<p>The use of positron-emitting tracers has been extended beyond tomographic facilities in the last few years, giving rise to a general positron-emitting tracing technique. The methodological part of the present thesis involved the evaluation of the performance of storage phosphor (SP) plates, with tracers labeled with high-energy, short-lived, positron-emitting radionuclides, using homogenized tissue specimens and autoradiography with frozen brain sections. The SP plates showed superior sensitivity and a linear response over a wide radioactivity range. Autoradioradiography provided reliable results due to (a) adequate sensitivity for low radioactivity concentration, b) an excellent linear range, and (c) satisfactory resolution. Though equilibration time of receptor-ligand interaction was dependent upon section thickness, quantification was possib with thinner sections.</p><p>An initial finding using frozen section autoradiography of rat brain and spinal cord showed preferential binding of [¹¹C]4-NMPB, a muscarinic acetylcholine (mACh) receptor antagonist, to the M4 subtype of mACh receptors. Further work to ascertain this specificity, by use of binding studies on cell membranes from CHO-K1 cells expressing individual subtypes of human mACh receptors, suggested lack of subtype selectivity. With respect to the possible cliinical use in glutamatergic neuropathology, [¹¹C]cyano-dizocilpine, as a potential PET tracer for the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, was studied. The <i>in vivo</i> visualization of specific binding could not be achieved, though <i>in vitro</i> binding demonstrated good specificity and preferential binding to the activated for of the NMDA receptors.</p><p>The use of the glucose analogue [¹⁸F]fluorodeoxyglucose (FDG) to study glucose utilization was evaluated in experimental traumatic brain injury (TBI). A trauma-induced increased uptake of FDG was seen, whereas the uptake of [1-¹⁴C]glucose remained unchanged. This discrepancy might be due to the increased postraumatic affinity of FDG for the endothelial glucose transporter proteins and/or to the hexokinase enzyme. [¹¹C]Cyano-dizocilpine, [¹¹C]4-NMPB, and [¹¹C]flumazenil were utilized in autoradiography to evaluate changes in NMDA, mACh, and GABA_A receptors, espectively, in experimental TBI. Observations showed a global decrease in the binding potential BP) of (i) [¹¹C]cyano-dizocilpine acutely and 12 hrs after TBI, and (ii) of [¹¹C]4-NMPB at 12 hrs after TBI, and (iii) a decrease in the BP of [¹¹C]flumazenil in the cortex and hippocampus ipsilateral to the site of injury. The demonstrated changes in receptor binding after TBI are indicative of a widely dissipated effect of TBI on the particular neurotransmitter receptor systems as compared with what would be expected from FDG studies after TBI, i.e., a local disturbed neurotransmission.</p>

The use of positron-emitting tracers has been extended beyond tomographic facilities in the last few years, giving rise to a general positron-emitting tracing technique. The methodological part of the present thesis involved the evaluation of the performance of storage phosphor (SP) plates, with tracers labeled with high-energy, short-lived, positron-emitting radionuclides, using homogenized tissue specimens and autoradiography with frozen brain sections. The SP plates showed superior sensitivity and a linear response over a wide radioactivity range. Autoradioradiography provided reliable results due to (a) adequate sensitivity for low radioactivity concentration, b) an excellent linear range, and (c) satisfactory resolution. Though equilibration time of receptor-ligand interaction was dependent upon section thickness, quantification was possib with thinner sections. An initial finding using frozen section autoradiography of rat brain and spinal cord showed preferential binding of [11C]4-NMPB, a muscarinic acetylcholine (mACh) receptor antagonist, to the M4 subtype of mACh receptors. Further work to ascertain this specificity, by use of binding studies on cell membranes from CHO-K1 cells expressing individual subtypes of human mACh receptors, suggested lack of subtype selectivity. With respect to the possible cliinical use in glutamatergic neuropathology, [11C]cyano-dizocilpine, as a potential PET tracer for the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, was studied. The in vivo visualization of specific binding could not be achieved, though in vitro binding demonstrated good specificity and preferential binding to the activated for of the NMDA receptors. The use of the glucose analogue [18F]fluorodeoxyglucose (FDG) to study glucose utilization was evaluated in experimental traumatic brain injury (TBI). A trauma-induced increased uptake of FDG was seen, whereas the uptake of [1-14C]glucose remained unchanged. This discrepancy might be due to the increased postraumatic affinity of FDG for the endothelial glucose transporter proteins and/or to the hexokinase enzyme. [11C]Cyano-dizocilpine, [11C]4-NMPB, and [11C]flumazenil were utilized in autoradiography to evaluate changes in NMDA, mACh, and GABAA receptors, espectively, in experimental TBI. Observations showed a global decrease in the binding potential BP) of (i) [11C]cyano-dizocilpine acutely and 12 hrs after TBI, and (ii) of [11C]4-NMPB at 12 hrs after TBI, and (iii) a decrease in the BP of [11C]flumazenil in the cortex and hippocampus ipsilateral to the site of injury. The demonstrated changes in receptor binding after TBI are indicative of a widely dissipated effect of TBI on the particular neurotransmitter receptor systems as compared with what would be expected from FDG studies after TBI, i.e., a local disturbed neurotransmission.

Drug dependence is a serious and increasing problem in our society, especially among adolescents. The use of the large variety of substances available can result in a range of physiological and psychological adverse effects on individuals and negative consequences on the society overall. Several different types of drugs induce neurotoxicological damages, which in turn can generate impairment in for example the reward system and affect cognitive parameters.  The drug gamma-hydroxybutyrate (GHB) is usually considered a harmless compound among abusers, but has now shown to be highly addictive. Furthermore, GHB can cause memory impairments in both humans and animals. On the contrary, growth hormone (GH) and its main mediator insulin-like growth factor 1 (IGF-1) have recently been suggested to improve memory and learning in several studies. The hormones exhibit certain neuroprotective capabilities and have also previously been demonstrated to reverse opioid induced apoptosis in hippocampal cells. These effects and the fact that GHB is shown to increase GH secretion, which attracted considerable attention among body builders, led us to initiate studies on GHB and its impact on relevant systems in the central nervous system (CNS). Thus, the main purpose of the present investigation was to elucidate some of the underlying mechanisms that could account for the effects exerted by GH and GHB in the CNS. We found that a) GH affects the density and functionality of GABAB-receptors and opioid receptors in the male rat brain, b) GHB induces cognitive deficits and down-regulates GABAB-receptors, c) GHB treatment creates an imbalance between the endogenous opioids Met-enkaphalin-Arg6Phe7 (MEAP) and dynorphin B and increases the levels of MEAP in regions of the brain that are associated with drug dependence, and d) GHB affects the expression of IGF-1 receptors but not the plasma levels of IGF-1. In conclusion, the present work demonstrates that GH interacts with both opioid and GABAB-receptors in the male rat CNS and that GHB has an impact on brain regions associated with cognition and the development of dependence. These observations may be of relevance in many aspects related to addiction and might be translated into humans.

The dopamine D2 receptor exists in vitro in two states of agonist affinity: a high-affinity state mediating dopamine’s physiological effects, and a physiologically-inert low-affinity state. Our primary goal was to determine the in vivo relevance of this two-affinity-state model for the agonist PET radiotracer [11C]-(+)-PHNO, developed for measurement of the D2 high-affinity state. Our second goal was to characterize the regional D2 versus D3 pharmacology of [3H]-(+)-PHNO binding and assess its utility for measuring drug occupancy at both receptor subtypes. Using ex vivo dual-radiotracer experiments in conscious rats, we showed that, contrary to the two-affinity-state model, the binding of [11C]-(+)-PHNO and the antagonist [3H]-raclopride were indistinguishably inhibited by D2 partial agonist (aripiprazole), indirect agonist (amphetamine) and full agonist ((-)-NPA) pretreatment. Furthermore, ex vivo [11C]-(+)-PHNO binding was unaffected by treatments that increase in vitro high-affinity state density (chronic amphetamine, ethanol-withdrawal), whereas unilateral 6-OHDA lesion, which increases total D2 receptor expression, similarly increased the ex vivo binding of [11C]-(+)-PHNO and [3H]-raclopride. These results do not support the in vivo validity of the two-affinity-state model, suggesting instead a single receptor state for [11C]-(+)-PHNO and [3H]-raclopride in conscious rat. Importantly, we also demonstrated that the increased amphetamine-sensitivity of the agonist radiotracers [11C]-(+)-PHNO and [11C]-(-)-NPA, commonly seen in isoflurane-anaesthetized animals and cited as evidence for the two-affinity-state model, is due to the confounding effects of anaesthesia. Using in vitro and ex vivo autoradiography in rat and the D3 receptor-selective drug SB277011, we found that [3H]-(+)-PHNO binding in striatum and cerebellum lobes 9 and 10 was due exclusively to D2 and D3 receptor binding, respectively, but in other extra-striatal regions to a mix of the two receptor subtypes. Surprisingly, the D3 contribution to [3H]-(+)-PHNO binding was greater ex vivo than in vitro. Also surprising, several antipsychotic drugs, at doses producing 80% D2 occupancy, produced insignificant (olanzapine, risperidone, haloperidol) or small (clozapine, ~35%) D3 occupancy, despite similarly occupying both receptor subtypes in vitro. These data reveal a significant discrepancy between in vitro and ex vivo measures of dopamine receptor binding and suggest that the D3 occupancy is not necessary for the therapeutic effect of antispychotic drugs.

The abuse of anabolic androgenic steroids (AAS) is becoming increasingly common and may result in a range of physiological as well as psychological effects such as altered behavior in terms of increased aggression, cognitive dysfunction and addictive behavior. AAS comprise testosterone and its derivatives, of which nandrolone is one of the more common. Previous studies have shown nandrolone-induced effects in male rats on peptide levels within the Substance P (SP) system and the dynorphinergic system; these effects may be linked to some of the reported behavior alterations. The studies presented in this thesis aimed to investigate the mechanisms underlying these peptide alterations and also to further investigate neuropeptidergic effects attributed to nandrolone administration. The results display significant effects on the enzymatic conversion of SP and Dynorphin A into their bioactive metabolites SP(1-7) and Leu-enkephalin-Arg6, respectively, as a result of nandrolone treatment. More profound investigations on the dynorphinergic system displayed effects on the kappa opioid receptor density in various brain regions. There was also a significant increase in the expression of the gene transcript of prodynorphin in the hippocampus, a brain region associated with cognitive processes. In addition, impaired spatial learning and memory in the Morris water maze task following nandrolone administration was encountered. The results provide further understanding regarding neuropeptidergic mechanisms underlying AAS-induced behavioral effects.

Made available in DSpace on 2015-07-22T20:50:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-26. Added 1 bitstream(s) on 2015-08-11T03:25:54Z : No. of bitstreams: 1 Publico-11764a.pdf: 1760987 bytes, checksum: 26371946d909a5525c0bd6c7cc6d7c33 (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:55Z : No. of bitstreams: 2 Publico-11764a.pdf: 1760987 bytes, checksum: 26371946d909a5525c0bd6c7cc6d7c33 (MD5) Publico-11764b.pdf: 969495 bytes, checksum: d87ae7194b036aaff67581e32d434f64 (MD5) / Objetivo: Verificar se indivíduos susceptíveis e não-susceptíveis às convulsões clônicas induzidas pelo DMCM, um agonista inverso benzodiazepínico, diferem: 1) na sensibilidade ao efeito hipnótico induzido pelo diazepam e por outros moduladores alostéricos positivos do receptor GABAA; 2) na marcação auto-radiográfica com o [3H]- flunitrazepam ao longo do encéfalo; 3) na marcação de [3H]-L-glutamato e do [3H]-MK 801 em membranas de regiões encefálicas; e 4) na atividade da enzima Na+/K+- ATPase, bem como na marcação da [3H]-ouabaína às isoenzimas Na+/K+- ATPase de alta e de baixa afinidade ao radioligante em membranas de regiões encefálicas. Métodos: Ratos Wistar, machos, adultos foram administrados intraperitonealmente duas vezes com uma DC50 de DMCM (com intervalo de uma semana entre as administrações), obtendo-se dois grupos distintos: o grupo susceptível às convulsões (SC), que apresentou convulsões clônicas em ambas as exposições à droga, e o grupo não-susceptível às convulsões (NSC), que não apresentou alterações motoras em ambas as exposições. Após cerca de 25 dias da segunda administração de DMCM, os grupos selecionados foram submetidos aos experimentos com os hipnóticos diazepam, pentobarbital e etanol, nos quais foram registrados o tempo e a latência de sono ou foram sacrificados e seus encéfalos retirados para os seguintes ensaios bioquímicos: 1) auto-radiografia com o [3H]-flunitrazepam; 2) marcação de [3H]-L-glutamato e de [3H]- MK 801 em membranas neuronais; e 3) atividade enzimática da Na+/K+- ATPase e marcação de [3H]-ouabaína em enzimas de alta e baixa afinidade em membranas neuronais. Resultados: O grupo SC apresentou menor tempo de sono induzido pelo diazepam com relação ao grupo NSC, embora não tenham se distinguindo no tempo de sono induzido pelo pentobarbital e pelo etanol. Com relação aos experimentos bioquímicos, observou-se uma menor marcação de [3H]-flunitrazepam na região CA2 ventral do hipocampo no grupo SC. Quanto à ligação de [3H]-L-glutamato foi menor no grupo SC nas regiões do córtex frontal, amígdala + córtex límbico e hipocampo, enquanto que a ligação de [3H]-MK 801 foi menor no córtex frontal, hipocampo e estriado. Embora os grupos não tenham se diferenciado na atividade enzimática da Na+/K+- ATPase, o grupo SC apresentou uma menor marcação da [3H]-ouabaína em isoenzimas de alta afinidade nas regiões do tronco encefálico, córtex frontal e hipocampo, bem como uma menor marcação de [3H]-ouabaína nas regiões do tronco encefálico e córtex frontal em isoenzimas de baixa afinidade. Conclusão: As diferenças entre os grupos quanto à sensibilidade ao efeito convulsivante do DMCM, à ansiedade observada em experimentos anteriores, bem como à sensibilidade ao efeito hipnótico do diazepam podem estar associadas a uma diferença nos sítios benzodiazepínicos da região CA2 ventral do hipocampo, na ix atividade glutamatérgica e em isoformas específicas da Na+/K+- ATPase em determinadas regiões encefálicas. / Objective: The aim of this work was to verify if rats susceptible and non-susceptible to clonic convulsions induced by DMCM, a benzodiazepine inverse agonist, differ: 1) in the sensitivity to the hypnotic effect induced by diazepam and by others positive allosteric modulators of GABAA receptors; 2) in auto-radiographical analysis of [3H]-flunitrazepam binding along the brain; 3) in the binding of [3H]-L-glutamate and of [3H]-MK 801 in membranes from discrete brain regions; and 4) in the Na+/K+-ATPase activity, as well as in the binding of [3H]-ouabain to Na+/K+-ATPase isoenzimes with high and low affinity to the radioligand in membranes from discrete brain regions. Methods: Adult, male, Wistar rats were administered with two intraperitoneal injections of a convulsant dose 50% (CD50) of DMCM (one-week interval between them), resulting in two distinct groups: the group susceptible to clonic convulsions (SC), which presented clonic convulsions in both the expositions to the drug, and the group nonsusceptible to clonic convulsions (NSC), which did not present any motor disturbance in both the expositions. After 25 days from the second exposition to DMCM, the selected groups were submitted to the experiments with the hypnotics diazepam, pentobarbital and ethanol, in which were registered the latency and the time of sleep or they were sacrified and their brains were removed to carry out the following assays: 1) autoradiography with [3H]-flunitrazepam; 2) binding with the [3H]-L-glutamate and with the [3H]-MK 801 in neuronal membranes; 3) enzymatic activity of Na+/K+-ATPase and binding of [3H]-ouabain to the isoenzimes with high and low affinity in neuronal membranes. Results: The SC group presented a lower sleeping time induced by diazepam compared to the NSC group, and did not differ in the sleeping time induced by pentobarbital and ethanol. Concearning the biochemical experiments, it was observed a lower binding of [3H]-flunitrazepam in the CA2 subregion of ventral hippocampus in the SC group. A lower binding of [3H]-L-glutamate was also observed in the SC group in the frontal cortex, amygdala plus limbic cortex and hippocampus, whereas the binding of [3H]-MK 801 was lower in the frontal cortex, hippocampus and striatum compared to the NSC group. Althougt the groups did not differ in the enzymatic activity of Na+/K+- ATPase, the SC group presented a lower binding of [3H]-ouabain to the high-affinity isoenzimes in the brainstem, frontal cortex and hippocampus, as well as a lower binding of [3H]-ouabain to the low-affinity isoenzimes in the brainstem and in the frontal cortex compared to the NSC group. Conclusion: The differences between the groups concerning the sensitivity to the convulsant effect of DMCM, the level of anxiety previously observed, as well as the sensitivity to the hypnotic effect of diazepam may be associated with the GABAA/benzodiazepine site in CA2 subregion of ventral hippocampus, with glutamatergic activity and with specific isoforms of Na+/K+-ATPase in rat brain regions. / TEDE / BV UNIFESP: Teses e dissertações

Made available in DSpace on 2014-10-09T12:53:57Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:09:29Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:53:57Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:09:29Z (GMT). No. of bitstreams: 0 / No presente trabalho foi desenvolvida uma técnica radiográfica para a inspeção de amostras finas, que emprega feixes de partículas - ? como radiação penetrante e, detectores de traços nucleares de estado sólido para o registro da imagem. O feixe é gerado em uma tela de boro natural quando irradiada com nêutrons térmicos e o detector utilizado foi o CR-39. A interação das partículas - ? com o detector geram danos, os quais sob a ação de uma solução química adequada transformam-se em traços, as unidades básicas formadoras da imagem. Foi desenvolvido um sistema digital próprio para a aquisição, análise e processamento dos dados e das imagens registradas nos detectores. As condições ótimas de irradiação e de revelação para a obtenção de uma radiografia são 1,3 horas e 25 minutos a 70°C, respectivamente. Nestas condições a técnica é capaz de analisar amostras com até 10?m de espessura, com uma resolução espacial de 32?m. O emprego do sistema digital permitiu uma redução significativa no tempo despendido para a aquisição e análise de dados e, uma melhoria na visualização das imagens radiográficas registradas. Além disto permitiu acesso a novos dados referentes aos traços registrados, o que abriu novas perspectivas de pesquisa pois, tem-se acesso aos parâmetros fundamentais que norteiam a teoria da formação da imagem em detectores de traços nucleares utilizada neste trabalho. São apresentadas algumas radiografias que demonstram a potencialidade da técnica desenvolvida. Quando comparada com as outras técnicas radiográficas usuais, empregadas para inspecionar amostras finas, a desenvolvida no presente trabalho permite um menor tempo para a obtenção da imagem, não é necessário a manipulação de materiais radioativos líquidos e, o filme empregado é praticamente insensível às radiações ?, ?, raios - X e luz visível. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

Abstract In the Nordic countries, event-free survival (EFS) exceeds 80% in certain groups of children treated for acute lymphoblastic leukaemia (ALL). With the improved cure rates, however, there are more children suffering from neurological late effects, especially due to therapy directed at the central nervous system (CNS). The aim of this study is to examine the changes taking place in the nervous system after leukemia treatment and to evaluate the role of treatment in these changes in patients and in an animal model. Twenty-seven ALL survivors and healthy controls were examined by means of motor evoked potentials (MEPs). ALL survivors were also examined clinically. The children with ALL continued to show decreased motor nerve conduction in the peripheral nerves, but not within the CNS, five years after the cessation of treatment. Clinical neurological findings were obtained in 33% of the cases. The MEP results indicated reversibility of the motor injury due to CNS effects. Nineteen patients underwent perfusion magnetic resonance imaging (MRI) at the cessation of treatment or 4-8 years after the treatment. Seventeen of them also underwent single-photon emission computed tomography (SPECT). The studies showed small perfusion defects in SPECT, which were not visible by perfusion MRI. Methotrexate (Mtx) neurotoxicity was studied in a swine model using functional MRI, brain perfusion SPECT, iodine-123 labelled 2β-carbomethoxy-3β-(4-iodophenyl) tropane ([123I]β-CIT) SPECT and whole-hemisphere autoradiography with [125I]β-CIT in ten Mtx-treated animals and five control animals. Mtx-related changes in the brain could be detected as reduced or negative blood-oxygen-level-dependent (BOLD) responses to somatosensory activation in BOLD contrast MRI, which indicates changes in flow metabolism coupling. Perfusion defects in brain SPECT were seen in the Mtx group and the control group, which suggests that the perfusion defects seen in brain SPECT are probably multifactorial. The change in dopamine transporter (DAT) density in the Mtx group was not different from that in the controls. The abnormalities in nerve conduction after treatment in survivors of ALL were partly reversible years after the treatment. The patients had perfusion defects in SPECT imaging which were not seen in perfusion MRI. The clinical significance of these defects remains obscure. The animal model suggested perfusion defects to be multifactorial.

The distribution of neurokinin receptors in rat kidney, renal artery, renal vein, and proximal ureter was evaluated by autoradiography after in vitro labeling of NK1 sites with [125I]Bolton-Hunter substance P (BHSP) or NK3 sites with [125I][MePhe7]neurokinin B ([MePhe7NKB). Film autoradiography using [125I][MePhe7]NKB revealed specific binding sites associated with the renal vein and its large branches, the renal pelvis, the inner strip of outer renal medulla, and the proximal ureter. High-resolution autoradiograms demonstrated that these sites were localized to the smooth muscle layer in the veins, pelvis, and ureter. Neither the renal arterial system nor the renal cortex contained specific [125I][MePhe7]NKB binding sites although a high level of nonspecific binding was associated with the renal artery. Specific binding of [125I]BHSP was associated with the renal artery and renal pelvis but not the renal veins. Arterial NK1 receptors appeared to be localized to the adventitia. The results indicate that at least two types of tachykinin receptor are present in the rat kidney. The distinct localization observed for most of the NK1 and NK3 receptors suggests that they have different functions.