Diabetes mellitus is associated with several clinically significant abnormalities in the oral cavity and salivary glands, the most common being periodontitis, salivary gland enlargement and a sensation of dry mouth. The pre­valence of dental caries in diabetics is mostly reported to be decreased or unaffected. Since there is a shortage of information concerning the influence of metabolic control and duration of diabetes on these abnormalities, the present studies were performed. Three-month-old rats were made alloxan-diabetic and investigated after one and twelve months’ duration of dia­betes for oral lesions and feeding behavior. They had free access to a standard pellet diet and tap water. Strepto­coccus mutans and lactobacilli were naturally occurring. In diabetic rats the proportion of the oral flora which was lactobacilli was positively correlated to the blood glucose level. Untreated long-term alloxan-diabetic rats de­veloped advanced periodontal disease and root surface caries in the molars at sites of interdental impaction of foreign material. The degree of alveolar bone loss was positively correlated to the blood glucose level. Diabetic rats were hyperphagic and had a longer total eating time, including day-time eating, mainly by having longer meals but no significant increase of meal frequency, as compared with non-diabetic animals. The untreated al­loxan-diabetic rats developed reduced salivary gland weight which was of the same degree in short- and long­term diabetic animals. Short- and long-term untreated alloxan-diabetic rats showed a similar degree of morpho- metrically estimated lipid accumulation in the acinar cells of the submandibular glands, and the degree was posi­tively correlated to the blood glucose level. However, lipid inclusion occurred only in rats with a morning non­fasting blood glucose level exceeding 15 mmol/L. The capillaries in the submandibular glands of the untreated long-term alloxan-diabetic rats had a significantly increased thickness of the basement membranes as compared with the observations in short-term diabetic and non-diabetic rats. Untreated alloxan-diabetic rats were also shown to have a decreased salivary flow rate compared with non-diabetic rats, and the decreased flow was negatively correlated to the blood glucose concentration. Salivary flow rate increased with the duration of the disease. The diabetic rats had increased salivary glucose levels, which were positively correlated to blood glucose values when the latter were above 15 mmol/L, suggesting a threshold mechanism for salivary glucose excretion. Insulin therapy reversed salivary flow rate and salivary glucose concentrations toward normal. Salivary investigations were performed in eleven diabetic patients on two occasions with different metabolic con­trol. Salivary flow rate showed marked interindividual differences but was not significantly changed by improv­ed metabolic control although several of the patients initially had severely deranged glucose metabolism. A posi­tive correlation between the glucose concentration in blood and saliva was seen in the parotid saliva during secre­tory stimulation. No significant change in electrolytes, amylase or antimicrobial factors was found. / <p>S. 1-48: sammanfattning, s. 49-90: 5 uppsatser</p> / digitalisering@umu

Submitted by Aelson Maciera (aelsoncm@terra.com.br) on 2018-01-30T17:09:56Z No. of bitstreams: 1 DissAA.pdf: 1196078 bytes, checksum: c96629c7fdc72285ebf9260f9394bbae (MD5) / Approved for entry into archive by Ronildo Prado (bco.producao.intelectual@gmail.com) on 2018-02-01T17:39:56Z (GMT) No. of bitstreams: 1 DissAA.pdf: 1196078 bytes, checksum: c96629c7fdc72285ebf9260f9394bbae (MD5) / Approved for entry into archive by Ronildo Prado (bco.producao.intelectual@gmail.com) on 2018-02-01T17:40:05Z (GMT) No. of bitstreams: 1 DissAA.pdf: 1196078 bytes, checksum: c96629c7fdc72285ebf9260f9394bbae (MD5) / Made available in DSpace on 2018-02-01T17:44:38Z (GMT). No. of bitstreams: 1 DissAA.pdf: 1196078 bytes, checksum: c96629c7fdc72285ebf9260f9394bbae (MD5) Previous issue date: 2017-07-10 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Water and sodium intake are fundamental behaviors for body fluid homeostasis. These behaviors are controlled by a neural circuitry involving facilitatory and inhibitory mechanisms, which are modulated by signals activated by changes in body fluid volume and composition. Important inhibitory mechanisms for water intake and particularly for sodium intake are located in the lateral parabrachial nucleus (LPBN), a pontine structure. It is known that LPBN inhibitory mechanisms are controlled by several neurotransmitters, some increasing, others reducing the action of this nucleus on sodium intake... / A ingestão de água e a ingestão de sódio são comportamentos fundamentais para a homeostase dos líquidos corporais. A manifestação destes comportamentos é controlada por um circuito neural constituído por mecanismos facilitatórios e inibitórios os quais são constantemente modulados por informações relativas aos líquidos corporais. Importantes mecanismos inibitórios para a ingestão de água e especialmente para a ingestão de sódio localizam-se no núcleo parabraquial lateral (NPBL), uma estrutura pontina. Sabe-se que os mecanismos inibitórios do NPBL são controlados por diversos neurotransmissores, alguns aumentando e outros diminuindo a ação deste núcleo sobre a ingestão de sódio .....

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Neste trabalho, estudos comportamentais, farmacolÃgicos e neuroquÃmicos em Ãreas cerebrais foram realizados em animais adultos (2 meses de idade) que apresentaram convulsÃo e estado epilÃptico (EP), atravÃs da administraÃÃo de pilocarpina 400mg/kg, s.c., P400, com a finalidade de investigar os mecanismos envolvidos durante a fase aguda (1 e 24h) do processo convulsivo. Os estudos comportamentais em animais mostraram que a administraÃÃo de P400 produziu sinais colinÃrgicos perifÃricos (SCP) e movimentos estereotipados (ME) em todos os animais em ambos os perÃodos de observaÃÃo. Os animais observados por 1h apresentaram parÃmetros comportamentais semelhantes ao grupo de 24h, somente o Ãndice de desenvolvimento de convulsÃes, EP, foi um pouco menor, e nÃo houve nenhuma morte. Os estudos farmacolÃgicos com antagonista colinÃrgico bloqueou todos os parÃmetros comportamentais observados. Das drogas gabaÃrgicas, os antagonistas dopaminÃrgico e glutamatÃrgico apresentaram melhor resultado, aumentado a latÃncia da primeira convulsÃo (LC), a latÃncia do EP (LEP) e reduzindo o nÃmero de mortes. Os antagonistas dopaminÃrgico D2, o opÃide, os antidepressivos e o agonista dopaminÃrgico reduziram a LC, LEP e aumentou o nÃmero de mortes, enquanto o antipsicÃtico testado aumentou a LC, a LEP e reduziu as mortes. O lÃtio potencializou os efietos da pilocarpina aumentado o nÃmero de mortes e reduzindo a LC e a LEP. Os estudos neuroquÃmicos revelaram que a atividade da acetilcolinesterase no hipocampo, cÃrtex frontal (CF) e corpo estriado dos animais sofreu uma reduÃÃo significativa apenas na primeira hora da fase aguda, enquanto que no perÃodo de 24h de observaÃÃo a atividade enzimÃtica praticamente se normalizou. O nÃvel de lipÃdio peroxidaÃÃo e o conteÃdo de nitrito e nitrato aumentaram, enquanto que, a concentraÃÃo de GSH diminui nos dois perÃodos do estudo nas Ãreas investigadas. A atividade da SOD aumentou durante 1h em todas a Ãreas. Por sua vez, no perÃodo de 24h aumentou apenas no CF. Na atividade da catalase em ambos os perÃodos e nas Ãreas estudadas verificou-se um aumento significativo. Estudos sobre a densidade mÃxima (Bmax) dos receptores colinÃrgicos muscarÃnicos (M1 e M2) e dopaminÃrgicos (D1 e D2) nas Ãreas analisadas durante 1 e 24h de observaÃÃo foram diminuÃdos e nÃo alterados, respectivamente. Em relaÃÃo aos receptores serotonÃrgicos (5-HT2) nÃo foi verificado alteraÃÃo no Bmax apÃs 1 e 24h de observaÃÃo, nas trÃs Ãreas estudadas. Nas trÃs Ãreas estudadas observou-se um aumento e uma diminuiÃÃo no Bmax dos receptores glutamatÃrgicos e GABAÃrgicos, respectivamente. O P400 alterou de forma diversificada a constante de dissociaÃÃo (Kd) dos receptores M1, M2, D1, D2, 5-HT2, GABAÃrgicos e glutamatÃrgicos nos animais que apresentaram convulsÃo, EP e que foram sacrificados 1 e 24 h depois do tratamento. Na determinaÃÃo de monoaminas e seus metabÃlitos com HPLC, o P400 alterou a concentraÃÃo de DA, DOPAC e HVA, bem como da NA e da 5-HT e seu metabÃlito o 5-HIAA nas diferentes regiÃes do cÃrebro apÃs 1 e 24 h de observaÃÃo. Nos experimentos de determinaÃÃo do conteÃdo dos aminoÃcidos com HPLC, o P400 alterou a concentraÃÃo de (glutamato) GLU estriatal, (glutamina) GLN hipocampal, (aspartato) ASP cortical e nÃo modificou o conteÃdo de tirosina com 1h nas trÃs Ãreas estudadas. JÃ com 24 horas de observaÃÃo nÃo houve alteraÃÃo em nenhuma das Ãreas nos aminoÃcidos GLU, GLN e ASP, mas o conteÃdo de tirosina aumentou de forma significativa nas Ãreas investigadas. / The present study was aimed at investigating behavioural and neurochemical alterations in brain areas of adults rat (2-month-old) which presented seizures and status epilepticus (SE) after treatment with a single dose of pilocarpine (400mg/kg, s.c., P400) in order to clarify the mechanisms of the acute phase in the convulsive process (1 and 24h). Behavioural studies have demonstrated that the P400 administration produced peripheral cholinergic signs and stereotyped movements in all of the animals in both periods of observation. The behavioural parameters assessed between 1 and 24 h were similar, but the seizure and SE development index was slightly lower in the 1h group and in the same group no case of fatality was observed. The pharmacological studies with antagonist cholinergic did not present any of the behavioural alterations. The drugs gabaergic, the antagonist dopaminergic D1, antipsychotic used and glutamatergic antagonist presented increased in the latency to first seizure (LS), latency of the SE (LSE) and decreasing the number of the death. The antagonists dopaminergic D2, the opioide, the antidepressants, and the dopaminergic agonist decreased the LS and a LSE and increased the number of the death. The lithium increased the effects of the pilocapine as well as the number of the death and decreased the LS and LSE. Neurochemical assessments revealed that acetylcholinesterase activity in hippocampus, frontal cortex and striatum decreased significantly only the first hour of the acute phase, meanwhile after 24h, the enzymatic activity remained unaltered. Lipid peroxidation level and nitrite e nitrate content were augmented whereas the GSH concentration was decreased in the areas investigated in both periods of observation. The SOD activity was increased during the first hour in the three areas. In turn, in the 24h period it was augmented in the frontal cortex alone. The catalase activity was significantly increased in both periods and in all areas. Works concerning maximum density (Bmax) of muscarinc cholinergic (M1 e M2) and dopaminergic (D1 e D2) receptors in the areas studied during 1h and 24h of observation were decreased and unaltered, respectively. Regarding the serotonergic receptors (5-HT2) was not verified alteration in the Bmax after 1 and 24h of observation in the three areas studied. In the areas studied the Bmax from glutamatergic and GABAergic receptors were increased and decreased, respectively. P400 altered the M1, M2, D1, D2, 5-HT2, GABAergic e glutamatergic receptors dissociation constant values (Kd) in distinct ways after 1 e 24h from the treatment. In the monoamine and their metabolites with HPLC determination P400 changes the DA, DOPAC and HVA as well as NA and 5-HT and its metabolite 5-HIIAA concentrations in the different cerebral areas after 1 and 24h of observation. In the experimental determinations of the amino acids contents, the P400 altered the content of (glutamate) GLU striatal, (glutamine) GLN hippocampal and the (aspartate) ASP cortical and no modified the concentration od tyrosine in the areas observed. However the TYR contents after 24 h of observation increas in striatum, hippocampus and frontal cortex, but GLU, GLN e ASP reamained unaltered in this period of the observation .

nÃo hà / Um estudo prospectivo e duplo-cego foi conduzido para verificar os efeitos do uso da pilocarpina no tratamento da xerostomia induzida por radiaÃÃo em pacientes oncolÃgicos, testando duas via de administraÃÃo: sublingual e bochechos. Os voluntÃrios foram randomicamente alocados em dois grupos: grupo para uso da soluÃÃo sublingual e grupo para uso de bochechos. Cada grupo foi subdividido, totalizando quatro amostras de resultados: duas para soluÃÃo sublingual (placebo e pilocarpina) e duas para bochechos (placebo e pilocarpina). As soluÃÃes foram formuladas na mesma concentraÃÃo de pilocarpina a 4% e administradas na dose total de 15 mg por dia, durante trÃs semanas. A amostra compreendeu quarenta voluntÃrios, de ambos os sexos, que foram submetidos à radiaÃÃo de cabeÃa e ou pescoÃo no serviÃo do CRIO (Centro Regional Integrado de Oncologia). Assim a distribuiÃÃo foi de dez pacientes em cada grupo. NÃo houve restriÃÃes quanto à idade nem ao grupo Ãtnico. Uma sialometria antes do estudo foi realizada em cada voluntÃrio, sendo selecionados os pacientes com fluxo salivar menor ou igual a 1 mL/min. Os resultados da sialometria apÃs uso das formulaÃÃes foram realizados semanalmente e anotados no formulÃrio de relato de caso (CRF). NÃo houve aumento quantitativo do fluxo salivar e nem diferenÃa estatisticamente significante entre as formulaÃÃes testadas. Portanto a soluÃÃo de pilocarpina a 4%, administrada na forma de bochechos ou pela via sublingual, nÃo aumentou o fluxo salivar nos pacientes com xerostomia induzida por radiaÃÃo / A prospective and double-blind study was conducted to verify the effects of the use of pilocarpine in the treatment of xerostomy induced by irradiation in oncologic patients, testing two administration routes: sublingual and mouthful. The volunteers were randomly allocated in two groups: a group for use of mouthfuls and a group for use of sublingual solutions. Each group was subdivided, totaling four samples of results: two for sublingual solutions (placebo and pilocarpine) and two for mouthfuls (placebo and pilocarpine). The solutions were formulated in the same pilocarpine concentration to 4% and administered in the total dose of 15 mg a day, for three weeks. The total group was formed by forty (40) volunteers, of both sex, treated with head and neck radiation in CRIO (Oncologic Integrated Regional Center) .The final distribuition was ten (10) patients in each subgroup. There was not restriction to age or etnic group.The volunteers who had the salivary flow less or equal to 1mL/min were selected to the study. The sialometry was measured weekly and whrote down in an appropriated formulary (CRF). The was not quantitative increase of the salivary flow or significant statistic difference between the two tested solutions. Therefore the pilocarpine solution 4% administrated sublingual or mouthful, did not increase the salivary flow in patients with xerostomy induced by radiation

Introdução: A esquizofrenia é um dos principais transtornos mentais e promove distúrbios comportamentais e cognitivos. Apesar do grande impacto social, pouco se conhece sobre a patofisiologia e etiologia da esquizofrenia. Pacientes com desordens psicóticas têm aumentado risco de desenvolver epilepsia, e pacientes com epilepsia do lobo temporal tem alta frequência de psicoses. A utilização de modelos experimentais animais de esquizofrenia e epilepsia pode ajudar a entender a neurobiologia dessas doenças e ter papel importante no desenvolvimento de novas estratégias terapêuticas. As interações complexas das crises epilépticas e quadros psicóticos tem sido aos poucos desvendadas. O óxido nítrico (NO) é um importante modulador da neurotransmissão e doadores de NO como o nitroprussiato de sódio (NPS) tem efeitos promissores em pacientes com esquizofrenia. Em modelos animais de epilepsia, NO pode ter efeito pró ou anticonvulsivante, sugerindo que o uso de NPS em pacientes com epilepsia e psicose pode não ser tão simples. Objetivos: Desenvolver um modelo animal de epilepsia e psicose comórbida, e compará-lo do ponto de vista comportamental e neuropatológico com modelos animais puros de epilepsia e esquizofrenia. Além disso, verificar a possível modulação desencadeada pelo tratamento com NPS nos modelos citados. Metodologia: Ratos Wistar machos (260-300g) foram divididos em grupos controle (SAL+SAL), epilepsia puro (PILO+SAL), esquizofrenia (SAL+QUET), epilepsia e psicose comórbida (PILO+QUET) e suas versões tratadas com NPS (SAL+SAL+NPS, PILO+SAL+NPS, SAL+QUET+NPS e PILO+QUET+NPS). Esses animais foram avaliados em diferentes aspectos comportamentais (campo aberto, teste de inibição pré- pulso, memória de reconhecimento de objeto e memória de trabalho), e filmados ao longo de 71 dias para o monitoramento de crises epilépticas espontâneas recorrentes. Também foram avaliadas a perda neuronal e a expressão de nNOS em diferentes regiões cerebrais. Resultados: NPS reduziu a frequência de crises nos animais com epilepsia e psicose comórbida quando comparados a animais não tratados. NPS também teve efeito ansiolítico no modelo animal de epilepsia com e sem psicose, e diminuiu os correlatos comportamentais de sintomas positivos dos animais no modelo de psicose e no de epilepsia + psicose, além de reverter o prejuízo no filtro sensório motor nos animais que receberam pilocarpina e quetamina. No reconhecimento de objeto o modelo de epilepsia e epilepsia+psicose o tratamento com NPS não interferiu no desempenho de memória de curto prazo, porém NPS melhorou a memória de longo prazo no modelo de psicose. NPS preservou regiões como o córtex entorrinal e subiculum nos animais epilépticos, mas a camada granular e córtex pré-límbico revelaram perda significativa nos animais controles. Alta expressão de nNOS em diferentes regiões cerebrais foram visualizadas nos animais com epilepsia, com destaque para aqueles que receberam tratamento com NPS. Conclusão: O tratamento com NPS foi efetivo na amenização de sintomas correlatos comportamentais de psicose no modelo puro de esquizofrenia e no comórbido VIII com epilepsia. Além disso, não exacerbou crises e contribuiu para diminuição delas nos animais do modelo de epilepsia e psicose, que apresentou grandes similaridades com o que é encontrado em casos clínicos. Nossos resultados sugerem que o uso do NPS pode ter resultados na melhoria dos sintomas da psicose interictal humana, assim como já observado para a esquizofrenia. / Introdution: Schizophrenia is a major mental disorder that affects 1% of young adults and has a great impact on quality of life, behavior, and cognition. Despite the high social burden, little is known about the pathophysiology and etiology of schizophrenia. Patients with psychotic disorders have increased risk of developing epilepsy, and patients with temporal lobe epilepsy have a high frequency of psychoses. The use of experimental animal models of epilepsy and schizophrenia may help to better understand the neurobiology of these diseases and play an important role in the development of potential new therapeutic strategies. The complex interactions of seizures and psychotic symptoms are being unveiled. Nitric oxide (NO) is an important modulator of neurotransmission and NO donors such as sodium nitroprusside (SNP) have shown promising effects in schizophrenic patients. In animal models of epilepsy, NO may exert pro- or anticonvulsant effects, suggesting that the use of SNP in patients with epilepsy and psychosis may not be so straightforward. Objectives: To develop an animal model of epilepsy and comorbid psychosis, and compare it from the behavioral and neuropathological point of view with pure animal models of epilepsy and schizophrenia. It was hoped, to verify the possible modulation triggered by the treatment with SNP. Metodology: Males Wistar rats weighing 260-300g were divided in controls group (SAL+SAL), pure epilepsy (PILO+SAL), schizophrenia (SAL+QUET), comorbid psychosis (PILO+KET) and their versions treated with SNP (SAL+SAL+SNP, PILO+SAL+SNP, SAL+KET+SNP and PILO+KET+SNP). These animals were evaluated in a variety of behavioral tests (open field, prepulse inhibition startle reflex, object recognition and working memory), and were filmed over 71 days to monitor spontaneous recurrent seizures. We also evaluated neuronal loss and nNOS expression in different brain regions. Results: SNP reduced seizure frequency in animals with epilepsy and psychosis when compared to those not treated with SNP. SNP also showed anxiolytic effects in the animal model of epilepsy with or without psychosis, decreased behavioral correlates of positive symptoms in the animal model of epilepsy + psychosis, and prevented sensorimotor gating deficits in epilepsy + psychosis IX model, The object recognition test showed that in epilepsy and epilepsy + psychosis models the treatment with SNP did not interfere with short-term memory performance, but SNP improved long-term memory in the psychosis model. SNP preserved brain regions like entorhinal cortex and subiculum in epileptic animals, but the granular layer and prelimbic cortex revealed significant loss in controls animals. High expression of NOS in same brain regions was observed in the animal model of epilepsy, especially in the animals receiving SNP. Conclusion: Treatment with SNP was effective in ameliorating symptoms of behavioral correlates of psychosis in the pure model of schizophrenia and the model comorbid with epilepsy. Moreover, SNP did not exacerbate seizures and reduced seizure frequency in the animal model of epilepsy + psychosis, which showed striking similarities to clinical cases. Our results suggest that the use of SNP could ameliorate symptoms of human interictal psychosis, such as those observed in schizophrenia.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Epilepsy is a disease that affects about 1-2% of the general population. Considering the high number of patients with epilepsy who are refractory to currently available drugs, it is important to search for new anticonvulsant drugs. For this it is important that reproduces model epilepsy, such as the pilocarpine model, a muscarinic agonist causing limbic seizures and status epilepticus, which after a latency period is characterized by a temporal lobe epilepsy. A potential drug target in the treatment of epilepsy is the Na+,K+-ATPase, which is characterized by being a plasma membrane protein having important role in the maintenance of cellular ionic homeostasis, changes in the Na+,K+-ATPase activity directly affect cell signaling via neurotransmitters and neuronal activity. In this context, a impair the functioning of the Na+,K+-ATPase leads to an increased or decreased neuronal excitability, depending on the degree of inhibition induced neuronal and type affected. Therefore, the present study searched for the role of Na+,K+-ATPase and the specific antibody that enhances the activity of Na+,K+-ATPase (DRRSAb) in the pilocarpine model in C57BL/6 mice challenged with pentylenetetrazol (PTZ). It was seen that the activity of Na+,K+-ATPase was decreased in hippocampus of epileptic mice, 60 days after status epilepticus (SE). Furthermore, the Michelis-Menten constant for different ATP concentrations increased in the SE. Reduced activity of Na+,K+-ATPase appears to involve the nitration of α subunit, but no changes in the expression or its phosphorylation state at Ser943 was found. Interestingly, activation of Na+,K+-ATPase intrahippocampal injection with a specific antibody (DRRSAb) produced against a regulatory site of the α subunit, decreases susceptibility to myoclonic seizures induced by PTZ in epileptic animals. On the other hand, the administration of DRRSAb in the hippocampus of naive animals facilitated the onset of seizures induced by PTZ. Quantitative analysis of hippocampal EEG recordings revealed that DRRSAb increased the percentage of total power contributed by delta frequency band (0-3 Hz) to large irregular amplitude pattern of hippocampal EEG. On the other hand, no DRRSAb-induced changes were found regarding the theta functional state. Therefore, activation of Na+,K+-ATPase activity as a novel approach in seizure disorders, may become a potential target for epilepsy. / A epilepsia é uma doença que atinge cerca de 1 % da população em geral. Embora vários tratamentos farmacológicos sejam utilizados, um elevado número de pacientes com epilepsia são refratários às drogas disponíveis atualmente o que torna importante à busca por novas drogas anticonvulsivantes. Para isso, é necessário um modelo que reproduza a epilepsia, como é o caso do modelo da pilocarpina, um agonista muscarínico que causa convulsões límbicas e status epilepticus (SE), que após crises recorrentes se caracteriza por uma epilepsia do lobo temporal. Um possível alvo farmacológico na terapia da epilepsia é a enzima Na+, K+-ATPase, que se caracteriza por ser uma proteína de membrana plasmática que tem um papel importante na manutenção da homeostase iônica celular cuja mudança na atividade da Na+, K+-ATPase afeta diretamente a sinalização celular via neurotransmissores e a atividade neuronal. Neste contexto, um prejuízo ao funcionamento da Na+, K+-ATPase ocasiona aumento ou diminuição da excitabilidade neuronal, dependendo do grau de inibição induzido e do tipo neuronal afetado. Portanto, o presente estudo, procurou o papel da Na+, K+-ATPase e de um anticorpo específico que aumenta a atividade da Na+, K+-ATPase (DRRSAb), no modelo da pilocarpina em camundongos C57BL/6 e sobre a susceptibilidade ao pentilenotetrazol (PTZ). Foi constatado que a atividade da Na+, K+-ATPase está diminuída no hipocampo de camundongos 60 dias após o SE. Além disso, a constante de Michelis-Menten para as diferentes concentrações de ATP aumentou no grupo pós-SE. A redução da atividade da Na+,K+-ATPase parece envolver a nitração da subunidade α, mas nenhuma alteração na expressão ou no estado de fosforilação na Ser943 foi encontrada. Interessantemente, a ativação da Na+, K+-ATPase, com uma injeção intrahipocampal do anticorpo DRRSAb produzido contra um local regulador da subunidade α, diminui a susceptibilidade para crises mioclônicas induzida por PTZ nos animais epilépticos. Por outro lado, a administração de DRRSAb no hipocampo de animais normais facilitou o aparecimento de convulsões induzidas por PTZ. A análise quantitativa do registro eletroencefalográfico (EEG) no hipocampo, revelou que o DRRSAb aumentou a porcentagem de poder total na frequência da banda delta (0-3 Hz), quando analisado padrão de atividade irregular de grande amplitude (LIA). Por outro lado, não houve alterações induzidas pelo DRRSAb sobre o estado funcional do ritmo teta. Portanto, a ativação Na+, K+-ATPase, como uma nova abordagem em distúrbios convulsivos, pode tornar-se um alvo farmacológico em potencial na epilepsia.

Background: Cystic fibrosis (CF) is a hereditary disorder caused by genetic mutations, which affect the chloride ion channels, leading to disrupted salt balance in different organs. A lack of properly functioning chloride ion channels can lead to formation of thick mucus, which hinders organ function, especially in the lungs where repeated inflammation occurs. Early diagnosis is critical to prevent further deterioration of the patient's condition. Current method of analysis of CF diagnostics uses conductivity meters to measure sweat electrolytes. However, current guidelines suggest using a chloridometer to directly measure chloride concentration, is the most reliable marker of cystic fibrosis. The aim of this project was to conduct a comprehensive evaluation of the new instrument's safety, reliability, validity, and conformity of the reference range to international chloride meter guidelines. Additional aims were to investigate the effect of storage conditions on sweat chloride concentration levels and examine the effect of increased salt intake on sweat test results. The study recruited healthy participants and took samples of their sweat by inducing sweat gland secretion. The chloride ion concentration was determined using a coulometric method. The results of the study found that the new method was reliable and matched international protocols. It also revealed that an increased salt consumption can impact chloride concentration in sweat, but not to an extent that it can affect medical decisions. Additionally, the study demonstrated that sweat samples can be frozen for up to two weeks without affecting the outcome of the chloride determination.

Introdução: A epilepsia do lobo temporal mesial (ELTM) é a forma mais comum de epilepsia na população adulta, tendo a esclerose hipocampal como principal substrato neuropatológico. Uma elevada proporção dos pacientes com ELTM apresentam história familiar positiva para a epilepsia, sugerindo a participação de fatores genéticos nesta síndrome. O citoesqueleto neuronal é essencial para os processos fisiológicos das células nervosas e crises epilépticas parecem afetar esta estrutura. O citoesqueleto neuronal possui importantes componentes regulatórios, dentre eles as proteínas associadas aos microtúbulos do tipo 2 (MAP2) e tau. Objetivos: Avaliar a densidade neuronal (células imunomarcadas com NeuN), neobrotamento das fibras musgosas (por histoquímica de neo-Timm) e a expressão imunohistoquímica das proteínas MAP2 e tau no hipocampo de casos cirúrgicos de ELTM (subtipos familiar e esporádico) e controles. As mesmas análises foram conduzidas no modelo animal de lítiopilocarpina. Métodos: Casos ELTM (n=38) foram divididos em ELTM familiar (n=20) e ETLM esporádica (n=18). Hipocampos controles (n=10) foram provenientes de necrópsias de sujeitos sem histórico de problemas neurológicos. No modelo animal de lítio-pilocarpina, animais submetidos ao status epilepticus (SE) foram sacrificados nos seguintes tempos pós-SE: 1, 7, 15 e 60 dias e classificados como grupos SE1, SE7, SE15 e SE60. Animais controle foram injetados com salina. Resultados: O grupo ELTM apresentou menor densidade neuronal que o controle nas regiões da camada granular, hilo, CA4, CA3, CA1 e prosubículo. O grupo ELTM mostrou maior valor de cinza da histoquímica de neo-Timm na camada granular, molecular interna e molecular externa e maior espessura do neobrotamento axonal na camada molecular interna em relação ao grupo controle. O grupo ELTM esporádica teve maior valor de cinza da histoquímica de neo-Timm e tendência à maior espessura do neobrotamento que o grupo ELTM familiar na camada molecular interna. O grupo ELTM apresentou menor expressão de MAP2 que o grupo controle no hilo, CA4, CA3, CA1 e prosubículo e maior expressão que o controle na camada granular, CA2 e parasubículo. O grupo ELTM esporádica teve maior expressão de MAP2 que o grupo ELTM familiar em CA1 e córtex entorrinal. O grupo ELTM apresentou maior expressão de tau que o grupo controle nas regiões da camada granular, hilo, CA3, CA2 e córtex entorrinal. O grupo ELTM esporádica demonstrou menor imunorreatividade para tau que o grupo ELTM familiar no córtex entorrinal. No modelo de lítio-pilocarpina, os grupos submetidos ao SE apresentaram menor densidade neuronal e maior neobrotamento que o grupo salina. Quanto à expressão de MAP2, os grupos SE60 e SE15 apresentaram maiores valores de expressão que os demais grupos em todas as regiões analisadas. A expressão de tau em CA3 não foi diferente entre os grupos animais analisados. Conclusão: Nossos resultados existem perda neuronal, reorganização sináptica das fibras musgosas e alterações na expressão de proteínas MAP2 e tau no hipocampo de pacientes com ELTM. Dentre os casos ELTM, encontramos diferenças no neobrotamento de fibras musgosas e na expressão de MAP2 e tau entre os grupos ELTM familiar e esporádica, indicando que estes grupos se apresentam de forma distinta em relação à epilepsia. Encontramos resultados II semelhantes em relação à densidade neuronal e o neobrotamento de fibras musgosas entre pacientes com ELTM e no modelo experimental. Porém, em relação à expressão das proteínas MAP2 e tau, encontramos algumas diferenças entre humanos e animais com ELTM. Nossos resultados sugerem que apesar do modelo de epilepsia induzida por lítio-pilocarpina ser relevante para o estudo da ELTM humana e mimetizar importantes aspectos neuropatológicos, a correspondência quanto à expressão de algumas proteínas estruturais não é completa. Estudos adicionais de expressão de proteínas do citoesqueleto em outros modelos animais de ELTM serão de grande importância para o melhor entendimento do processo epileptogênco. / Introduction: Mesial temporal lobe epilepsy (MTLE) is the most common form of epilepsy in adults and has hippocampal sclerosis as the main pathological substrate. A high proportion of patients with MTLE have positive familial history for epilepsy, suggesting the involvement of genetic factors in this syndrome. Seizures may affect the neuronal cytoskeleton, an structure that is essential in the physiological processes of nerve cells. Components of the neuronal cytoskeleton include microtubule-associated protein type 2 (MAP2) and tau. Objectives: Our aim was to evaluate neuronal density, mossy fiber sprouting and immunohistochemical expression of MAP2 and tau in the hippocampus of surgical cases of MTLE (familial and sporadic subtypes) and controls. The same analysis were conducted in the MTLE lithiumpilocarpine animal model.Methods: MTLE cases (n = 38) were divided into familial MTLE (n = 20) and sporadic MTLE (n = 18). Control hippocampi (n = 10) were obtained from autopsies of subjects without history of epilepsy. In the lithium-pilocarpine animal model, male Wistar rats were submitted to status epilepticus (SE) and were killed at the following post-SE days: 1, 7, 15 and 60, and were further classified SE1, SE7, SE15 and SE60. Control animals were injected with saline. Results: MTLE showed decreased neuronal density than controls in the granular layer, hilus, CA4, CA3, CA1 and prosubiculum. MTLE group showed increased neo-Timm gray value in the granular layer, inner and outer molecular layer, as well as increased mossy fiber length of mossy fiber in the inner molecular layer when compared to controls. Sporadic MTLE specimens exhibited increased inner molecular layer gray value than familial MTLE. MTLE hihppocampi showed decreased MAP2 expression in the hilus, CA4, CA3, CA1 and prosubiculum. In the granular layer, CA2 and parasubiculum, MAP2 expression was higher in MTLE specimens than in controls. CA1 and entorhinal cortex from sporadic MTLE hippocampi showed increased MAP2 expression than familial MTLE. Tau expression was increased in the granular layer, hilus, CA3, CA2 and entorhinal cortex of MTLE specimens. Sporadic MTLE exhibited lower tau immunoreactivity in the entorhinal cortex than familial MTLE. In the lithiumpilocarpine animal model, rats submitted to SE presented lower values of neuronal density and mossy fiber sprouting than controls. SE15 and SE60 showed increased MAP2 expression in all hippocampal subfields. Tau expression in CA3 was not different among the groups. Conclusion: Our results indicate that besides neuronal loss and axonal sprouting, MTLE specimens also exhibit dendritic abnormalities related to MAP2 expression. Increased tau immunoreactivity in epileptogenic hippocampi indicates possible abnormal expression related to mossy fiber sprouting in chronic MTLE. We found differential sprouting, MAP2 and tau expression between sporadic and familial MTLE. It is possible that different genetic background might result in somehow distinct neuropathological substrates between the two MTLE subtypes, although their clinical manifestation is quite similar. While neuronal loss and axonal sprouting profiles in human MTLE and chronic phase of the lithium-pilocarpine animal model are comparable, we could not find corresponding results regarding MAP2 and tau expression. IV Several studies have shown that chemo-convulsant as pilocarpine and kainic acid result in widespread brain epileptic discharges, which are different from the more focal hippocampal discharges seen in human MTLE. Despite its limitations, the lithium-pilocarpine model stands as an important and widely used animal model of epilepsy. Besides that, other animal models in which ictal discharges and lesions are more limited to the hippocampal formation might better mimic what we see in human MTLE.

Da die Arzneistoffpermeation durch okulare Gewebe einen entscheidenden Einfluss auf die Heilung vieler Augenleiden hat, wurde die in vitro-Permeation hydrophiler und lipophiler Arzneistoffe durch okulare Gewebe und Zellkulturen in dieser Arbeit untersucht. Die Dissertation befasst sich vorrangig mit der Permeation des hydrophilen Modellarzneistoffs Pilocarpinhydrochlorid (P-HCl) durch isolierte Schweinecornea (SC), Schweinesklera, Kaninchenkonjunktiva und corneale bzw. konjunktivale Kaninchenepithelzellkultur. Der Einfluss verschiedener Formulierungsparameter wie Benzalkoniumchlorid (BAC), Natriumedetat, pH-Wert und Tonizität auf die P-HCl-Permeation wurde untersucht. Dabei konnte eine gute Korrelation zwischen isolierten Geweben und Zellkulturen in Reaktion auf die Variation der Formulierungsparameter festgestellt werden. Unter den getesteten Parametern zeigte BAC den größten Enhancereffekt. Weiterhin wurden vergleichende Permeationsstudien an gelaserter SC mit P-HCl und dem relativ lipophilen Diclofenac-Natrium (D-Na) durchgeführt. Das Entfernen von Epithelschichten der SC mittels Excimer-Laser sollte den Heilungsverlauf, vor allem aber die zeitabhängige Reepithelisierung der Cornea nach erfolgter Photorefraktiver Keratektomie, simulieren. Die Ergebnisse dieser Studie zeigten, dass unterschiedliche Epitheldicken einen signifikanten Einfluss auf die P-HCl-Permeation haben. Im Gegensatz dazu blieb die D-Na-Permeation nahezu unbeeinflusst. Ein weiteres Anliegen dieser Arbeit bestand darin, eine okular applizierbare Formulierung des Immunsuppressivums Mycophenolatmofetil (MMF) zu entwickeln. Sowohl für das Prodrug MMF als auch für dessen aktiven Metaboliten Mycophenolsäure (MPA) wurde die Permeabilität von SC getestet. Ausgewählt wurde eine Zubereitung, die 1% MMF in Glutathion-Bicarbonat-Ringerlösung enthält und mit 10% Hydroxypropyl-beta-Cyclodextrin versetzt ist. Diese Suspension wurde bei 121° C und 200 kPa für 15 min autoklaviert, um das schwerlösliche MMF in Lösung zu bringen. Da der Ester MMF bei der Herstellung der Testlösung einer teilweisen Hydrolyse zu MPA unterliegt, außerdem eine minimale in vitro-Verfügbarkeit aufweist und sehr schwer wasserlöslich ist, sollte MPA in Augentropfenformulierungen der Vorzug gegeben werden. Die Korrelierbarkeit mit in vivo-Resultaten ist jedoch im Rahmen dieser Arbeit nicht untersucht worden, so dass die Ergebnisse als Grundlage für Permeationsstudien am in vivo-Modell zu bewerten sind. / The permeation of drugs through ocular tissues plays an important role in healing of various eye diseases. The objective of this work was to investigate the in vitro permeability of hydrophilic and lipophilic drugs through ocular tissues and cell cultures. Mainly, the permeability of the hydrophilic model drug pilocarpine hydrochloride (P-HCl) through isolated pig cornea (PCr) and sclera, rabbit conjunctiva, and rabbit conjunctival or corneal epithelial cell culture was compared. Additionally, the study included investigations about the influence of the formulation parameters benzalkonium chloride (BAC), ethylene diamine tetra acetic acid disodium salt, pH value and tonicity on the permeability of the small drug. In summary, a good correlation between the isolated tissues and cell cultures with regard to P-HCl transport could be observed. In general, BAC caused the most facilitated drug transport within the formulation parameters. Furthermore, the permeation of P-HCl and the lipophilic diclofenac-sodium (D-Na) through lasered PCr was studied. To investigate the effects of photorefractive keratectomy on drug permeation, excimer laser ablations with varying depths were performed on isolated pig eyes. As a result, P-HCl demonstrated a significant enhancement of permeation in relation to the ablation depth. In contrast, corneal epithelial thickness scarcely influenced the permeation rate of D-Na. Another aim of this work was to develop an appropriate application form for topical ocular use of the immunomodulating substance mycophenolate mofetil (MMF) and to investigate the in vitro permeability of PCr for the prodrug MMF and its parent substance mycophenolic acid (MPA). The test formulation consisted of Glutathion-bicarbonate-Ringer's solution, 10% hydroxypropyl- beta-cyclodextrin and 1% MMF. To reach a concentration of 1% of the poor soluble MMF a treatment under autoclaving conditions at 121° C and 200 kPa for 15 min was needed. MPA should be preferred in eye drops because of an higher in vitro availability compared to MMF, an hydrolysis of MMF to MPA in the cornea during permeation and the poor water solubility of MMF. The correlation with in vivo results was not studied in this work but the findings can be assumed as basis for investigations on in vivo models.

INTRODUÇÃO: As relações entre sono e epilepsia são complexas e de grande importância clínica. A melhor compreensão das inúmeras lacunas que permeiam essa relação reforçaria os alicerces para o desenvolvimento de abordagens terapêuticas mais eficazes que pudessem contribuir para o bem-estar do paciente portador de epilepsia e transtornos do sono. OBJETIVO: O presente estudo teve como principal objetivo o estudo comportamental e a caracterização eletrofisiológica do ciclo vigília-sono (CVS) de ratos adultos tornados epilépticos por pilocarpina. MÉTODO: Ratos Wistar machos (N=6), tornados epilépticos após status epilepticus (SE) induzido por pilocarpina e não epilépticos (N=6) foram submetidos à cirurgia extereotáxica para implante de elétrodos bipolares nas áreas corticais (A3, somatosensorial) e hipocampais (CA1) de ambos os hemisférios. Registros contínuos de 24 horas foram submetidos à minuciosa análise visual e os seguintes parâmetros foram analisados: identificação e quantificação dos padrões eletrofisiológicos das fases do ciclo CVS; duração dos episódios oníricos ocorridos durante o sono dessincronizado (SD); padrão de ocorrência do CVS assim como do ciclo de sono (CS), e análise do volume do núcleo supraquiasmático. Os estudos da distribuição do CVS e comportamento onírico foram submetidos à Análise de Variância Multivariada - MANOVA, ao passo que as análises da ocorrência dos ciclos (CVS e CS) e volume do núcleo supraquiasmático foram submetidas ao teste da Análise de Variância (ANOVA) de dois fatores e ao teste de Mann- Whitney, respectivamente. RESULTADOS: Todas as fases do CVS foram identificadas nos ratos epilépticos. As fases da vigília e do sono eram permeadas por espículas e outros grafoelementos epileptiformes, como ondas delta espiculadas no SS e potenciais de alta frequência e baixa voltagem durante VA e o SD. Ao contrário do padrão de ocorrência típico das fases de vigília e sono em ratos não epilépticos, o grupo epiléptico apresentou diferenças significativas quanto à distribuição dessas fases em função do período. Foi observada redução significativa de VA (p<0,002) com concomitante aumento de SS (p<0,005) e vigília relaxada (VR) (p=0,021) no escuro, sendo que a VR era preponderante apenas na primeira metade da noite. Durante o dia, a quantidade de SS era maior no período da manhã (p<0,001), ao passo que houve redução do SD (p=0,002) concomitante com aumento de VA (p<0,001) no período da tarde.Os animais tornados epilépticos por pilocarpina apresentaram redução no padrão de ocorrência do CVS e CS (p=0,004 e p=0,003, respectivamente). Não houve diferença estatística na duração dos episódios oníricos, assim como no volume do núcleo supraquiasmático entre os grupos analisados (p>0,63 e p=0,47, respectivamente). CONCLUSÃO: Os animais epilépticos apresentaram alterações na arquitetura do CVS, bem como nos padrões de ciclicidade evidenciado pelas alterações de comportamento, especialmente no ciclo escuro. Esses fatos sugerem possível comprometimento estrutural e/ou funcional das circuitarias responsáveis pela geração e manutenção das fases de vigília e sono, assim como dos sistemas de temporização do CVS. Tomados em conjunto, os dados reproduziram anormalidades do CVS observadas em pacientes epilépticos, sugerindo que o presente modelo pode ser uma importante ferramenta para o estudo de mecanismos subjacentes à epilepsia do lobo temporal e sono. / INTRODUCTION: Relationships between sleep and epilepsy are complex and have great clinical importance as well. The full understanding of the various gaps present in this relationship would pave the ground for new studies that could generate new clinical approaches aiming to contribute to the well-being of the patient suffering from epilepsy and sleep disorders. OBJECTIVE: The present study aimed to carry out a behavioral analysis and electro-oscillographic characterization of the phases of sleep-wake cycle (SWC) of pilocarpine- induced epilepsy in adult rats. METHODS: Male Wistar rats that became epileptic after 60 days of pilocarpine-induced status epilepticus (SE) (N=6) and non epileptic ones (N=6) were submitted to extereotaxic surgery for implantation of bipolar electrodes in cortical (A3, somestesic) and hippocampal (CA1) areas in both hemispheres. Twenty-four hour continuous registers were submitted to detailed visual analysis and the following parameters were studied: identification and quantification of electrophysiological parameters of phases of SWC, duration of oniric episodes during desynchronized sleep (DS), the pattern of occurrence of SWC and cycles of sleep (CS). In addition, the volume of suprachiasmatic nuclei was investigated. To analyze the architecture of sleep-wake phases and oniric behavior, Multivariate Analysis of Variance-MANOVA was utilized, whereas the pattern of cycles (SWC and CS) and volume of suprachiasmatic were submitted to Analysis of Variance with 2 factors-Two-way ANOVA and Mann-Whitney test, respectively. RESULTS: In the epileptic rats all phases of SWC were identified. The phases of wake and sleep were permeated by spikes and graph elements epileptiforms such as spiked delta waves in SS and low frequency waves with high voltage during AW and SD phases. In contrast to the pattern of normal rhythmic activity evident in non-epileptic rats the epileptic group presented significant differences concerning distribution of the phases of SWC according to the period. In the dark cycle significant reduction of AW (p<0.002) was observed concomitantly with an increase of SS (p<0.005), while the relaxed wakefulness (RW) showed an increase during the first half of the night (p=0.021). In the light cycle, the SS was more prominent in the morning period (p<0.001), following by a reduction of DS (p=0.002) concomitantly with an increase of AW (p<0.001) during the afternoon in the epileptic group. The number of cycles with a regular sequence of each phase from awake to sleep (SWC) was significantly decreased (p=0.004), as was the number of cycles of sleep (p=0.003) in epileptic rats. No significant differences were found in duration of oniric episodes and volume of suprachiasmastic nuclei (p>0.63 e p=0.47, respectively) between non epileptic and epileptic groups. CONCLUSION: The data obtained revealed that after SE the epileptic animals presented some alterations in the SWC architecture as well as in the cyclicity patterns mainly in dark cycle. Such facts suggest a possible functional and/or structural impairment in the circuitry responsible for the generation of sleep and wake phases and in the SWC timing system. Taken together the data reproduced the abnormalities observed in patients, suggesting that the pilocarpine model is a suitable one to study sleep dysfunctions in temporal lobe epilepsy.