Nicotine is a major psychoactive ingredient in tobacco that is thought to promote smoking behavior via nicotinic acetylcholine receptors (nAChRs) in the brain. Given reports that people smoke to relieve anxiety and that anxiety precipitates relapse, the overarching goal of this dissertation research is to assess beta 2 subunit containing nAChR (beta2*nAChR) contributions to anxiety-like behavior. Nicotine’s activity at beta2*nAChRs is concentration-dependent, with high concentrations facilitating activation followed by rapid desensitization and low concentrations preferentially desensitizing beta2*nAChRs; hence, activation or inhibition of beta2*nAChRs may support smoking behavior. Rodent studies reveal that nicotine affects anxiety-like behavior dose-dependently: low doses promote anxiolysis- and high doses support anxiogenic-like behavior. These pharmacological and genetic studies in mice test the hypothesis that nicotine administration promotes anxiolysis via inactivation of beta2*nAChRs and begin to identify which subunits, namely alpha 4 and alpha 6, work in concert with beta 2 to affect anxiety-like behavior. Low dose nicotine and inhibition of beta2*nAChRs supported anxiolysis-like behavior in a number of tasks with predictive validity for anxiolysis efficacy. These studies further suggest that activation of alpha6beta2*nAChRs is sufficient to produce anxiogenic-like behavior and that inhibition of alpha4beta2*nAChRs supports anxiolysis-like behavior. A secondary goal of these studies is to assess if beta2*nAChRs affect anxiety-like behavior during aging. Dysregulation of cholinergic tone can increase anxiety in the elderly, but little is known regarding beta2*nAChR contributions to anxiety in this population or where in the brain this may take place. These studies show that alpha4beta2*nAChR expression differentially affects anxiety-like behavior in adult and aged mice. With a focus on the lateral septum, a GABA-ergic limbic nucleus thought to regulate anxiety-like responses to external stimuli, a third goal of these studies is to elucidate the neuroanatomical and intracellular underpinnings of anxiety-like behavior that are affected by beta2*nAChR inhibition and expression. Previous studies demonstrate that exposure to stressors reduces phosphorylation of extracellular regulated kinase (ERK) in the lateral septum. In these studies, levels of pERK in the lateral septum were inversely associated with alpha4beta2*nAChR expression as well as anxiogenic-like behavior. In sum, these preclinical studies suggest that inhibition alpha4beta2*nAChRs may support cessation in those who smoke to relieve anxiety.

Neuronal nicotinic acetylcholine receptors modulate both cholinergic and non-cholinergic synaptic transmission. Our research concerns α6 and α4 neuronal nicotinic subunits because they often co-assemble with the β2 subunit, which has abundant expression in the CNS and previous work has demonstrated that β2* nAChRs are involved in nicotine and cocaine reward. α6β2* and α4β2* nAChRs are highly expressed in midbrain, which is known to be critical for the incentive salience associated with natural and artificial (drug) reward. Our goal was to assess the role of α6β2* and α4β2* nAChRs in nicotine and cocaine reward using an unbiased conditioned place preference (CPP) test in mice. Adult male C57BL/6J mice or male mice null for the α6 or α4 nicotinic receptor subunit were used. For CPP: On day 1, pre-conditioning scores were recorded; Days 2-4 mice underwent conditioning, where they were randomly assigned to either the black or the white compartment paired with drug, and the opposite chamber paired with saline; Day 5 was a drug-free test day where post conditioning scores were recorded. α-Conotoxin MII[H9A;L15A], a selective antagonist of α6β2* nAChRs, was given centrally either into the lateral ventricle or the nucleus accumbens on conditioning days, which tested for acquisition of CPP, or it was given only once into the lateral ventricle on test day which tested for expression of CPP. Antagonizing α6*nAChRs resulted in a significant attenuation of both nicotine and cocaine place preference. This was complemented with diminished nicotine and cocaine place preference in α6 KO mice compared to WT littermates. Studies with α4 KO mice showed significantly reduced nicotine place preference scores compared to WT littermates. In contrast, α4 KO and WT mice showed significant place preference for 20mg /kg cocaine, suggesting that the α4 subunit is not required for the reward-like effects of cocaine in our behavioral test. Our results implicate α6β2* and α4β2* nAChR involvement in nicotine and cocaine CPP, but only α6β2* nAChR involvement in cocaine CPP. Lithium conditioned place avoidance and food reward were not altered in α6 KO mice or by α-Conotoxin MII[H9A;L15A], thereby validating the specificity of hedonics of targeting α6* nAChRs in CPP. Our studies suggest that α6β2* and α4β2*nAChR should be further characterized for future nicotine cessation therapies, and α6β2* could provide a new target for treating cocaine addiction.

Excitotoxic calcium influx and activation of calcium-regulated systems is a common event in several types of neuronal injury. This mechanism has been the focus of intense research, with the hope that a more complete understanding of how neuronal injury affects calcium-regulated systems will provide effective treatment options. This study examines one such calcium-stimulated enzyme, calcineurin, in the context of two common neurological pathologies, status epilepticus and traumatic brain injury.Status epilepticus was induced by pilocarpine injection. NMDA-dependent increases in calcineurin activity were observed in cortical and hippocampal homogenates. Upon closer examination, the most profound increases in activity were found to be present in crude synaptoplasmic membrane fractions isolated from cortex and hippocampus. A concurrent status epilepticus-induced increase in calcineurin concentration was observed in membrane fractions from cortex and hippocampus. Immunohistochemical analysis revealed an increase in calcineurin immunoreactivity in apical dendrites of hippocampal pyramidal neurons. We examined a cellular effect of increased dendritic calcineurin activity by characterizing a calcineurin-dependent loss of dendritic spines. Increased dendritic calcineurin led to increased dephosphorylation and activation of cofilin, an actin-depolymerizing factor. Calcineurin-activated cofilin induced an increase in actin depolymerization, a mechanism shown to cause spine loss in other models. Finally, via Golgi impregnation, we demonstrated that status epilepticus-induced spine loss is blocked by calcineurin inhibitors.To demonstrate that the increase in dendritic calcineurin activity was not model-specific, we examined a moderate fluid-percussion model of brain injury. Calcineurin activity was significantly increased in hippocampal and cortical homogenates. This increased activity persisted for several weeks post-injury, and may be involved in injury-induced neuronal pathologies. Also similar to the SE model, calcineurin immunoreactivity was dramatically increased in synaptoplasmic membrane fractions from cortex and hippocampus, and immunohistochemistry revealed increased calcineurin content in dendrites of hippocampal CA1-3 pyramidal neurons. These changes in calcineurin distribution also persisted for several weeks post-injury.These studies demonstrate a novel, cellular mechanism of calcium-mediated pathology in two models of neuronal injury. Elucidation of cellular events involved in the acute and chronic effects of brain trauma is essential for the development of more effective treatment options.

Foram estimados custos diretos de diferentes opções de tratamento para artrite reumatóide com base em dados de eficácia obtidos por revisão de literatura. O modelo analítico de tomada de decisão para o tratamento e desfechos durante um período de 48 meses baseado no modelo de Markov foi fundamentado em protocolos clínicos recomendados pela Sociedade Brasileira de Reumatologia, com esquemas alternativos para cinco ciclos de tratamento durante o período de quatro anos. O paciente pode permanecer em algumas das etapas ou migrar entre elas, de acordo com a resposta à terapia. Foram analisados custos diretos (medicamentos, materiais médico-hospitalares e exames laboratoriais). As doses dos medicamentos e o monitoramento foram baseados no Consenso Brasileiro para o Diagnóstico e Tratamento da Artrite Reumatóide, considerando-se o peso médio do paciente de 70 kg. Na comparação entre o custo das cinco etapas de tratamento os resultados mostram que a etapa que usa o medicamento infliximabe tem um custo superior às outras, este causado pelo preço de aquisição do medicamento. O custo do monitoramento tem impacto nas etapas que utilizam os medicamentos com preço de aquisição menor. O ciclo com a menor razão custo/efetividade foi o 1 (respondedor ao MTX). Aquele que usa o biológico desde o início do tratamento é responsável pela maior razão de custo/efetividade. A variação do custo entre os outros ciclos não foi muito grande, mas todas devem ser consideradas quando da escolha do prescritor. Estes resultados se mostraram robustos com a análise de sensibilidade. / The costs of the different rheumatoid arthritis therapy options were estimated and they were compared by cos/efficacy reason, through the development of an analytical model for a 48-month period. For the calculation of these costs, it was developed an analytical decision model based on the Markov Analysis, where five different therapy stages were elaborated based on clinical protocols recommended by the Brazilian Society of Rheumatology, and then five therapy cycles, where patients may continue in some of those stages or shift between them according to the therapy response, for a time horizon of 4 years. Only direct costs with drugs, medical-hospital materials for drug administration and laboratory examinations required for the patient monitoring because of the use of some drugs, were comprised in the analyzed data. The doses of drugs and the monitoring were based on the Brazilian Consensus for the Diagnosis and Treatment of Rheumatoid Arthritis, considering the average patient weight of 70 kg. When comparing the cost of the five treatment stages, the results show that the stage in which the infleximabe drug is used has a cost higher than that of the other stages and that the impact is caused by the drug acquisition price. The monitoring cost impacts the stages that employ drugs with smaller acquisition price. The cost-effectiveness cycle was the number 1 (good response with MTX). When we use de biologic in the beginning, it is responsible for the higher cost-effectiveness reason. The cost variation between cycles 2, 3 and 4 was smaller, but they have to be considered for the decision maker. The sensitivity analysis confirm this results.

Stroke is cerebrovascular injury that has been reported to be the third leading cause of death and the first leading cause of disability in the world (W. H.O. 2007). Currently, there is only one FDA approved treatment for stroke which is recombinant tissue plasminogen activator. This treatment has a narrow therapeutic window of three hours after ischemic stroke and can adversely cause the production of oxygen free radicals and intracranial hemorrhage. These limitations result in only 2-3% of all stroke victims as being candidates for this therapy as many patients do not arrive at the hospital in time to receive treatment, are not properly diagnosed, or do not know that they have had a stroke within this three hour time period. The purpose of these experiments was to elucidate alternative targets of stroke for the benefit of developing new treatments that stimulate neuroprotective and anti-inflammatory effects at the site of injury. It has been shown that transfusion of human umbilical cord blood cells up to 48 hours after stroke significantly reduces infarction and we have examined other targets that mimic these effects. We have shown that sigma receptor activation by DTG, a high affinity universal sigma agonist, reduces infarct volume when administered 24 hours after stroke. This suggests that modulation of neurodegenerative and inflammatory responses can extend the therapeutic window of treatment. For the first time, evidence is provided that shows that the spleen enhances the neurodegeneration caused by stroke as splenectomy prior to stroke profoundly decreased infarction volume. Finally, we studied signaling mechanisms of the splenic reaction to stroke and determined that this response is not directly dependent on neurotransmission via the splenic nerve. Denervation of the spleen prior to stroke showed no changes in neurodegenerative load at the site of injury in rat brains when compared to those subjected to stroke only. Overall, these experiments provide evidence showing that targets mediating neuroprotective and anti-inflammatory effects can lead to novel therapeutic interventions of stroke.

Pharmacogenetics is the study of genomic-guided individualized drug prescription that plays an important role in preventing the severe adverse effects of drugs, decreasing the time and cost of therapeutic choices, and directing healthcare professionals to choose medications that are effective and safe. It is noteworthy that this approach becomes highly beneficial in patients suffering from chronic diseases or disorders, since these conditions may require multiple and long term pharmacological therapies, as in children with autism spectrum disorder (ASD). However, public acceptance is a major challenge when implementation of pharmacogenetics merges into clinical practice. The purpose of this study is a) to investigate, among small cohort group of children with ASD, several genetic variants of enzymes that influence the metabolism of commonly prescribed drugs to treat ASD and b) to inspect the knowledge of, attitude towards and future expectations with regards to pharmacogenetics among parents of children with ASD. A group of 15 school-aged participants with ASD were recruited for the study. Approximately 5 ml of venous blood was drawn for each participant to analyze the genotype of enzymes implicated in drug metabolism via pharmacogenetics testing. Thereafter, the parents of these children attended a training session to help them gain a better understanding of the pharmacogenetics results depicted in the drug panel results. A pre-training and post-training survey was conducted to assess the knowledge of, attitude towards and future expectations of pharmacogenetics among the children’s parents.

Abdominal aortic aneurysms (AAAs), a permanent dilation in the abdominal region of the aorta, is a highly sexually dimorphic disease. AAAs prevalence is ranging from 4-10 fold higher in males than females. Defining the mechanistic basis for reduced (in females) or increased (in males) AAA formation and progression may uncover potential therapeutic targets. The majority of studies examining sexual dimorphism focus on the role of sex hormones. However, genes residing on sex chromosomes, in addition to sex hormones, may contribute to sexual dimorphism of AAAs. For example, the X chromosome contains about 5% of the whole genome, but the role of sex chromosomes genes to sexual dimorphism of cardiovascular diseases such as AAAs is largely unknown. The purpose of this study was to determine the role of sex chromosomes as mediators of sex differences for angiotensin II (AngII)-induced AAAs in hypercholesterolemic mice. We used the four core genotype murine model, which enables the creation of phenotypically normal male and female mice with an XX versus XY sex chromosome complement, to test the hypothesis that an XY sex chromosome complement promotes AngII-induced AAAs. Transgenic male mice expressing the Sry gene on an autosome, but not on the Y-chromosome, were bred to female low-density lipoprotein receptor deficient mice to create male and female mice with an XX or an XY sex chromosome complement. In females, an XY sex chromosome complement doubled the incidence and markedly increased the severity of AngII-induced AAAs. To define mechanisms, we examined gene expression patterns in abdominal aortas and demonstrated elevated expression of inflammatory genes that were linked to increased MMP activity and oxidative stress in aortas from XY females. Moreover, administration of testosterone to XY females, to mimic males, resulted in a striking level of aneurysm rupture. In males, transcriptional profiling of abdominal aortas revealed 450 genes that were influenced by sex chromosomes. Infusion of AngII to XY males resulted in diffuse pathology along the length of the aorta, while XX males developed focal AAAs, with pathology reduced by orchiectomy in both genotypes. Thoracic aortas of XY males exhibited adventitial thickening which was not exist in thoracic aortas from XX males. Following a prolonged period (3 months) of AngII infusions XY males had AAAs with expanded aortic walls, while XX males had thin walled dilated AAAs. In summary, our findings demonstrate a remarkable effect of sex chromosome complement to regulate aortic vasculature and disease development. Aside from demonstrating mechanisms of sexual dimorphism of aortic diseases, these findings indicate that chronic sex hormone therapy in the aging and transgender population may have cardiovascular ramifications. Moreover, identification of targets influenced by sex chromosomes and/or sex hormones in a manner that predicts disease development may identify sex-specific approaches to cardiovascular therapy.

Inflammation or nerve injury sensitizes several populations of nociceptive neurons in the dorsal horn of the spinal cord, including those that express the neuropeptide Y (NPY) Y1 receptor (Y1R). Our overall hypothesis is that after tissue or nerve injury, these Y1R-expressing neurons enter a state of latent sensitization (LS) that contributes to vulnerability to the development of chronic pain; furthermore, LS is under the tonic inhibitory control of endogenous Y1R signaling. First, we evaluated the intracellular signaling pathways that become activated in Y1R-expressing neurons and participate in LS. To do this, we established behavioral models of inflammatory or neuropathic pain, allowed pain hypersensitivity to resolve, and then during this period of pain remission we administered the Y1R receptor antagonist, BIBO3304, by intrathecal injection. As observed previously with mu-opioid receptor antagonists/inverse agonists, we found that BIBO3304 reinstated pain hypersensitivity via an N-methyl-D-aspartate receptor (NMDAR)- and adenylyl cyclase type 1 (AC1)-dependent mechanism. Our subsequent behavioral pharmacological experiments then established two signaling pathways downstream of AC1 that maintain LS. The first pathway involves protein kinase A (PKA) and transient receptor potential cation channel A1 (TRPA1) and channel V1 (TRPV1). The second pathway involves exchange proteins activated by cAMP (Epac 1 and Epac 2). We next found that nerve injury decreases the co-expression of Y1R with markers of excitatory interneurons, suggesting that Y1R-expressing neurons acquire a pain-enhancing phenotype after peripheral nerve injury. In a separate set of experiments that utilized Y1R-receptor internalization as an index of NPY release, we found that nerve injury increased stimulus-evoked NPY release. We conclude that injury induces pain-facilitatory mechanisms of LS in the dorsal horn involving PKA→TRPA1 and PKA→TRPV1 at the central terminals of primary afferent neurons. Whether Epac mechanisms are located on these same presynaptic terminals and/or at Y1R-expressing excitatory interneurons remain to be determined. We also conclude that injury-induced LS is masked by a compensatory up-regulation of spinal NPY release that tonically inhibits pain. These results present a novel mechanism of injury-induced LS and endogenous control of the transition from acute to chronic pain by the NPY-Y1R system. Our work sheds light on novel targets for the treatment of chronic pain.

While cancer chemotherapy continues to significantly contribute to the number of cancer survivors, exposure to these drugs can often result in chemotherapy-induced peripheral neuropathy (CIPN), a consequence of peripheral nerve fiber dysfunction or degeneration. CIPN is characterized by sensory symptoms in the hands and feet, such as numbness, burning, and allodynia, resulting in an overall decrease in quality of life. Paclitaxel (Taxol), a microtubule poison that is commonly used to treat breast, lung, and ovarian cancers, has been found to cause CIPN in 59-78% of cancer patients. There is currently no effective preventative or therapeutic treatment for this side effect, which can be a dose-limiting factor for chemotherapy or delay treatment. Our collaborators in the laboratory of Dr. M. Imad Damaj have shown that nicotine, a nicotinic acetylcholine receptor (nAChR) agonist, and R-47, an α7 nAChR silent agonist, can prevent and reverse paclitaxel-induced peripheral neuropathy in mice. With regard to cancer, this work demonstrates that nicotine and R-47 do not enhance A549 and H460 human non-small cell lung cancer cell viability, colony formation, or proliferation alone, and they do not attenuate paclitaxel-induced growth arrest, apoptosis, or DNA fragmentation. Most importantly, nicotine and R-47 do not increase the growth of A549 tumors or interfere with the antitumor activity of paclitaxel in tumor-bearing mice. These data suggest that targeting nAChRs may be a safe and efficacious approach for the prevention and treatment of CIPN in cancer patients.

Elevation of blood pressure leads to structural and functional alterations in vasculature, resulting in increased arterial stiffness, which in turn is a predictor of future hypertension and cardiovascular risks. Angiotensin II (Ang II) plays a crucial role in blood pressure regulation. In addition to its hemodynamic effects, Ang II activates both innate and adaptive immunity. The objective of this study is to define the roles of CD4⁺ T lymphocyte subsets in the progression of vascular remodeling and stiffening induced by Ang II. A mouse model of Ang II infusion was used to induce hypertension and vascular diseases. In the WT mice, Ang II infusion led to an increased aortic stiffness within 7 days of the treatment as well as an increase in aortic remodeling within 14 days of the treatment. Interestingly, RAG1(-/-) mice, lacking functional T and B lymphocytes were prevented from the vascular stiffening and remodeling caused by Ang II. Characterization of T cell subsets in the perivascular aortic infiltrates showed that there was a sequential activation of peri-arotic Th1 and Th17 during the time course of Ang II treatment, which was associated with the initial increased aortic stiffness and the subsequent remodeling, respectively. To extend the concept, roles of suppressive regulatory T cells (Tregs) were further examined. Proliferation of Tregs was successfully induced in vivo using a cytokine complex of IL-2 and anti-IL-2 mAb clone JES6-1. Ang II-infused mice that received the IL-2/anti-IL-2 complex exhibited a reduced vascular remodeling and stiffening caused by Ang II. Stimulation of Tregs with the IL-2/anti-IL-2 complex also suppressed the Th1 and Th17 responses and reduced immune cells infiltrates in the aortas. Since hypertension is closely related to the kidney and renal homeostasis is also tightly regulated by Ang II, the kidney function was determined in this Ang II-hypertensive model. In the wild type mice, two weeks infusion of Ang II resulted in an increased glomerular filtration rate (GFR) whereas immunodeficient RAG1(-/-) mice exhibited a marked decrease in GFR. Subsequent experiments showed that Th17 was crucial in renal hemodynamic response to Ang II, partly by regulating secretion of vasodilatory prostaglandin E₂.