-01) Chaudhari, Kiran; Nathalie Sumien; Michael J. Forster; Eric B. GonzalesApolipoprotein E4 (APOE4) is a well-established and extensively prevalent genetic risk factor for the development of Alzheimer’s disease (AD). The presence of APOE4 allele accelerates the pathophysiology and symptomology of AD. A large set (36%) of the population suffering from AD expresses APOE4. Being a chronic progressive disease with very few pharmaco-therapeutic agents approved by FDA, non-drug lifestyle modifications have been an important part of management of AD. People often eat healthy diet rich in antioxidants and focus on healthy living habits such as exercise. Health care providers frequently suggest combining antioxidants with physical activity for higher benefits. Antioxidants have been beneficial in counteracting oxidative stress and improving learning and memory. Similarly, different regimens of exercise also improved cognition and delayed development of AD. However, the nature of the interaction between antioxidants and exercise remain elusive and complicated. While some studies reported additive effects, others have also shown a concerning antagonistic action of the antioxidants on the beneficial effects of exercise. In the context of APOE genotype, we set our study to determine the nature of such interaction between antioxidants and exercise. Using vitamins C and E and a treadmill-based forced exercise in a genetically modified mouse model expressing human APOE3 and APOE4 (GFAP-APOE3, GFAP-APOE4), we explored the nature of that interaction on functional and biochemical outcomes. We examined the mice for spatial learning and memory, working memory and executive function, coordinated running performance, muscular reflexes, spontaneous locomotor activity, anxiety and muscle strength. Interestingly, we observed that the young adult mice expressing E4 allele performed better on higher brain functions including spatial learning and memory and short term memory in contrast to middle age mice, which developed a cognitive deficit as expected. Motor functions, reflexes and coordination were poor among all the mice carrying E4 allele irrespective of age. Antioxidants and exercise interventions led to outcomes that were dependent on genotype, age and the brain function under consideration. There was additive beneficial effect of combination of antioxidants and exercise on cognitive outcomes but not on motor outcomes in middle age groups. However, in young adults, an antagonistic interaction was observed on motor outcomes but no such interaction was observed on cognitive outcomes. Hence we can conclude that, combination of antioxidants and exercise is not a “fit for all” approach and needs to be tailored base on individual’s age and genotype.Item Cross-Tolerance Between the Discriminative Stimulus Properties of Ethanol, Diazepam and Pentobarbital(1995-12-01) Lytle, Douglas A.; Michael Forster; Glenn Dillon; Thomas YorioLytle, Douglas A., Cross-Tolerance Between the Discriminative Stimulus Properties of Ethanol, Diazepam and Pentobarbital. Doctor of Philosophy (Biomedical Sciences), December, 1995, 132 pp., 8 tables, 19 figures, bibliography, 176 titles. Ethanol, benzodiazepine agonists and barbiturates all facilitate GABA-mediated CT flux. The present experiments tested the hypothesis that, because these agents share this common action, tolerance to discriminative stimulus properties of one of these drugs would result in cross-tolerance to the others. Rats were trained to detect either ethanol (EtOH; 1.0 g/kg), the benzodiazepine diazepam, (DZP; 5.6 mg/kg), or the barbiturate pentobarbital (PB; 10.0 mg/kg) from vehicle using a two-lever choice procedure where food was available under a fixed-ration ten schedule of reinforcement. Subsequently, dose-effect curves for EtOH (0.1-1.78 g/kg), DZP (0.56-17.8 mg/kg), or PB (1.0-17.8 mg/kg) were tested before and after chronic administration of EtOH 96.0 g/kg/12hrs for seven days), DZP (20.0 mg/kg/8hrs for seven days), or PB (32.0 mg/kg/8hrs for seven days). The chronic administration of EtOH conferred tolerance to itself in all cases and cross-tolerance to DZP and PB in subjects trained to detect EtOH, but did not confer cross-tolerance to these agents in their respective discriminations. The chronic administration of DZP conferred tolerance to itself substituting for DZP. Although tolerance developed to DZP substituting for PB after treating animals with chronic DZP, this regimen on DZP did not confer tolerance to itself substituting for EtOH. This regimen of DZP failed to confer significant cross-tolerance to either EtOH or PB under any conditions. The chronic administration of PB conferred tolerance to itself substituting for PB. Although tolerance developed to PB substituting for DZP after treating animals with chronic PB, this regimen of PB did not confer tolerance to itself substituting for EtOH. This regimen of PB failed to confer significant cross-tolerance to either EtOH or DZP under any conditions. In summary, EtOH was found to confer cross-tolerance to DZP and PB only in animals trained to detect EtOH. The chronic administrations of DZP and PB failed to confer tolerance to themselves substituting for EtOH. These results are parsimonious with the heterogeneous nature of the GABA receptor. Finally, tolerance to either DZP or PB does not result in cross-tolerance to the discriminative stimulus properties of the other drug. These results suggest that the mechanisms mediating tolerance to BZs and barbiturates are not linked.Item Discriminative and Negative Reinforcing Properties of the Periaqueductal Gray and the Medial Hypothalamus(1994-12-01) Jung, Marianna E.; Emmett-Oglesby, Michael W.; Yorio, ThomasMarianna Eunsun, Jung, Discriminative and Negative Reinforcing Properties of Electrical brain stimulation of the Periaqueductal Gray and the Medial Hypothalamus. Master of Science [Biomedical Sciences, (Pharmacology)], December, 1994, 123 pp., 24 figures, references, 137 titles. Electrical brain stimulation (EBS) of the periaqueductal gray (PAG) and the medial hypothalamus (MH) is known to serve as a discriminative and a negative reinforcing stimulus (NRS). Using a two-lever food reinforced discrimination paradigm and a switch-off paradigm, the present study investigated the effects of anxiolytic drugs and an anxiogenic drug on these stimulus effects. A prototypic anxiogenic, pentylenetetrazole (PTZ) potentiated both discriminative stimulus and NRS effects, whereas the full benzodiazepine (BZD) agonist diazepam (DZP), the partial BZD agonist abecarnil (ABC) and 5-HT1A agonist buspirone (BUS, chronic regimen) attenuated a NRS effect. A BZD antagonist, flumazenil (FLU) blocked the effects of DZP and ABC on the NRS effects. DZP failed to attenuate the discriminative stimulus effect. Thus, present study extended the use of a switch-off paradigm to detect novel anxiolytic ABC (putative) and BUX as well as an anxiogenic PTZ. In addition, under the condition used in this study, the use of NRS in a switch-off paradigm more reliably detected both anxiolytic drugs and an anxiogenic drug than the use of discriminative stimulus in a two-lever food reinforced paradigm.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 Elucidation of the Mechanism of Action of Carisoprodol at GABAA Receptors(2009-05-01) Gonzalez, Lorie A.; Dillon, Glenn H.Carisoprodol is an increasingly abused, centrally-acting muscle relaxant. Its sedative effects, which contribute to its therapeutic and recreational use, are attributed to its metabolite, meprobamate, a controlled substance with barbiturate-like activity at GABAA receptors (GABAARs). GABAARs are ion channel-coupled protein complexes underlying the majority of fast synaptic inhibition in the central nervous system. Recent evidence suggests carisoprodol may act independently of meprobamate. Thus, we used behavioral and pharmacological approaches to investigate carisoprodol’s effects on GABAAR function with the ultimate goal of elucidating its mechanism of action at these receptors. In mice, the time course of locomotor depression was comparable for carisoprodol (intraperitoneal or oral) versus meprobamate (intraperitoneal). GABAergic ligands substituted for carisoprodol in drug discrimination studies using carisoprodol trained rats. As observed in vitro, carisoprodol’s effects were antagonized by bemegride, a barbiturate antagonist, but not by the benzodiazepine site antagonist flumazenil, suggesting carisoprodol produces barbiturate-like effects in vivo. Moreover, whole-cell patch clamp recordings were obtained from HEK293 cells expressing human α1β2 and αxβzγ2 (where x = 1-4 and z = 1-2) GABAARs. Each receptor configuration was directly activated and allosterically modulated by carisoprodol in a barbiturate-like manner. Carisoprodol efficacy, but not potency, was subunit-dependent with α and β isoforms contributing to carisoprodol site(s) of action. Notably, carisoprodol was more efficacious at α1-containing receptors, consistent with its sedative effects and abuse potential. Homomeric glycine α1 and GABA ρ1 receptors were carisoprodol-insensitive. Despite similarities between carisoprodol and barbiturates, their sites of action are likely not equivalent as barbiturate-sensitive ρ1W328M subunits were carisoprodol-insensitive. However, chimeric ρ1/α1 receptors gained sensitivity to modulation, but not direct activation by carisoprodol. Our findings indicate carisoprodol modulates GABAARs in a subunit- and receptor-dependent manner, contributing to its pharmacological profile and possibly its abuse potential. Furthermore, partial restoration of modulation, but not direct gating by carisoprodol suggests this drug may mediate its effects via multiple sites on GABAARs.Item Intermittent hypoxia induced opioidergic protection of the heart(2015-08-01) Estrada, Juan A.; Robert T. Mallet; Steve W. Mifflin; J. Thomas CunninghamNormobaric intermittent hypoxia conditioning (IHC) induces a robust cardioprotected phenotype in dogs that is remarkably resistant to ischemia and reperfusion induced myocardial infarction and lethal arrhythmias. Previous studies demonstrated that IHC induced cardioprotection requires β1-adrenergic receptor activity. Cardiac opioid systems are stimulated by, and counteract the harmful effects of, excessive stressors such as sympathetic activity. Additional modes of hypoxic conditioning have been shown to induce synthesis of cardiac enkephalins and delta opioid receptors (DOR). The hypothesis that DOR mediates IHC cardioprotection was examined in two studies conducted in intermittent hypoxia conditioned and non-hypoxic sham dogs. For the first study dogs were assigned to groups subjected to non-hypoxic sham conditioning, IHC, IHC plus the aminothiol antioxidant N-acetylcysteine (NAC), and IHC plus the DOR antagonist naltrindole. After IHC or sham conditioning, the dogs were subjected to an left anterior descending coronary artery occlusion/reperfusion protocol and incidence of reperfusion arrhythmias and myocardial infarct size were measured and adjusted for coronary collateral flow. Naltrindole and NAC abolished the anti-infarct and anti-arrhythmia effects of IHC, in a manner independent of collateral blood flow. Intermittent hypoxia conditioning is thus dependent on DOR activity as well as formation of reactive oxygen species (ROS) during cylic hypoxia-reoxygenation. Whether ROS are generated upstream, downstream, or in parallel to DOR activation remains to be determined. DORs are abundant on dog parasympathetic nerves and therefore are ideally positioned to stimulate cardioprotective cholinergic activity. However it is unknown in what direction IHC modulates bimodal DORs, i.e. modulation of synaptic inhibitory or excitatory activity. In the second study dogs were assigned to sham conditioned, IHC, and IHC plus naltrindole groups. IHC resulted in a profound enhancement of vagal bradycardia, in the absence and presence of increasingly vagolytic doses of the DOR agonist MEAP. This result demonstrated that IHC shifts DOR signaling in favor of the vagotonic DOR-1 receptor subtype. However, the fate of the vagolytic DOR-2 receptors was unknown. Immunolabeling of atrial tissue revealed that IHC increased content of the monosialoganglioside GM1 in autonomic nerve fibers associated with parasympathetic fibers, an effect which may shift DOR signaling in favor of the DOR-1 subtype. In addition, IHC increased the number of fibers containing the vesicular acetylcholine transporter within the sinoatrial node. However, DOR positive fibers in both the atria and SAN were decreased after IHC, perhaps reflecting redistribution or intracellular trafficking of DOR1 and/or DOR2 receptors. Immunoblotting revealed decreased content of adrenergic protein tyrosine hydroxylase in the left ventricle following IHC. Collectively, these results indicate IHC is dependent on opioidergic activity to induce cardioprotection by enhancing cholinergic signaling components at the expense of adrenergic proteins, suggesting IHC-induced shifting of autonomic balance in favor of parasympathetic control of the heart.Item Intravenous Pyruvate to Prevent Renal Injury Following Cardiac Arrest and Resuscitation(2014-08-01) Hollrah, Roger A.; Robert T. Mallet; Myoung-Gwi Ryou; Rong MaIntroduction: Cardiac arrest followed by resuscitation and recovery of spontaneous circulation (ROSC) produces systemic ischemia reperfusion (I/R), affecting all internal organs, including the kidney. This type of stress generates both a robust increase in reactive oxygen and nitrogen species (RONS) and an intense inflammatory response, which can result in renal cell death. The glycoprotein erythropoietin (EPO) has been shown to combat renal I/R injury by offering cyto-protection against inflammation and oxidative damage, as well as inhibiting apoptosis. The endogenous intermediary metabolite pyruvate has been observed to stabilize specific genetic machinery responsible for the production of EPO. This study was conducted to test the efficacy of intravenous pyruvate in exploiting these endogenous mechanisms of EPO to protect the kidney from cardiac arrest-induced, I/R injury. Hypothesis: Pyruvate administration during cardiopulmonary resuscitation (CPR), defibrillation, and ROSC will protect the kidneys from I/R injury by suppressing oxidative stress and inflammation via increased EPO production at the renal corticomedullary border. Methods: Yorkshire swine underwent 10 minutes of cardiac arrest, CPR effected by precordial compressions, and defibrillation, and were recovered for either 4 hours (acute) or 3 days (chronic). The animals were randomly assigned to 1 of 4 groups. Two groups underwent the cardiac arrest protocol described above: one group received intravenous infusion of 2M sodium pyruvate at a rate of 0.1 mmol∙kg-1∙min-1 during CPR and the first 60 minutes of recovery; the other group received an equimolar infusion of NaCl. The other two groups were surgically prepared and infused with NaCl or sodium pyruvate, but were not subjected to cardiac arrest, CPR, or defibrillation. For the acute protocol (n=28), animals were sacrificed 4hr after cardiac arrest, while in the chronic protocol (n=18), animals recovered for 3d before sacrifice. To evaluate the impact of cardiac arrest and pyruvate treatment on renal metabolism and antioxidant defense, proteins were extracted from snap-frozen renal corticomedullary border tissue for spectrophotometric activity assays of a panel of 10 metabolic and antioxidant enzymes; myeloperoxidase (MPO), an enzyme marker of pro-inflammatory leukocytes, was analyzed to assess inflammation. Plasma was sampled before cardiac arrest and at the time of biopsy to measure creatinine concentration, an indirect measure of glomerular filtration rate (GFR). Enzyme-linked immunosorbent assay (ELISA) kits were used to measure EPO content and Kidney Injury Molecule-1 (KIM-1) content, a receptor expressed on renal tubular cells that plays an important role in apoptosis. Tissue sections were stained with hematoxylin and eosin (H&E) and examined under light microscopy to count neutrophils and monocytes and to compare structure integrity across the different treatment groups and protocols. Results: In this study global I/R stress imposed on the kidneys by reversible cardiac arrest did not appreciably alter the activity of the 10 panel enzymes. Despite having no histological evidence of neutrophil infiltration (H&E stained slides), an increase in renal MPO activity was evident at 4 h recovery in the NaCl group which was prevented by pyruvate treatment (P [less than] 0.05). There was no evidence of ultrastructural damage to renal cortical and outer medullary structures. There was a noticeable increase in renal EPO content at 4 h ROSC vs. the sham group. An apparent, albeit not statistically significant, increase in KIM-1 content was observed in the two CPR groups vs. the NaCl-infused sham group. Plasma creatinine concentrations did not change appreciably between pre-arrest baseline and 3 d recovery. Interpretation and Conclusion: The I/R stress produced by the present cardiac arrest-resuscitation failed to alter appreciably the activities of the 10 panel enzymes, suggesting the oxidative stress was not sufficient to overwhelm the kidney’s endogenous antioxidant defenses. Plasma creatinine concentrations were also stable, implying the GFR was maintained and the glomerular ultrastructures were unaffected by I/R. The increase in MPO activity at 4 h ROSC implied a transient infiltration of inflammatory leukocytes, although none were visible on histological examination. The increase in KIM-1 content, though not statistically significant, suggests modest renal apoptotic activity after cardiac arrest and reperfusion. The transient increase in renal EPO content in the NaCl-infused post-arrest vs. sham pigs supports the possibility that even a brief period of renal ischemia by cardiac arrest can evoke renal EPO production. Collectively, these results indicate the renal I/R imposed by cardiac arrest and resuscitation does not inflict appreciable damage on the kidneys or its enzyme systems, at least within the first 3 d of post-arrest recovery. Abbreviations: AKI: acute kidney injury; ARF: acute renal failure; CK: creatine kinase; CPR: cardiopulmonary resuscitation; CS: citrate synthase; EPO: erythropoietin; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; G6PDH: glucose 6-phosphate dehydrogenase; GFR: glomerular filtration rate; GP: glutathione peroxidase; GR: glutathione reductase; HIF-1: hypoxia-inducible factor 1; I/R: ischemia-reperfusion; KIM-1: kidney injury molecule 1; LDH: lactate dehydrogenase; MPO: myeloperoxidase; PFK: phosphofructokinase; PHD: prolyl hydroxylase; RONS: reactive oxygen and nitrogen species; ROSC: recovery of spontaneous circulation.Item Intravenous pyruvate to protect heart and brain during closed-chest resuscitation and recovery from cardiac arrest(2014-08-01) Cherry, Brandon H.; Mallet, Robert T.; Olivencia-Yurvati, Albert H.; Raven, Peter B.Cardiac arrest is a leading cause of death in the United States and Western Europe. Cardiopulmonary resuscitation (CPR) is the only means of sustaining the victim until application of defibrillatory countershocks. Although it has been over 50 years since its advent, CPR remains a work in progress. Many initially resuscitated victims later die from the damage sustained from ischemia-reperfusion, and treatments to combat the extensive ischemia-reperfusion injury sustained during cardiac arrest-resuscitation remain elusive. The major mechanism of injury underlying ischemia-reperfusion is the intense overproduction of reactive oxygen and nitrogen species (RONS) that accumulate during reperfusion and compromise normal cell function. RONS formed during resuscitation trigger lipid peroxidation, disable enzymes vital for cell metabolism and survival and, ultimately, induce cell death within affected organs. In order to prevent extensive damage to the central nervous system culminating in permanent neurocognitive disability and death, prospective treatments must possess robust antioxidant properties, traverse the blood-brain barrier between the cerebral circulation and brain parenchyma, and be non-toxic at effective doses. Pyruvate is a natural intermediary metabolite, energy-yielding substrate and antioxidant. Pyruvate neutralizes RONS, thereby dampening oxidative stress and preventing covalent oxidative modification of enzymes and lipid membranes, and generates ATP to support brain function. Pyruvate readily traverses the blood-brain barrier and is non-toxic over a wide range of doses, including those previously demonstrated to protect the heart during cardiopulmonary bypass and the brain during stroke, thereby supporting oxygen and fuel delivery to the recovering brain. Moreover, pyruvate has been shown to promote cardiac electromechanical and metabolic recovery following cardiac arrest and open-chest CPR. This study tested whether infusion of pyruvate during, CPR and early recovery can decrease the biomarkers of oxidative stress after cardiac arrest. Isoflurane-anesthetized pigs were subjected to 6 min electrically-induced, untreated ventricular fibrillation, followed by 4 min closed-chest CPR, defibrillation and either 1 or 4 h recovery. Beginning at 5.5 min arrest, either sodium pyruvate or NaCl control were infused iv for the duration of CPR and for the first 60 min after recovery of spontaneous circulation (ROSC). Arterial blood was sampled pre-arrest and at 5, 15, 30, 60, 120, 180, and 240 min ROSC for analyses of blood gases and plasma constituents. At either 1 h (i.e. end of treatment infusion) or 4 h ROSC, a craniotomy was performed, the pig was euthanized, the brain was removed, and biopsies from hippocampus and cerebellum were snap-frozen in liquid nitrogen for biochemical analysis. The first phase of this project tested the hypothesis that intravenous administration of sodium pyruvate during precordial compressions and the first 60 min ROSC restores hemodynamic, metabolic, and electrolyte homeostasis in a closed chest porcine model of cardiac arrest. Resuscitation with pyruvate sharply decreased the incidence of lethal pulseless electrical activity (PEA) following defibrillatory countershocks, and lowered the dosage of vasoconstrictor phenylephrine required to maintain systemic arterial pressure. Pyruvate also enhanced glucose clearance, elevated arterial bicarbonate, and raised arterial pH. The second phase of this project tested the hypothesis that pyruvate prevents the decrease in activity of the brain’s antioxidant enzymes following cardiac arrest and hyperoxic (100% O2). Activities of glutathione peroxidase and glutathione reductase were decreased at 60 min ROSC vs. sham in both the hippocampus and cerebellum. Pyruvate partially preserved glutathione peroxidase activity at 1 h ROSC, but by 4 h, after 3 h of pyruvate clearance from the circulation, the enzyme’s activity fell to the same extent as in NaCl-infused pigs. Interestingly, the glutathione peroxidase/reductase activity fell sharply in non-arrested sham pigs between the time points corresponding to 1 and 4 h ROSC, suggesting that hyperoxia resulting from ventilation with 100% produced sufficient oxidative stress to inactivate the enzymes. Similarly, lactate dehydrogenase activity fell between 1 and 4 h ROSC in hippocampus and especially cerebellum. In sham pigs, lactate dehydrogenase activity decreased from the time points corresponding to 1 and 4 h ROSC, and pyruvate had no effect on lactate dehydrogenase in either region of the brain. Thus, cardiac arrest and hyperoxic ventilation disabled a critical antioxidant system in two ischemia-sensitive brain regions. Pyruvate afforded partial protection of these enzymes which waned after pyruvate cleared from the circulation. We conclude that 1) Pyruvate infusion during cardiac arrest, CPR and early recovery promotes conversion from ventricular fibrillation to a productive sinus rhythm instead of lethal PEA; 2) Pyruvate hastened glucose clearance, a prognostic measure used clinically; 3) Pyruvate elevated the arterial bicarbonate concentration and raised arterial pH, which combats the acidemia normally observed following ROSC; 4) Cardiac arrest-resuscitation and hyperoxic ventilation disabled the glutathione peroxidase-reductase system, a critical component of the brain’s antioxidant defenses, in hippocampus and cerebellum; and 5) Pyruvate delayed oxidative inactivation of glutathione peroxidase in the cerebellum, but this effect subsided as pyruvate elevated. These investigations demonstrate the therapeutic effects and limitations of pyruvate as a resuscitative treatment to hasten electrocardiographic and metabolic recovery post cardiac arrest.Item Lifelong vs. Late Life Tocopherol on Learning and Memory in Mice(2004-05-01) McDonald, Shelley R.; Michael Forster; Glenn DillonMcDonald, Shelley R., Lifelong vs. late life tocopherol on learning and memory in mice. Doctor of Philosophy (Biomedical Sciences), May, 2004, 132 pp., 1 table, 14 figures, bibliography, 122 titles. The purpose of these studies was to determine if vitamin E supplementation, a well-studied antioxidant, could improve the cognitive functions of old mice either by preventing age-dependent impairments or reversing age-related dysfunction. Cellular oxidative stress is believed to be a causal factor in senescence, and the brain appears to be particularly susceptible to oxidative damage because of a relatively high rate of reactive oxygen species generation without commensurate levels of antioxidant defenses. If oxidative stress indeed plays a role in age-related brain dysfunction, then it can be predicted that experimental interventions capable of lowering oxidative stress would either prevent or restore function. This was tested using apolipoprotein E-deficient mice, which have an increased susceptibility to neuronal oxidative damage, maintained on 3 different doses (2 mg/kg, 20 mg/kg, or 200 mg/kg/day) of dl-α-tocopheryl acetate (vitamin E) via supplemented food pellets from 8 weeks of age throughout behavioral testing when 6 or 18 mo of age. A separate experiment used wild type mice 24 months of age to examine whether or not a combination of vitamin E (123 mg/kg/day) with coenzyme Q10 (200 mg/kg/day) which leads to higher tissue levels of vitamin E, could improve brain functions in old mice. Mice were tested on multiple behavioral tasks that required utilization of various components of memory and learning, as well as sensorimotor testing. The highest dose of vitamin E prevented the decline of spatial memory in old apolipoprotein E-deficient mice, but did not prevent age-related impairments in learning and memory for discriminated escape. When old wild type mice were treated with the combined vitamin E and coenzyme W10, the mice learned and remembered to avoid a preemptive shock significantly more than old mice treated with vitamin E or coenzyme Q10 alone. A followup experiment with higher doses of coenzyme Q10 alone (250 or 500 mg/kg/day) resulted in no cognitive improvements. No treatments improved sensorimotor performance.Item Mechanism of Action and Clinical Efficacy of AL-3789, an Angiostatic Steroid(1996-12-01) Defaller, Joseph M.; Clark, Abbot F.; Quist, Eugene E.; Yorio, ThomasDeFaller, Joseph M., Mechanism of Action and Clinical Efficacy of AL-3789, an Angiostatic Steroid. Doctor of Philosophy (Pharmacology), December, 1996, 115 pp., 11 tables, 31 illustrations, reference list, 115 titles. Angiogenesis, the growth of new blood vessels, is important in cancerous tumor growth, diabetes, and degenerative diseases. During angiogenesis, proliferating vascular endothelial cells from blood vessels secrete proteinases, including urokinase (uPA) and stromelysin-1 (MMP-3), which dissolve the extracellular matrix and allow them to migrate and form new blood vessels. In this investigation the angiostatic effects of AL-3789, an angiostatic steroid, were investigated in in vitro human cell models and human clinical trials. The angiostatic mechanism of action of this agent at 10^-5 molar concentration was demonstrated to include: 1) inhibition of lipopolysaccharide-induced uPA and MMP-3 production by human microvascular endothelial cells (HMVEC-L), 2) dose-dependent inhibition of HMVEC-L proliferation, and 3) alteration in the expression of approximately 1% of HMVEC-L proteins visualized by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). These protein changes are similar in magnitude to those caused by glucocorticoids, which act via an intracellular/intranuclear glucocorticoid receptor. The clinical efficacy of AL-3789 in preventing re-neovascularization and recurrence of malignant pterygia following surgical excision in humans was demonstrated by topical ocular dosing of a 1.0% suspension three times daily in a double-masked, randomized, prospective, placebo-controlled trial. Pterygium is a chronic condition in which neovascularized fibrotic tissue grows over the cornea to eventually obstruct the visual axis in some patients. Computer image analysis of serial photographs for each patient following surgery showed a re-neovascularization growth rate following pterygium excision of 1.58 mm^2/week for patients treated with placebo compared to 0.78 mm^2/week exhibited by the AL-3789 treated group (p [less than] 0.05, ANOVA). Pterygia recurred in 71% of the placebo group compared to 42% of the AL-3789 treated group (p [less than] 0.05, Chi-square contingency test). In conclusion, the angiostatic steroid AL-3789 inhibits neovascularization in part by decreasing vascular endothelial cell proliferation and proteinase (urokinase and stromelysin-1) secretion. AL-3789 treatment significantly inhibits re-neovascularization and recurrence rates following malignant pterygium excision in humans.Item Modulation of GABAA Receptor Function by Tyrosine Phosphorylation(1998-05-01) Fang, Mingjun; Glenn Dillon; Thomas Yorio; Eugene E. QuistMingjun, Fang. Modulation of GABAA Receptor Function by Tyrosine Phosphorylation. Master of Science (Biomedical Sciences), May, 1998, 32 pp., 6 illustrations, bibliography, 42 titles. The goal of this study was to determine the modulation of GABAA receptor function by tyrosine kinase phosphorylation, and to detect which subunit is phosphorylated to alter the GABA-induced chloride currents. From previous studies, we suggested that protein tyrosine phosphorylation may maintain GABAA receptor function. Here we tested the hypothesis that tyrosine phosphorylation modulates other GABAA receptor subtypes e.g., α1β2γ2 and α6β2γ2, and subsequently attempted to determine which subunit(s) may be phosphorylated. Our results support the hypothesis that PTK phosphorylation may maintain GABAA receptor function. In addition, we suggest this tyrosine phosphorylation occurs at the γ2 subunit of the receptor.Item Neuroprotective Effects of Brn3b in PC12 Cells and Retinal Ganglion Cells under Glaucomatous Conditions(2015-08-01) Phatak, Nitasha R.; Raghu R. Krishnamoorthy; Weiming Mao; John V. PlanzGlaucoma is a group of chronic progressive optic neuropathies commonly characterized by elevated intraocular pressure (IOP) (a subset of glaucoma patients display neurodegenerative effects at ‘normal’ IOP) leading to axonal degeneration, optic nerve head cupping and apoptosis of retinal ganglion cell death (RGCs), which result in visual field defects and blindness. While there are medications available to lower IOP, there is an unmet need for neuroprotective treatments for glaucoma, since some neurodegenerative effects persist despite lowering IOP. The main focus of this study was on the class 4 POU domain transcription factor, Brn3b, which has been shown to play a key role in the development of RGCs. Two previous studies from other labs showed that a decrease in Brn3b expression occurs in animal model of glaucoma. A recent publication from our laboratory demonstrated neuroprotective effects of adeno-associated virus (AAV) mediated expression of Brn3b in a rat model of ocular hypertension. This research project identified some mechanisms of Brn3b-mediated neuroprotection in cultured PC12 cells (under the condition of hypoxia) and also in vivo in the Morrison’s model of ocular hypertension in rats. In the first part of the study, we demonstrated the effect of overexpression of Brn3b on various markers of synaptic plasticity in PC12 cells under conditions of normoxia as well as hypoxia. Immunoblot as well as immunocytochemical analyses revealed an increase in expression of neurite growth markers, GAP-43 and ac-TUBA, by Brn3b upregulation both under conditions of normoxia as well as hypoxia. . This suggests that transcription factor Brn3b has the ability to upregulate expression genes contributing to synaptic plasticity genes both under ‘normal’ conditions and during a glaucomatous insult (hypoxia). In the concluding part of this study, cell survival factors including, Bcl-2, Bcl-xL and p-AKT were studied as potential targets of Brn3b-mediated neuroprotection. Adeno-associated virus-mediated expression of Brn3b in rat eyes with elevated IOP promoted an upregulation of Bcl-2, Bcl-xL and p-AKT in RGCs, as determined by immunohistochemistry. Taken together, the evidence suggests that Brn3b has the potential to be developed as a therapeutic agent for neuroprotection during ocular neurodegenerative diseases like glaucoma.Item Psalmotoxin-1 and nonproton ligand interactions with acid-sensing ion channels(2015-05-01) Smith, Rachel N.; Gonzales, Eric B.; Dillon, Glenn H.; Sumien, NathalieAcid-sensing ion channels (ASICs) are trimeric, sodium-selective channels activated by extracellular protons. Although ASICs are intriguing molecular targets for pharmacological agents, there remains a lack of selective compounds that differentiate ASIC subtypes. The peripherally located ASIC3 activates with the simple removal of calcium. Additionally, nonproton ligands, like 2-guanidine-4-methylquinazoline (GMQ), have been identified to selectively activate ASIC3 via the nonproton ligand sensor domain (NPLSD). A pair of glutamates in rat ASIC3 (E79 and E423) responsible for GMQ activation is present in ASIC1, despite having no direct modulation effect on the channel. We proposed that nonproton ligand activation of ASIC1 may be state dependent, and relies on expansion of the NPLSD in order for GMQ to reach the binding site and exert its effects. We utilized two features of ASICs in order to test our hypothesis with whole cell and outside out patch-clamp electrophysiology. First, we induced a persistent current in chicken ASIC1 (cASIC1) via a natural venom toxin, Psalmotoxin-1 (PcTx1). We determined that GMQ acts as a molecular wedge by prying apart the transmembrane domains of the cASIC1-PcTx1 protein complex and potentiating ASIC-current. This led us to better understand that the NPLSD is intact in cASIC1 and is sensitive to GMQ additions, albeit in a state-dependent manner. We then theorized that direct activation of rASIC3 by GMQ is possible due the channel’s interaction with extracellular calcium, and were interested in introducing feature into the cASIC1 channel. We generated a chimeric ASIC combining the extracellular, transmembrane, and intracellular domains of rASIC3 and cASIC1 in order to individually isolate the calcium and nonproton ligand effects on channel activation. This chimera, termed cASIC1 (rASIC3-TM/ITC), is comprised of the extracellular domain of cASIC1 and the transmembrane/intracellular domains of rASIC3, and can be activated by GMQ in the absence of calcium similarly to wild-type rASIC3. Thus, GMQ activation was introduced in cASIC1 by replacing the transmembrane domains with those of ASIC3 suggesting that the ASIC3 TM domains dictate NPLSD influence on channel activity. Taken together, we identified that channel state influences nonproton ligand interaction with ASICs, and the transmembrane domains are critical for this interaction.Item Regulation of Human Macrophage Colony-Stimulating Factor Transcription(2001-05-01) Kamthong, Pisate John; Lad Dory; Richard Easom; Stephen R. GrantThe role of macrophage colony-stimulating factor (M-CSF) in hematopoiesis has been firmly established mainly by using bone marrow cell cultures. Semi-solid culture of bone marrow cells that were independently developed by Bradley and Metcalf in 1966 and Pluznik and Sachs in 1965, has been the standard method to study proliferation and differentiation of hematopoietic cells since the mid-1960s. It supports the clonal expansion of the hematopoietic colonies in vitro. Thus provides the means to functionally assay the hematopoietic colonies in vitro. Thus provides the means to functionally assay the hematopoietic progenitor cells and aides the discovery of growth factors regulating the progenitor cell differentiation. Macrophage colony-stimulating factor (M-CSF) was initially identified as a hematopoietic growth factor that stimulates the proliferation, differentiation and survival of monocytes, macrophages, and their progenitors (Robinson et al. 1969; Stanly et al. 1971). M-CSF is produced by a large variety of cells throughout the body. It can be purified from various body fluids as well as the conditioned media of several cell lines and tissues, such as leukocytes, placenta, lung, pancreatic cancer cells and spleen (Metcalf 1984; Stanley and Guilbert 1981; Yunis 1983). The sources of M-CSF recently have been extended to include liver parenchymal cells (Ezure et al. 1997) and thyrocytes (Kasai et al. 1997). It was also previously called colony-stimulating factor from human urine (CSF-HU) attributing the source of human urine growth factor that stimulated the formation of small aggregates consisting of granulocyte clusters in a soft agar culture system of human bone marrow cells (Metcalf 1974). At first without the knowledge about biochemical structures of CSFs, several colony-stimulating factors, later proven to be M-CSF, were recognized by their sources, i.e. mouse L-cell CSF, mouse uterus CSF, human lung-conditioned-medium CSF. Later, researchers in the field adopted the reclassification of CSF subtypes using the predominant colony types stimulated by the factor in semi-solid bone marrow cell cultures. M-CSF is for CSF that predominantly stimulates macrophage colony formation. Granulocyte colony-stimulating factor (G-CSF) refers to a granulocyte-active material, such as peritoneal cell-conditioned medium CSF (Horiuchi and Ichikawa 1977). CSF stimulating both types of colonies is called granulocyte-macrophage colony-stimulating factor, GM-CSF. M-CSF was also termed colony stimulating factor 1 (CSF-1), described as the first CSF to be purified (Stanley 1977). G-CSF was later called CSF-2. The existence of these two biochemically distinct CSFs was first identified in this lab (Wu et al. 1981). By the same virtue, GM-CSF sometimes was referred to as CSF-3. All of the three CSFs were later identified as distinct peptides encoded by different genes. Sachs and Pluznick group at Rehovot introduced alternative nomenclature for CSFs. The term macrophage granulocyte inducer-1M, MGI-1M, refers to M-CSF, MGI-1G is equivalent to G-CSF, and MGI-GM is used for GM-CSF. M-CSF stimulates differentiation of progenitor cells (colony-forming unit macrophage, CFU-M) to mature monocytes, and prolongs the survival of monocytes (Motoyoshi et al. 1997). It enhances expression of differentiation antigens (Hashimoto et al. 1997) and stimulates chemotactic, phagocytic and the killing activities of monocytes (Wang et al. 1988.) It also stimulates production of several cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and interleukin (IL)-6 by priming monocytes, and directly stimulates production and secretion of IL-8 and reactive nitrogen intermediates (Motoyoshi and Takaku 1991).Item Sensing and imaging of hyaluronidase activity using a long-lived fluorophore(2016-05-01) Chib, Rahul; Gryczynski, Zygmunt; Fudala, Rafal; Borejdo, JulianThis dissertation explores the synthesis, characterization and biomedical applications of a fluorescent probe for sensing and imaging of hyaluronidase activity. The enzyme hyaluronidase is overexpressed in various cancer including bladder cancer, prostate cancer, melanoma, head and neck carcinoma etc. Fluorescence-based sensing and imaging have tremendous applications in biomedical sciences. A fluorescent probe specific to a disease biomarker can help in the diagnosis and treatment of various diseases like cancer. Fluorescence emission in the red region of the electromagnetic spectrum provides the best optical window for sensing and imaging, as the contribution of autofluorescence decreases in this region. To distinguish the signal from the fluorophore and autofluorescence, efforts have been focused on developing red-emitting fluorophores, preferentially with a long fluorescence lifetime (significantly longer than autofluorescence). This improves signal-to-noise ratio and opens the possibility for time-gated detection. However, the commercially available red fluorophores have a very short fluorescence lifetime. The groups of currently developed triangulenium fluorophores like Azadioxatriangulenium (ADOTA), which emits in the orange/red region with a long fluorescence lifetime and high quantum yield presents great opportunities for sensing and imaging applications. The goal of this study was to characterize the photophysical properties of Azadioxatriangulenium (ADOTA) fluorophore and explore its properties for biomedical sensing and imaging. A sensor for the enzyme hyaluronidase was developed by using ADOTA fluorophore. This sensor was developed by heavy labeling of hyaluronic acid with ADOTA fluorophore. Results from these studies show the applications of ADOTA in fluorescence-based sensing, as a contrast agent in fluorescence-lifetime imaging microscopy (FLIM), and its application in time-gated detection for background-free cellular imaging.Item Sensitization to Cocaine: Behavioral and Genetic Characterization(1998-04-01) Odom, Linda Ann; Michael Forster; Glenn Dillon; Harbans LalOdom, Linda Ann, Sensitization to Cocaine: Behavioral and Genetic Characterization. Doctor of Philosophy (Pharmacology). April 1998, 141 pages, 2 tables, 23 figures, 89 references. Conditioned associations between environmental context and cocaine effects may play a significant role in acquisition and maintenance of cocaine dependence. Conditioning may also contribute significantly to cocaine sensitization, a leftward shift in the cocaine dose-response curve that is attributable to cocaine pre-exposure. Both studies examined the sensitization of cocaine’s behavioral effects after one or four prior exposures to cocaine in two distinct environments, allowing evaluation of the acquisition and magnitude of sensitization to cocaine and the contribution of conditioning to sensitization. An extinction component was added to the second study to allow determination of persistence of context-dependent sensitization in C57BL/6 and DBA/2 mice. The purpose of the first study was to fully characterize the quantity and quality of the sensitized behavioral response to cocaine in Swiss Webster mice and to determine parameters for sensitization in the second study. Results of this study indicated that pairing cocaine to the testing environment resulted in a leftward shift of the dose-response curves for both horizontal and stereotypy measures and a concurrent decrease in maximal effect of cocaine on horizontal distance and an increase in maximal effect of cocaine on horizontal distance and an increase in maximal effect of cocaine on stereotypy. The multivariate behavior profile indicated that the sensitized response to cocaine was best observed in response to 1 to 5 mg/kg cocaine, and that the conditioned response elicited by saline following cocaine pre-exposure closely resembled the 10 mg/kg acute cocaine response. The overall purpose of the second study was to determine if genetic differences in various aspects of such conditioned associations could contribute to individual differences in cocaine dependence. It was determined that, although DBA/2 mice had a faster rate of acquisition of context-dependent sensitization to cocaine than C57/BL6 mice, the multivariate behavior profile of the conditioned response of C57BL/6 mice resembled the behavior observed with a higher dose of acute cocaine and had greater magnitude and greater persistence than that of DBA/2 mice, which may explain in part the susceptibility of the C57BL/6 mice to cocaine dependence.Item Sexually Dimorphic Anxiety-Like Interoceptive Discriminative Stimuli(1997-12-01) Jung, Marianna E.; Walls, Cleatus; Downey, H. Fred; Forster, MichaelJung, Marianna E., Sexually Dimorphic Anxiety-Like Interoceptive Discriminative Stimuli. Doctor of Philosophy (Biomedical Sciences), December 1997, 150 pp, introduction, 2 chapters, discussion, bibliography, 109 titles. This study compared gender differences in the anxiogenic stimuli induced by either a GABA-A antagonist, pentylenetetrazol (PTZ) or by a 5-HT1b/2 agonist, m-chlorophenylpiperazine (m-CPP) before and during ethanol withdrawal (EW). Rats were trained to discriminate either PTZ (16mg/kg, IP) or m-CPP (1.2 mg/kg, IP) from saline in a two lever choice task for food reward. Male and female rats were gonadectomized or sham-operated, and ovariectomized (OVX) female rats were tested during replacement treatment with 17β estradiol (2.5 mg, 21 day release, sc). The dose-response for the discrimination of the interoceptive stimulus (IDS) produced by PTZ (0-16 mg/kg) or m-CPP (0 to 1.2 mg/kg) was measured under all hormonal conditions. For m-CPP trained rats, latency to first lever-press response was also tested. Results: sham and estradiol-replaced female rats had higher ED50s for discrimination of the PTZ or m-CPP IDS than intact males or OVX rats. There is a dose-related impairment of operant responding after mCPP injection. Sham and estradiol replaced OVX rats showed an increased delay to the initiation of response after m-CPP injection as compared to sham or castrated male rats or OVX rats that showed no effect at the doses tested. Rats then received a chronic ethanol diet (6.5%) for 10 days. At twelve hours of ethanol withdrawl, they were tested for lever selection after saline injection. Fewer sham female and estradiol-replaced female rats responded on the drug lever during acute EW as compared to sham male, castrated or OVX rats. In general, the anxiogenic drug lever selection of OVX rats resembled that of male rats but was restored toward that of sham female rats by estradiol replacement. Castration did not alter the response of male rats to either PTZ or mCPP. Serum β –estradiol concentrations were determined by radioimmunoassay for sham, OVX, and estradiol-replaced female rats. The concentration was significantly higher in hormone-replaced female rats than in OVX. The estradiol concentration in sham female rats showed a cyclic pattern over 4 consecutive days, but this pattern did not correlate with any difference in IDS. Blood ethanol concentration (BEC) was determined using head space gas chromatography. BEC was higher in intact female rats than in intact male rats after ethanol injection (2 g/kg, ip), but did not differ during EW. Conclusions: females produce less anxiogenic IDS in response to either GABA inhibition or 5-HT1b/2 activation, but are more impaired by m-CPP in their ability to initiate operant responses than male rats. In addition, fewer intact females developed a spontaneous IDS during EW than males which is not the result of lower BEC. Estrogen appears to play a trophic role in altering responsiveness to anxiogenic stimuli.