Esse projeto de pesquisa teve como objetivo estudar simultaneamente as concentrações da ocitocina no líquido cefalorraquidiano (LCR) e no plasma de ovelhas multíparas durante a ordenha, estabelecendo suas possíveis correlações entre os dois compartimentos corporais, bem como em relação à produção e ejeção do leite. Para tanto, foram utilizadas 10 ovelhas multíparas (Ovis aries) da raça Santa Inês apresentado peso médio de 40 Kg. Os animais foram divididos em quatro grupos, sendo eles: ordenha mecânica exclusiva (OME; cordeiros apartados 3 dias após o parto), manejo misto com ordenha mecânica (MMom; cordeiros permanecem com as mães durante o período diurno, após a ordenha mecânica, sendo apartados durante a noite), manejo misto com ordenha manual (MMoma) e amamentação exclusiva (AE; cordeiros apartados durante o período noturno). Para a obtenção de LCR foram realizadas a punção do espaço subaracnóideo e implantação de um cateter epidural, enquanto que o plasma foi colhido com o auxílio de um scalp. As amostras de LCR e sangue foram coletadas simultaneamente antes (-0,5 min), durante (0,5; 1 e 4 min) e após (10; 15 min) a amamentação ou ordenha para posterior quantificação das concentrações de ocitocina por ensaio imunoenzimático. Foi obtido um total de 503 amostras, sendo 241 de LCR e 262 de plasma. As estimativas de média, desvio padrão, coeficiente de variação, mínimo e máximo para as concentrações de ocitocina no LCR e plasmáticas foram 257,880 ± 265,90 pg/ml; 103,11%; 11,70 pg/ml e 1000,00 pg/ml, respectivamente. A análise estatística não revelou correlação significante entre LCR e plasma para os quatros grupos experimentais avaliados. O coeficiente de correlação para os grupos OME, AE, MMom e MMoma foram, respectivamente: -0,26, -0,19, 0,05 e 0,04. Com relação ao LCR, não ocorreu diferença significante entre os 4 grupos experimentais em relação às concentrações de ocitocina. Já para o plasma, os animais do grupo MMom (679,80 ± 25,63) e MMoma (591,82 ± 30,56) apresentaram maiores concentrações plasmáticas médias de ocitocina em relação a OME e AE. Assim também, o grupo OME (381,04 ± 22,09) apresentou maior concentração média de ocitocina em relação ao grupo AE (218,82 ± 27,04). Conclui-se que, não existe correlação positiva entre as concentrações de ocitocina central e na circulação periférica durante a ordenha ou amamentação. Os padrões de liberação de ocitocina plasmática diferem de acordo com o tipo de manejo ao qual o animal é submetido, o que pode ter conseqüências para a ejeção do leite e conseqüentemente, para a produção. Finalmente, as concentrações de ocitocina presentes no LCR não sofrem influência do tipo de manejo de ordenha ao qual o animal foi submetido, ao contrário daquilo que foi observado para o plasma. / The aim of the present work was to study a possible relationship between central and peripheral oxytocin release and its consequences to milk production during milking in experimental ewes. Ten multiparous Santa Ines ewes (Ovis aries) were divided in 4 groups according to milk ejection stimuli: exclusive machine milking (OME), mixedmanagement of milking and suckling (MMom: lambs separated during night and reunited to their mother after morning milking; MMoma: mixed-management with manual milking) and exclusive suckling (AE: lambs separated also during night). Cerebrospinal fluid (CSF) was collected through a implanted sub-arachnoid catheter and plasma was collected from the jugular vein. imultaneous sampling was performed at -0.5, 0.5, 1, 4, 10 and 15 min (0 min was teat attachment to either machine or manual milking system or lamb suckling). A total of 241 samples of CSF and 262 plasma samples were processed and oxytocin concentrations were quantified by immunoenzimatic assays. Estimated means, standard deviation, variation coefficient and minimum and maximun values of CSF and plasma oxytocin concentrations were, respectively: 257.880 ± 265.90 pg/ml; 103.11%; 11.70 pg/ml e 1000.00 pg/ml. No statistical strong positive correlations (OME= -0.26, AE= -0.19, MMom= 0.05 and MMoma= 0.04) were found between CSF and plasma samples. Also, CSF was not influenced by milk ejection stimuli, although plasmatic oxytocin was higher in MMom (679.80 ± 25.63) and MMoma (591.82 ± 30.56) compared to OME and AE. In addition, OME (381.04 ± 22.09) plasmatic oxytocin concentration was higher when compared to AE (218.82 ± 27.04). In conclusion, no positive correlations between central and peripheral oxytocin concentrations were observed during milking or suckling. Plasma oxytocin oncentrations differ as a function of management and have consequences to milk ejection as well as to milk production Also, plasma, but not CSF oxytocin, was influenced by different milk ejection stimuli.

No presente trabalho, foi realizada uma MULTIPLEX-PCR para a detecção do DNA bacteriano de S. agalactiae, S.pneumoniae, N. meningitidis, H. influenzae e outros possíveis agentes etiológicos bacterianos das meningites. Este ensaio combina cinco diferentes iniciadores que detectam simultaneamente o gene crtA de N. meningitidis, o gene p6 de H. influenzae, o gene fbsA de S. agalactiae, o gene lytA de S. pneumoniae e o gene universal 16S rDNA para identificar a presença de agente bacteriano. Foram analisadas 447 amostras de LCR, o ensaio detectou 27 amostras positivas para cultura bacteriana e 13 amostras com resultado de cultura negativa. Estas amostras com cultura negativa apresentavam alterações bioquímicas, hematológicas, imunológicas ou microbiológicas (bacterioscopia) sugestivas de meningite, estes dados auxiliaram na análise dos resultados do MULTIPLEX-PCR. Este ensaio não apresentou reações inespecíficas com fungos, vírus e com outros agentes bacterianos testados (amplificando somente o gene 16S rDNA). A MULTIPLEX-PCR é um ensaio rápido, confiável, de fácil execução e facilmente implementável para a confirmação de meningite bacteriana. E este método pode auxiliar no diagnóstico de meningite com cultura de LCR negativa, particularmente para pacientes que previamente iniciaram antibioticoterapia e no diagnostico diferencial de meningite bacteriana ou viral. / In this work, a MULTIPLEX-PCR has conducted for the bacterial DNA detection of S. agalactiae, S.pneumoniae, N. meningitidis, H. influenza and other possible etiologic bacterial meningitis agents. This test combines five different primers that detect simultaneously the crtA gene of N. meningitides, the p6 gene of H. influenza, the fbsA gene of S. agalactiae, the lytA gene of S. pneumoniae and the 16S rDNA universal gene to identify the presence of bacterial agent. From the 447 samples of CSF that were analyzed, the test detected 27 positive samples for bacterial culture and 13 samples with the result of negative culture. These negative culture samples presented biochemical changes, hematological, immunological or microbiological (bacterioscopy) suggestive of meningitis, these data helped in the analysis of the MULTIPLEX-PCR results. This test showed no nonspecific reactions with fungi, viruses and other bacterial agents tested (only amplifying the gene 16S rDNA). The MULTIPLEX-PCR test is a fast, reliable, easy to implement and easily implementable for of bacterial meningitis confirmation. And this method can aid in the meningitis diagnosis with negative culture of CSF, particularly for patients who previously started antibiotic therapy and in the differential diagnosis of bacterial or viral meningitis.

The biological basis of the clinical heterogeneity in Parkinson's Disease (PD) is unclear. It is likely to involve complex interactions between genetic and environmental factors and between a range of pathological processes, including protein homeostasis and immune system function. Microglial activation in the brain and peripheral innate immune changes are known to occur in PD. Recently genetic, animal and cellular studies have linked several innate immune related genes and proteins (e.g. HLADR, TREM2, TLR2, TLR4, caspase-1) to PD and provided evidence that they may have a role in PD pathogenesis. Alpha-synuclein is central to PD, with evidence from neuropathology, genetics and animal/cell models indicating that it plays a significant pathogenic role. There is developing evidence directly linking innate immune activity and alpha-synuclein pathology. For example, inflammation, particularly in response to microbial infection, is associated with increased alpha-synuclein accumulation in the periphery and activation of the innate immune inflammasome related caspase-1 leads to increased cleavage and aggregation of alpha-synuclein. Overall Hypothesis- "Parkinson's disease (PD) and its clinical heterogeneity are associated with systemic changes in innate immune and associated microbial factors and in alpha-synuclein". This was investigated from the perspective of an epidemiological study, a study of peripheral blood monocyte, innate immune/microbial markers and a cerebrospinal fluid (CSF) study in PD patients. *The epidemiological study, involved the longitudinal PICNICS cohort of 290 Idiopathic PD patients, and showed that the use of medication known to influence alpha-synuclein and immune function is associated with motor heterogeneity in PD. *The peripheral immune study involved 41 early PD patients and 41 age, gender and MAPT genotype matched paired controls, with the PD patients categorised into 2 groups based on the presence of previously identified clinical and genetic risk factors for the development of an early dementia (impaired semantic fluency, pentagon copying and MAPT H1/H1 haplotype). This study demonstrated that the phenotypic profile of peripheral monocytes and the level of serum alpha-synuclein and relevant innate immune and microbial markers do differ in early PD compared to controls and that there are differential changes in those patients at higher versus lower risk for early dementia. The systemic alpha-synuclein related changes appear to be present overall in PD patients compared to controls, while the more microbial/innate immune related changes appear to be more prominent in the dementia higher risk group. *The CSF study involved samples from 35 PD patients and has demonstrated evidence of relationships between neurodegeneration-linked CSF tau species and inflammatory cytokines, and between CSF alpha-synuclein and cognitive function, suggesting that these factors may be involved in PD heterogeneity within the central nervous system as well. Overall, these studies provide evidence that variations in alpha synuclein/ tau homeostasis and innate immune and microbial factors are related to PD and its clinical heterogeneity.

The intracranial system consists of three main basic components - the brain, the blood and the cerebrospinal fluid. The physiological processes of each of these individual components are complex and they are closely related to each other. Understanding them is important to explain the mechanisms behind neurostructural disorders such as hydrocephalus. This research project consists of three interrelated studies, which examine the mechanical properties of the brain at the macroscopic level, the mechanics of the brain during hydrocephalus and the study of fluid hydrodynamics in both the normal and hydrocephalic ventricles. The first of these characterizes the porous properties of the brain tissues. Results from this study show that the elastic modulus of the white matter is approximately 350Pa. The permeability of the tissue is similar to what has been previously reported in the literature and is of the order of 10-12m4/Ns. Information presented here is useful for the computational modeling of hydrocephalus using finite element analysis. The second study consists of a three dimensional finite element brain model. The mechanical properties of the brain found from the previous studies were used in the construction of this model. Results from this study have implications for mechanics behind the neurological dysfunction as observed in the hydrocephalic patient. Stress fields in the tissues predicted by the model presented in this study closely match the distribution of histological damage, focused in the white matter. The last study models the cerebrospinal fluid hydrodynamics in both the normal and abnormal ventricular system. The models created in this study were used to understand the pressure in the ventricular compartments. In this study, the hydrodynamic changes that occur in the cerebral ventricular system due to restrictions of the fluid flow at different locations of the cerebral aqueduct were determined. Information presented in this study may be important in the design of more effective shunts. The pressure that is associated with the fluid flow in the ventricles is only of the order of a few Pascals. This suggests that large transmantle pressure gradient may not be present in hydrocephalus.

<p>Radio-frequency (RF) lesioning is a method utilising high frequency currents for thermal coagulation of pathological tissue or signal pathways. The current is delivered from an electrode with a temperature sensor, permitting control of the current at a desired target temperature. In the brain RF-lesioning can e.g. be used for severe chronic pain and movement disorders such as Parkinson’s disease. This thesis focuses on modelling and simulation with the aim of gaining better understanding and predictability of the lesioning process in deep brain structures. The finite element method (FEM) together with experimental comparisons was used to study the effects of electrode dimensions, electrode target temperature, electric and thermal conductivity of the brain tissue, blood perfusion and cerebrospinal fluid (CSF) filled cysts. Equations for steady current, thermal transport and incompressible flow were used together with statistical factorial design and regression analysis for this purpose.</p><p>Increased target temperature, electrode tip length and electrode diameter increased the simulated lesion size, which is in accordance with experimental results. The influence of blood perfusion, modelled as an increase in thermal conductivity in non-coagulated tissue, gave smaller simulated lesions with increasing blood perfusion as heat was more efficiently conducted from the rim of the lesion. If no consideration was taken to the coagulation the lesion became larger with increased thermal conductivity instead, as the increase in conducted heat was compensated for through an increased power output in order to maintain the target temperature. Simulated lesions corresponded well to experimental in-vivo lesions.</p><p>The electric conductivity in a homogeneous surrounding had little impact on lesion development. However this was not valid for a heterogeneous surrounding. CSF-filled cysts have a much higher electric conductivity than brain tissue focussing the current to them if the electrode tip is in contact with both. Heating of CSF can also cause considerable convective flow and as a result a very efficient heat transfer. This affected simulated as well as experimental lesion sizes and shapes resulting in both very large lesions if sufficient power compared to the cysts size was supplied and very small lesions if the power was low, mitigating the heat over a large volume.</p><p>In conclusion especially blood perfusion and CSF can greatly affect the lesioning process and appear to be important to consider when planning surgical procedures. Hopefully this thesis will help improve knowledge about and predictability of clinical lesioning.</p>

<p>In the demyelinating autoimmune disease multiple sclerosis (MS) there is a great need for validated prognostic biomarkers that can give information about both prognosis and disease course. So far only clinical parameters have been shown to predict future outcome. CXCL13 is a potent B cell chemoattractant that has been suggested to be a potential biomarker candidate. The aim of this study was to investigate the usefulness of CXCL13 as a prognostic biomarker for MS.</p><p>Clinical, paraclinical, laboratory and MRI data about a large group of MS patients and controls were collected. CXCL13 levels in cerebrospinal fluid (CSF) samples from these patients were determined by standard enzymelinked immunosorbent assay (ELISA).</p><p>In general CXCL13 were increased in CSF in MS, especially in relapsing-remitting MS during relapses, i.e. with ongoing inflammations in the central nervous system. CXCL13 is a good candidate prognostic marker for MS, since newly diagnosed MS with high CXCL13 levels showed worsened disease course within five years. Most importantly, MS conversion occurred in higher rate in possible MS patients with high concentrations of CXCL13 in CSF, and in a shorter time point. This observation may support an early treatment decision in these patients.</p><p>In conclusion, this study provides support for an association between CXCL13 levels in the CSF and later development of disease severity in MS.</p>

<p>The nasal administration of drugs offers advantages over administration by intravenous injection. Drugs can be rapidly absorbed through the nasal mucosa, resulting in a rapid onset of action, and also avoiding degradation in the gastrointestinal tract and first-pass metabolism in the liver. The olfactory receptor cells, which are in direct contact with both the environment and the central nervous system (CNS), are potential routes for drugs into the CNS. The olfactory pathway thus circumvents the blood brain barrier (BBB) which prevents many systemically administered drugs from entering the brain.</p><p>The studies used compounds active in the CNS and the experiments were performed in rodents. The nasal bioavailability of (S)-UH-301, NXX-066 and [³H]-dopamine was investigated in a rat model; uptake into the cerebrospinal fluid (CSF) was compared after nasal and intravenous administration. The concentrations of S-UH-301 and NXX-066 in plasma and CSF were measured with high performance liquid chromatography. The possible transfer of dopamine and neurotensin along the olfactory pathway after nasal administration to mice was studied using brain tissue sampling and autoradiography. The radioactivity content in blood, CSF and dissected brain tissue samples after administration of [³H]-dopamine and [³H]-neurotensin was assessed using liquid scintillation, and thin layer chromatography (TLC) was used to investigate the metabolic fate of [³H]-dopamine.</p><p>The results of this thesis suggest that nasal administration of CNS-active compounds with low oral bioavailability is an interesting and workable alternative to intravenous injection. The small lipophilic compounds (S)-UH-301 and NXX-066 were rapidly and completely absorbed after nasal administration, although hard evidence of direct transfer from the nose remains elusive. Radioactivity measurements in the olfactory bulb following nasal administration of</p><p>[³H]-dopamine and [³H]-neurotensin indicate that transfer occurred. The TLC results showed the presence of unchanged dopamine in the olfactory bulb but it is less clear from initial results with neurotensin, which radioactive products of this molecule reached the olfactory bulb, and further studies are required.</p>

The nasal administration of drugs offers advantages over administration by intravenous injection. Drugs can be rapidly absorbed through the nasal mucosa, resulting in a rapid onset of action, and also avoiding degradation in the gastrointestinal tract and first-pass metabolism in the liver. The olfactory receptor cells, which are in direct contact with both the environment and the central nervous system (CNS), are potential routes for drugs into the CNS. The olfactory pathway thus circumvents the blood brain barrier (BBB) which prevents many systemically administered drugs from entering the brain. The studies used compounds active in the CNS and the experiments were performed in rodents. The nasal bioavailability of (S)-UH-301, NXX-066 and [3H]-dopamine was investigated in a rat model; uptake into the cerebrospinal fluid (CSF) was compared after nasal and intravenous administration. The concentrations of S-UH-301 and NXX-066 in plasma and CSF were measured with high performance liquid chromatography. The possible transfer of dopamine and neurotensin along the olfactory pathway after nasal administration to mice was studied using brain tissue sampling and autoradiography. The radioactivity content in blood, CSF and dissected brain tissue samples after administration of [3H]-dopamine and [3H]-neurotensin was assessed using liquid scintillation, and thin layer chromatography (TLC) was used to investigate the metabolic fate of [3H]-dopamine. The results of this thesis suggest that nasal administration of CNS-active compounds with low oral bioavailability is an interesting and workable alternative to intravenous injection. The small lipophilic compounds (S)-UH-301 and NXX-066 were rapidly and completely absorbed after nasal administration, although hard evidence of direct transfer from the nose remains elusive. Radioactivity measurements in the olfactory bulb following nasal administration of [3H]-dopamine and [3H]-neurotensin indicate that transfer occurred. The TLC results showed the presence of unchanged dopamine in the olfactory bulb but it is less clear from initial results with neurotensin, which radioactive products of this molecule reached the olfactory bulb, and further studies are required.

In Europe, Lyme disease is caused by the species Borrelia (B.) burgdorferi sensu stricto, B. garinii and B. afzelii. The disease is the most common vector-borne infection in Europe and the United States, and the resulting manifestation can involve the skin, nervous system, heart and joints. The symptoms that arise are associated with the Borrelia species causing the infection. The species most associated with neuroborreliosis is B. garinii whilst B. burgdorferi sensu stricto is associated with arthritis and B. afzelii is associated with dermatological symptoms. Lyme disease normally has three phases in untreated patients. The first phase is characterised by erythema migrans, a reddening of the skin around the area of the tick bite. If the disease develops to the second phase the patient will suffer from neuroborreliosis which is characterised by neurological symptoms such as headache and peripheral facial paralysis. Cerebrospinal fluid (CSF) analysis is used to diagnose neuroborreliosis. The diagnosis is complicated by variations between the different Borrelia species and that many healthy individuals have antibodies directed against Borrelia. Antibodies in CSF can be found in different diseases. The antibodies can be produced in the central nervous system or come across the blood-brain barrier and thus derive originally from the blood. By measuring the concentration of total albumin in serum and in CSF it can be determined if the antibodies present in the CSF have been produced in the central nervous system or if they originate from the blood. The typical manifestation in the last phase of Lyme disease is severe arthritis. The aim of this examination project was to compare two ELISAs for detection of antibodies directed to Borrelia. Indirect ELISAs from DAKO and Euroimmun were compared for the diagnosis of neuroborreliosis in 100 individuals. Borrelia specific antibodies of class IgM or IgG were found in 16 of 100 patients by DAKO’s ELISA and in 20 of the same 100 patients by Euroimmun’s ELISA. The reason that Euroimmun’s method detected more cases of neuroborreliosis is probably that this method detects antibodies directed to all three pathological species of Borrelia while DAKO’s method only detects antibodies directed to B. burgdorferi. In conclusion, this study indicates that Euroimmun’s method to detect antibodies of class IgM and IgG directed to Borrelia is superior to DAKO’s method. The obtained results were confirmed by Western blot analysis which gave results in accordance with those of Euroimmun’s ELISA.

The symptoms in Idiopathic Adult Hydrocephalus Syndrome (IAHS) – gait disturbance, incontinence, and cognitive deficit – correlate anatomically to neuronal dysfunction in periventricular white matter. The pathophysiology is considered to include a cerebrospinal fluid (CSF) hydrodynamic disturbance, including pressure oscillations (“B waves”), in combination with cerebrovascular disease. IAHS and Subcortical Arteriosclerotic Encephalopathy (SAE) show clinical similarities, which constitutes a diagnostic problem. The aim of this thesis was to investigate biochemical markers in CSF, possibly related to the pathophysiology, and their usefulness in diagnosis, to investigate the effect of ICP changes on glucose supply and metabolism in periventricular deep white matter, and to present criteria for objective, computerised methods for evaluating the content of B waves in an intracranial pressure (ICP) registration. CSF samples from 62 IAHS patients, 26 SAE patients, and 23 controls were analysed for sulfatide, total-tau (T-tau) hyperphosphorylated tau (P-tau), neurofilament protein light (NFL), and beta-amyloid-42 (Aß42). In ten IAHS patients, recordings of ICP, brain tissue oxygen tension (PtiO2), and samplings of brain extracellular fluid from periventricular white matter by way of microdialysis were performed, at rest and during a CSF infusion and tap test. Microdialysis samples were analysed for glucose, lactate, pyruvate, glutamate, glycerol, and urea. Patterns before and after spinal tap were analysed and changes from increasing ICP during the infusion test were described. The long term ICP registration was used to evaluate two computerised methods according to optimal amplitude threshold, monitoring time, and correlation to the manual visual method. In CSF, NFL was elevated in both IAHS and SAE patients, reflecting the axonal damage. In a multinominal logistic regression model, the combined pattern of high NFL, low P-tau and low Aß42 in CSF was shown to be highly predictive in distinguishing between IAHS, SAE and controls. Analysis of microdialysis samples for glucose, lactate, and pyruvate showed, in combination with PtiO2, a pattern of low-grade ischemia. After the spinal tap of CSF, the pattern changed, indicating increased glucose metabolic rate. During the infusion test, there were prompt decreases in the microdialysis values of glucose, lactate and pyruvate during ICP increase, but no sign of hypoxia. The values normalised immediately when ICP was lowered, indicating that the infusion test is not causing damage. One of the computerised methods, with an amplitude threshold set to 1 mm Hg, was shown robust in evaluating B wave content in an ICP registration. At least 5 hours registration time was needed. The highly predictive pattern of biochemical markers in CSF indicates a possibility of identifying simple tests in diagnosing and selecting patients for surgical treatment. The results of microdialysis and PtiO2 indicate low-grade ischemia in the periventricular white matter, which is ameliorated from CSF removal, and that glucose supply and metabolism are sensitive to short-term ICP elevations, thus proposing a link between ICP oscillations and symptoms from neuronal disturbance. A computerised method for evaluation of B waves is a prerequisite for evaluating the impact of pressure oscillations in the pathophysiology of IAHS.