Estudos anteriores em nosso laboratório demonstraram que pacientes portadores de DAC apresentam diferenças no metabolismo do CL e CE de uma nanoemulsão artificial rica em colesterol (LDE), nos quais o CL apresentou maior remoção plasmática e depósito arterial. Dando continuidade a esta linha de pesquisa, neste trabalho foram avaliadas a cinética plasmática, representada pela taxa fracional de remoção (TFR), e a captação do 3H-colesterol livre (3H - CL) e 14C - colesterol esterificado (14C - CE) da LDE por segmentos arteriais e por órgãos de coelhos normais (n=17) e coelhos submetidos à indução de aterosclerose por dieta rica em colesterol (1%) (n=13). Além disso, avaliou-se a captação in vitro do 3H CL e do 14C CE da LDE por células endoteliais aórticas de coelhos. Por último, foi avaliada a influência da inibição da enzima lecitina-colesterol aciltransferase (LCAT), e indiretamente, a esterificação do CL em ratos normais (n=9) e tratados com diazepam (n=9). Em coelhos que receberam dieta normal, não houve diferença entre a remoção plasmática do 3H - CL e do 14C - CE. Em coelhos que desenvolveram hiperlipidemia e aterosclerose através de dieta rica em colesterol, o 3H - CL foi removido mais rapidamente da circulação do que o 14C - CE (p<0,05), entretanto houve maior captação de 14C - CE do que de 3H - CL no arco aórtico (p<0,05). Em ambos os grupos, os principais órgãos captadores de colesterol da LDE foram fígado, pulmão, adrenais e baço (p<0,05). Tanto a TRF quanto a captação hepática de 3H - CL e 14C - CE foram menores no grupo que recebeu a dieta rica em colesterol. Em células endoteliais aórticas de coelhos, a captação de 3H - CL foi maior que a de 14C CE independente da massa de LDE incubada (p<0,01). Em ratos, não houve diferença entre a captação das duas formas de colesterol da LDE pela aorta no grupo controle, entretanto, quando a atividade da LCAT foi diminuída pelo tratamento com diazepam, a captação arterial de 3H - CL foi maior do que a de 14C - CE (p< 0,01). A hiperlipidemia e distúrbios no processo de estabilização do colesterol, favorecem a dissociação entre o CL e o CE das lipoproteínas, e podem elevar o risco de desenvolvimento da aterosclerose, assim como agravar o processo de aterogênese. / I n previously studies, it was shown that free cholesterol (FC) and cholesterol ester (CE) of a cholesterol-rich nanoemulsion (LDE) behaves differently in patients with coronary artery disease (CAD). The FC plasma clearance and arterial deposition is greater than CE. In the present study we evaluate the plasma kinetics, estimated by the fractional clearance rate (FCR), and the tissue uptake of 3H-free cholesterol (3H FC) and of 14C cholesterol ester (14C - CE) of LDE by arterial segments and organs of rabbits with (n=13) and without atherosclerosis (n=17). Furthermore, it was evaluated the in vitro uptake of 3H FC and 14C - CE by rabbit aortic endothelial cells. Finally, it was evaluated the inhibition of the enzyme lecithin-cholesterol acyltransferase (LCAT), and indirectly, the FC esterification in rats non-treated (n=9) and treated with diazepam (n=9). In rabbits without atherosclerosis that received an standard diet there was no difference between the plasma clearance of 3H FC and 14C CE. In rabbits with hyperlipidemia and atherosclerosis induced by the cholesterol-rich diet the 3H - FC was removed faster than 14C - CE (p<0.05), however the arch aortic uptake of 14C CE was greater than of 3H - FC (0p<0.05). In both groups, liver, lungs, adrenals and spleen were the principal sites of LDE cholesterol uptake. The FCR and tissue uptake were smaller in rabbits with than those without atherosclerosis. In rabbit aortic endothelial cells the 3H - FC uptake was greater than 14C CE independently of incubated LDE mass (p<0.01). In control rats there was no difference on the arterial uptake of both cholesterol forms of LDE, but when the LCAT activity was diminished by the diazepam treatment, the arterial uptake of 3H FC were greater than 14C CE (p< 0.01). The hyperlipidemia and cholesterol stability alterations may lead to dissociation between lipoproteins FC and CE. This dissociation may increase the risk for atherosclerosis and likewise enhance the severity of atherosclerosis.

Abnormality of dopamine D2 receptor (D2R) function, often observed as D2R supersensitivity (D2RSS), is a commonality of schizophrenia and related psychiatric disorders in humans. Moreover, virtually all psychotherapeutic agents for schizophrenia target D2R in brain. Permanent D2RSS as a feature of a new animal model of schizophrenia was first reported in 1991, and then behaviorally and biochemically characterized over the next 15–20 years. In this model of schizophrenia characterized by production of D2RSS in ontogeny, there are demonstrated alterations of signaling processes, as well as functional links between the biologic template of the animal model and ability of pharmacotherapeutics to modulate or reverse biologic and behavioral modalities toward normality. Another such animal model, featuring knockout of trace amine-associated receptor 1 (TAAR1), demonstrates D2RSS with an increase in the proportion of D2R in the high-affinity state. Currently, TAAR1 agonists are being explored as a therapeutic option for schizophrenia. There is likewise an overlay of D2RSS with substance use disorder. The aspect of adenosine A2A-D2heteroreceptor complexes in substance use disorder is highlighted, and the association of adenosine A2Areceptor antagonists in discriminative and rewarding effects of psychostimulants is outlined. In summary, these new animal models of schizophrenia have face, construct, and predictive validity, and distinct advantages over earlier models. While the review summarizes elements of D2RSS in schizophrenia per se, and its interplay with substance use disorder, a major focus is on presumed new molecular targets attending D2RSS in schizophrenia and related clinical entities.

Foi investigado o curso temporal da orientação da atenção numa tarefa de orientação encoberta adaptada para ratos. A tarefa envolveu a avaliação do efeito de pistas auditivas centrais e periféricas, preditivas ou não preditivas, sob o tempo de reação a alvos visuais apresentados à direita ou à esquerda do campo visual. Foram utilizados tempos entre pista e alvo (SOAs) entre 50 a 1200 ms. Pistas centrais consistiram em bipes de 5 ou 8 kHz reproduzidos em ambos ouvidos concomitantemente. Neste caso a frequência do bipe indicava o lado para o qual a atenção deveria ser orientada. Pistas periféricas constituíram-se de bipes de 5 ou 8 kHz, apresentados individualmente no ouvido esquerdo ou direito. Neste caso o lado da apresentação do estímulo indicava a direção para a qual o animal deveria orientar a atenção. Esses estímulos foram apresentados de forma preditiva - em 80% das tentativas as pistas indicavam corretamente a localização do alvo (tentativas válidas) e em 20% o indicavam incorretamente (tentativas inválidas) - ou não preditiva - 50% de tentativas válidas e 20% de tentativas inválidas - a depender do grupo. Vinte e quatro ratos Wistar machos, com 3 meses no início dos experimentos, divididos em quatro grupos experimentais independentes - Central Preditivo (CP), Central Não Preditivo (CNP), Periférico Preditivo (PP) e Periférico Não Preditivo (PNP) - foram empregados. Os resultados mostram que animais dos grupos preditivos (CP e PP) respondem mais rapidamente e de forma mais precisa em tentativas válidas do que em tentativas inválidas, ao passo que animais em grupos não preditivos (CNP e PNP) respondem da mesma maneira em tentativas válidas ou inválidas. Esses resultados indicam de estes animais foram capazes de orientar a atenção de forma endógena, de forma análoga a seres humanos, sugerindo que ratos podem ser empregados amplamente como modelo animal na avaliação das orientações endógena e exógena da atenção / Orienting of visual attention was investigated in rats using a 3-hole nose-poke task analogous to the covert attention task for humans. The task involved evaluation of the effects of either central or peripheral auditory cues, presented either predictively or non-predictively on reaction times to a visual target presented either to the left or to the right sides of the visual field; stimuli-onset asynchronies (SOAs) varied from 50 to 1200 ms. Central cues were either 5 or 8 KHz auditory stimuli, released on both ears concomitantly. In this case, the frequency indicated the side to which attention should be oriented to. Peripheral cues were 5 and 8 KHz auditory stimuli presented individually either to the left or to the right ears. In this case, the sound source indicated to which side attention should be oriented to. These stimuli were presented either predictively - 80% of cues predicted the target\'s location correctly (valid trials) and 20% of cues predicted it incorrectly (invalid trials) - or non-predictively - 50% of valid trials and 50% invalid trials - depending on the group. Twenty-four male Wistar rats, 3 month-old at the beginning of the experiment, divided in four independent groups - Central Predictive (CP), Central Non-Predictive (CNP), Peripheral Predictive (PP) and Peripheral Non-Predictive (PNP) - were employed. Animals on predictive groups (CP and PP) showed faster and more accurate responses on valid trials than on invalid trials, while non-predictive groups (CNP and PNP) didn\'t show any differences on reaction times and accuracy in valid as compared to invalid trials. These results indicate that rats do orient attention endogenously, analogously to humans. This suggests that these animals may be employed as animal model to the study of endogenous and exogenous orienting of attention

A problematic connection has been reported between those who use nicotine related products alone or in combination with ADHD medications, like methylphenidate (MPH), in late childhood or early adolescence and the increased likelihood of later marijuana abuse in adulthood. Pre-clinical studies have found that the use of nicotine during the early adolescence period produces enduring changes to the endocannabinoid system in the brain. Since CB agonists, like marijuana, exert their effect through the eCB system, it is possible that early nicotine use may alter the rewarding nature of CB agonists in adulthood. In addition, MPH has also been shown to increase nicotine self-administration and abuse related behaviors of nicotine in rats. Thus, the current study consisted of two experiments looking at the effects of early nicotine and methylphenidate exposure on adult CB-agonist place conditioning in rats. In the first experiment, rats were pre-exposed to either saline or nicotine (0.16, 0.32, or 0.64 mg/kg) from PD 31 to PD 40. On PD 60, rats began a 13-day biased CPP procedure with the CB agonist, CP 55,940 (10, 20 or 30 μg/kg), or vehicle. No significant group differences were found, suggesting that early nicotine exposure does not influence the rewarding nature of CB agonists. Additional individual subgroup comparisons were conducted to determine if any subgroups significantly differed from 0 or no mean change in preference from preconditioning to testing. These analyses revealed that rats pre-exposed to the moderate (0.32 mg/kg) dose of nicotine showed a significant aversion to the high (30 μg/kg) dose of CP 55,940, suggesting that early nicotine exposure may reduce the rewarding nature of CB agonists in adulthood. In the second experiment, rats were pre-exposed to either saline or MPH (0.5, 2, 0r 5 mg/kg) from PD 21 to PD 30. Similar to the first experiment, rats began a 13-day biased CPP procedure on PD 60 with CP 55,940 (10, 20 or 30 μg/kg) or vehicle. Rats conditioned with the moderate (20 μg/kg) dose of CP 55,940 showed a significant preference for the CB agonist as compared to rats conditioned with the high (30 μg/kg) dose of CP 55,940. CP 55,940 exposed rats did not significantly differ from control rats. There was no significant effect of MPH or a MPH x CP 55,940 interaction, suggesting that early MPH exposure does not alter the rewarding nature of CB agonists in adulthood. Together these findings suggest that early nicotine, but not MPH, exposure may influence the rewarding nature of CB agonists in adulthood, suggesting an additional risk factor of early nicotine use. However, future studies should evaluate the effects of persistent nicotine and MPH exposure starting in early adolescence or childhood through adulthood to determine whether the effects of nicotine and MPH are altered if use is continued into adulthood.

Chronic musculoskeletal pain is a leading cause of disability worldwide yet the mechanisms of chronification and neural responses to effective treatment remain elusive. Non-invasive imaging techniques are useful for investigating brain alterations associated with health and disease. Thus the overall goal of this dissertation was to investigate the white (WM) and grey matter (GM) structural differences in patients with musculoskeletal pain before and after psychotherapeutic intervention: cognitive behavioral therapy (CBT). To aid in the interpretation of clinical findings, we used a novel porcine model of low back pain-like pathophysiology and developed a post-mortem, in situ, neuroimaging approach to facilitate translational investigation. The first objective of this dissertation (Chapter 2) was to identify structural brain alterations in chronic pain patients compared to healthy controls. To achieve this, we examined GM volume and diffusivity as well as WM metrics of complexity, density, and connectivity. Consistent with the literature, we observed robust differences in GM volume across a number of brain regions in chronic pain patients, however, findings of increased GM volume in several regions are in contrast to previous reports. We also identified WM changes, with pain patients exhibiting reduced WM density in tracts that project to descending pain modulatory regions as well as increased connectivity to default mode network structures, and bidirectional alterations in complexity. These findings may reflect network level dysfunction in patients with chronic pain. The second aim (Chapter 3) was to investigate reversibility or neuroplasticity of structural alterations in the chronic pain brain following CBT compared to an active control group. Longitudinal evaluation was carried out at baseline, following 11-week intervention, and a four-month follow-up. Similarly, we conducted structural brain assessments including GM morphometry and WM complexity and connectivity. We did not observe GM volumetric or WM connectivity changes, but we did discover differences in WM complexity after therapy and at follow-up visits. To facilitate mechanistic investigation of pain related brain changes, we used a novel porcine model of low back pain-like pathophysiology (Chapter 6). This model replicates hallmarks of chronic pain, such as soft tissue injury and movement alteration. We also developed a novel protocol to perform translational post-mortem, in situ, neuroimaging in our porcine model to reproduce WM and GM findings observed in humans, followed by a unique perfusion and immersion fixation protocol to enable histological assessment (Chapter 4). In conclusion, our clinical data suggest robust structural brain alterations in patients with chronic pain as compared to healthy individuals and in response to therapeutic intervention. However, the mechanism of these brain changes remains unknown. Therefore, we propose to use a porcine model of musculoskeletal pain with a novel neuroimaging protocol to promote mechanistic investigation and expand our interpretation of clinical findings.

Mice with human immune systems, generated by transplanting human CD34+ cells into immunodeficient mice, are essential tools for studying phenomena unique to the human immune system or poorly reproduced in existing mouse models. Human immune tolerance induction, function and autoimmunity have been poorly modeled in conventional murine models, which often have poor predictive value for preclinical development. Models that allow the study of human immune cells with the reproducibility and flexibility of small animal models are required. In our lab, humanized mouse models have been used to study preclinical protocols for human xenotolerance induction and to better understand the immunological underpinnings of human autoimmunity. These are each areas of critical unmet medical need. Xenotolerance-inducing protocols may be necessary to allow long-term survival of a transplanted pig organ in a human patient, and, with more than 113,000 Americans currently waiting for a life-saving organ, the need to expand the pool available for transplantation is urgent. Additionally, clinical options for patients with autoimmune diseases are limited. Currently, most patients with autoimmunity are only diagnosed after significant immune damage of target organs. Predicting who will develop autoimmunity – and who will not – before damage occurs would be very useful but is currently very difficult. Small animal models that can better help us understand how human autoimmunity develops could help us develop protocols for early detection and even prevention. We have developed a personalized immune model to study the development of an individual patient’s immune system in a transplanted mice to better understand immune abnormalities that underlie autoimmunity. We have used existing humanized mouse models to answer important questions related to human xenotolerance induction and autoimmunity, but in the studies described here we have worked to extend our capacity to use these models to study human T cell development and peripheral function. We would like to be able to study both the initial selection of T cell receptors (TCRs) in the thymus based on their ability to recognize antigen in the context of presenting MHC without reacting unduly to self-antigen, as well as in the periphery, where T cells interact with peripheral antigen-presenting cells (APCs) to maintain homeostasis and respond to antigen. First, we have incorporated TCR transgenesis into our humanized mouse models to allow greater precision in studying thymic selection in our humanized mice. Developing a system for this would allow us to study in greater detail mechanisms of human xenotolerance induction, including confirming that a swine thymus can support positive selection of T cells with human-restricted TCRs to allow a future xenotransplantation patient to maintain immune competence, while also robustly tolerizing human T cells expressing pig-reactive TCRs. We will also expand this system to study the thymic selection of human T cells with autoreactive TCRs to better understand mechanisms of central tolerance and understand how they fail in autoimmunity. Finally, while processes of thymic selection are critical for human T cell development and function, peripheral interactions also have a large impact on human T cell function and homeostasis and may contribute to the development of autoimmunity. For these interactions to occur appropriately requires robust engraftment and reconstitution of APCs, especially of myeloid and B cell lineages, in transplanted immunodeficient mice. APC reconstitution tends to be suboptimal in humanized mice and is even more so in mice transplanted with patient-derived CD34+ cells. Better characterization of human APC populations and their progenitors could allow us to develop approaches to improve long-term human APC reconstitution in patient-derived humanized mice, allowing us to more fully model patient peripheral T cell function.

The nasal route has primarily been used to deliver drugs for the treatment of local diseases such as nasal infections, nasal congestion and allergies. The nasal route can also be used as a non-invasive alternative route to deliver drugs systemically when a rapid onset of action and/or avoidance of hepatic metabolism are desired. Moreover, there is a growing interest in the use of this route for direct transport of drugs from the nose to the brain. Most of the drugs that have been studied for nasal delivery are either small molecules which are lipophilic enough to passively diffuse through the nasal epithelia or macromolecules where bioavailabilities less than 1% are clinically effective and acceptable. This study focused on identifying carrier proteins or transporters in the nasal mucosa that could improve the absorption of specific drug substrates across the nasal respiratory and olfactory epithelia. The presence of drug transporters in the nasal mucosa of humans and commonly used animal models were investigated. DNA microarray results for nasal samples from humans and two commonly used models, mice and rats, were obtained from GenBank and were analyzed in collaboration with the University of Iowa Center for Bioinformatics and Computational Biology. While cow tissues are frequently used in in-vitro nasal permeability analyses, there is limited information available in GenBank for this species. Both DNA microarray analysis and RT-PCR were performed on bovine nasal explants to determine transporter expression. Good agreement between the microarray and RT-PCR results was observed. While human and three animal species commonly used as models in nasal drug delivery research (mouse, rat, and cow) show similar patterns of expression for several transporters, interspecies differences in the level of expression were observed. Therefore, the expression level of transporters remains a factor to consider when translating results obtained using animal models to humans. The nucleoside transporter family was selected for further evaluation of the potential to improve the nasal absorption of substrates. Nucleoside transporters are integral proteins responsible for mediating and facilitating the flux of nucleosides across cellular membranes; they are also known to be responsible for the uptake of nucleoside analog drugs such as anti-cancer and anti-viral agents. RT-PCR and Western blotting were used to verify the presence of two transporter subtypes, ENT1 and CNT3, in the bovine nasal respiratory and olfactory mucosa. The expression level of both transporters in the respiratory mucosa was comparable to that in the olfactory mucosa. Using immunohistochemistry, ENT1 and CNT3 were found to be localized primarily at the apical surface of the nasal epithelial cells. This indicates that the nasal epithelium likely absorbs exogenous nucleosides for intracellular uses such as nucleic acid synthesis and regulating other cellular activities. The contribution of the nucleoside transporters to the permeation of a nucleoside analogue drug, alovudine, across the nasal epithelia was also studied. The transport of alovudine showed a non-linear increase with increasing donor concentration over the range of 50 to 3000 µM which suggests that nucleoside transporters play a role in its uptake. Polarized transport was not observed suggesting that the facilitative nature of ENT1 plays a major role in alovudine transport. S-(4-nitrobenzyl)-6-thioinosine (NBMPR), an ENT1 inhibitor, incompletely decreased alovudine permeability across the nasal mucosa. This demonstrates that at least one transporter, ENT, plays a significant role in the uptake of this nucleoside drug across the nasal mucosa.

Frequency selectivity is one of the most important functions of the mammalian hearing organ – the cochlea. The interaction of fluid mass and organ of Corti compliance sets a traveling wave along the basilar membrane (BM), which is longitudinally tuned to different frequencies. Beyond this passive tuning process, cochlear amplification locally enhances the vibration of the best frequency peak by factors of hundreds to boost the frequency selectivity and sensitivity of the cochlea. This amplification is achieved by a positive feedback loop between BM motion and outer hair cell (OHC) electrical-mechanical response. However, this active mechanism is vulnerable to damage and cannot be fully recovered in vivo. As the instruments of cochlear amplification, the frequency response of BM and OHCs are of great importance to understand cochlear tuning process. This thesis used animal models, aimed to understand cochlear tuning and investigate possibilities to manipulate the cochlear amplifier, by testing the cochlear amplifier’s sensitivity to operating conditions. The first project tested whether the cochlear amplification can adjust to a lower endocochlear potential (EP), which controls OHC electromechanical force by providing part of the voltage source to drive OHC transduction current. To investigate this possibility, we use intraperitoneal (IP) and intravenous (IV) injection of furosemide to reversibly reduce EP, while monitoring the EP and cochlear amplification simultaneously. Cochlear amplification was monitored by measuring the local cochlear microphonic (LCM) and distortion product emission (DPOAE). With IV injection, the cochlear amplification observed in LCM could attain nearly full or even full recovery with reduced EP. This showed the cochlea has an ability to adjust to diminished operating condition. Furthermore, the cochlear amplifier and EP recovered with different time courses: cochlear amplification just started to recover after the EP was nearly fully recovered and stabilized. Using a Boltzmann model and the 2nd harmonic of the LCM to estimate the mechanoelectric transducer channel operating point, we found that the recovery of cochlear amplification occurred with re-centering of the operating point. The second project studied the physiological and anatomical effects of perfusing the cochlea with a viscous fluid, for better understanding cochlear fluid mechanics. Perilymphatic perfusion was applied with artificial perilymph and viscous sodium hyaluronate (Healon, HA) in four different concentrations. Using compound action potential (CAP) thresholds as an indicator of cochlear condition, our results and analysis indicated that the cochlea can sustain, without a significant CAP threshold shift, up to a 1.5 Pa shear stress. Histology of the cochleae perfused with higher shear stress showed the Reissner's membrane was torn. These data also indicated that the cochlea mechanics remains normal within increased perilymphatic fluid viscosity up to an increase of a factor of 50. Beside these findings, a temporary CAP threshold shift was observed, perhaps due to the presence and then clearance of viscous fluid within the cochlea, or to a temporary position shift of the organ of Corti. The last project was to test the effect of OHC intracellular Cl- concentration on cochlear amplification. Chloride is known to enable the electromotility of the OHC by binding its motor protein, prestin. By locally perfusing high chloride perilymph and the chloride ionophore tributyltin, this study investigated whether increasing intracellular chloride concentration can restore cochlear sensitivity in a cochlea that was slightly damaged. This had been shown by others in guinea pig. However, we did not observe recovery in several attempts in gerbil.

It is a major challenge to understand functional neuronal circuits across the whole brain. Existing methods for observing neuronal activity represent a major bottleneck in addressing biological problems. In our lab, we developed Swept Confocally Aligned Planar Excitation (SCAPE) microscopy, which offers the ability to image a large 3D volume (e.g. 1000x800x250um) at speeds exceeding 10 volumes per second. Used with different genetically encoded fluorescent indicators, SCAPE enables us to observe neuronal activity across the whole brain of different small animal models, or a much larger volume of intact cortex/tissue compared to traditional approaches. The unique single objective design and flexible system layout of SCAPE makes it simple to image different samples without complex sample preparation and restraint. During this thesis work, I collaborated with biology and neuroscience labs to develop and optimize a range of novel in-vivo/in-vitro neuroimaging applications using SCAPE microscopy. In particular, my research has focused on using SCAPE to image freely crawling Drosophila Melanogaster larvae, intact mouse olfactory epithelium, head fixed behaving adult Drosophila, larval zebrafish brain and beating heart, and the neuronal system of behaving C. elegans, all in collaboration with experts in these models from Columbia University and other research institutions. I also developed and optimized different sample preparations and experimental procedures to take full advantage of the high-speed 3D imaging capabilities and flexibility of SCAPE microscopy. Finally, I optimized computational and image analysis techniques for large scale 5D SCAPE imaging datasets, including 3D cell tracking, large scale 3D data motion correction/registration, and cellular level neuronal activity extraction with different dimensionality reduction methods. The experiments I have performed in different animal models have enriched the long-term development of SCAPE by providing valuable feedback for system improvement and dissemination, and pushing the SCAPE design towards a more interchangeable platform with diverse capabilities suitable for routine uses by our collaborators and the wider neuroscience community.

Abstract not available