<p>Improving the functionality and reducing revision rates are important driving forces in the development of orthopaedic implants. FibMat is a fibrinogen based matrix developed towards commercialisation by the company Optovent AB. This matrix can be coated on implants and act as a local drug delivery system for bisphosphonates (BPs). BPs are drugs inhibiting bone resorption, and applied with FibMat to improve stability of implants in bone, e.g. when fixing bone fractures. In this thesis, FibMat loaded with BP (FibMat/BP) was coated on stainless-steel screws and titanium screws in order to investigate some technology properties relevant to its clinical applicability. Bone-mimicking materials were used to study scrape-off effect upon insertion. The coagulation properties of fibrinogen as well as the structural properties of BPs were studied after exposure to gamma radiation.</p><p>The screws were coated with FibMat and BP (alendronate and 14C-alendronate) using standard coupling techniques. The total amount and distribution of BP after insertion was measured by liquid scintillation and autoradiography. Coagulation assays were performed in order to determine the coagulation properties of fibrinogen, exposed to doses up to 35 kGy, mixed with thrombin. The structural properties of four different BPs (alendronate, pamidronate, zoledronate and ibandronate), exposed to doses up to 35 kGy were analysed by transmission infrared spectroscopy.</p><p>The results show that FibMat/BP coating on porous stainless-steel screws is virtually unaffected by insertion into bone materials. The anodised, planar titanium screws are more affected by the insertion process, but an even BP distribution in the cancellous material is indicated. The coagulation assays show that gamma-irradiated fibrinogen has a slower coagulation process compared to non-irradiated fibrinogen and form interrupted network unable to clot. The chemical structures of the BPs seem unaffected by exposure to gamma irradiation. In conclusion, the FibMat/BP is a promising technology for local distribution of BP in conjunction with bone implants.</p>

Epilepsy is a common neurological disease affecting 6 million people in Europe. Early prevention and accurate diagnosis and treatment are of importance to obtain seizure freedom. In this thesis new applications of carbon-11-labelled tracers in PET and autoradiographic studies were explored in focal epilepsy. Patients with low-grade gliomas often experience epileptic seizures. A retrospective PET-study assessing seizure activity, metabolic rate measured with 11C-methionine and other known prognostic factors was performed in patients with glioma. No correlation was found between seizure activity and uptake of methionine. The presence and termination of early seizures was a favourable prognostic factor. Activation of the neurokinin-1 (NK1) receptor by substance P (SP) induces epileptic activity. PET with the NK1 receptor antagonist GR205171 was performed in patients with temporal lobe epilepsy (TLE) and healthy controls. In TLE patients an increased NK1 receptor availability was found in both hemispheres, most pronounced in anterior cingulate gyrus ipsilateral to seizure onset. A positive correlation between NK1 receptors and seizure frequency was observed in ipsilateral medial structures consistent with an intrinsic network using the NK1-SP receptor system for transmission of seizure activity. The uptake of 18F-fluoro-deoxy-glucose (FDG) is related to cerebral blood flow (CBF). Previously, methods to estimate blood flow from dynamic PET data have been described. A retrospective study was conducted in 15 patients undergoing epilepsy surgery investigation, including PET with 11C-FDG and 11C-Flumazenil (FMZ). The dynamic FMZ dataset and pharmacokinetic modeling with a multilinear reference tissue model were used to determine images of relative CBF. Agreement between data of FDG and CBF was analyzed showing a close association between interictal brain metabolism and relative CBF. Epilepsy often occurs after traumatic brain injuries. Changes in glia and inhibitory neuronal cells contribute to the chain of events leading to seizures. Autoradiography with 11C-PK11195, 11C-L-deprenyl and 11C-Flumazenil in an animal model of posttraumatic epilepsy studied the temporal and spatial distribution of microglia, astrocytes and GABAergic neurons. Results showed an instant increase in microglial activity that subsequently normalized, a late formation of astrogliosis and an instant and prolonged decease in GABA binding. The model can be used to visualize pathophysiological events during the epileptogenesis.

Made available in DSpace on 2015-07-22T20:50:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-01-01 / No processo da sensibilização comportamental, que se desenvolve a algumas drogas psicoativas, participam diversos sistemas de neurotransmissão, entre eles o sistema colinérgico, que modula de maneira importante o funcionamento de vias dopaminérgicas. Neste estudo avaliamos os efeitos da escopolamina, um antagonista colinérgico muscarínico, no desenvolvimento e expressão da sensibilização ao efeito estimulante do etanol (Estudo I) e os níveis de ligação dos receptores colinérgicos muscarínicos M1 em animais classificados como apresentando Alta (AS) ou Baixa Sensibilização (BS) ao efeito estimulante do etanol (Estudo II). No Estudo I, quatro grupos camundongos suíços albinos machos receberam durante 21 dias, respectivamente: salina+salina (sal/sal); 1,0 mg/kg de escopolamina+salina (escop/sal); salina+2,2 g/kg de etanol (sal/2,2EtOH) ou 1,0 mg/kg escopolamina+2,2 g/kg de etanol (escop/2,2EtOH). A atividade locomotora dos animais foi registrada por 20 minutos no 1°, 7°, 14° e 21° dias de tratamento. Agudamente, tanto etanol como escopolamina não alteraram a atividade locomotora dos animais, porém, a co-administração das duas drogas induziu um significativo efeito depressor, ao qual se desenvolveu tolerância com o tratamento. Apenas o grupo sal/2,2EtOH desenvolveu sensibilização. Após o tratamento foram realizados 3 desafios (28°, 31° e 34° dias), nos quais metade do grupo sal/sal recebeu salina e a outra metade recebeu a droga-desafio (etanol nos desafios 1 e 2 e escopolamina no desafio 3). Nos desafios 1 e 3, realizados nas caixas de atividade, somente os animais dos grupos sal/2,2EtOH e escop/2,2EtOH expressaram sensibilização, sugerindo "sensibilização cruzada" entre etanol e escopolamina. No desafio 2, realizado em um ambiente novo para eles (campo aberto), a expressão da sensibilização foi bloqueada. No Estudo II os camundongos foram tratados por 21 dias com salina ou 2,2 g/kg de etanol (i.p.), sendo estes classificados como AS ou BS, com base na atividade do 21° dia. Os animais foram sacrificados para análise auto-radiográfica da densidade de receptores M1, não tendo sido observadas diferenças significativas entre os animais classificados como AS, BS ou controles (salina), em nenhuma das 20 regiões encefálicas analisadas. Em resumo, a escopolamina influenciou o processo de sensibilização ao efeito estimulante do etanol, sugerindo que o sistema colinérgico é importante neste processo. Porém, as neuroadaptações que ocorreram com o tratamento crônico com etanol parecem não afetar os níveis de receptores M1. / Various neurotransmission systems have influence on the behavioral sensitization process developed after repeated administration of some drugs of abuse, among them the cholinergic system, which modulates the dopaminergic pathway’s functioning. In this study we evaluated the influence of scopolamine (an antagonist of cholinergic muscarinic receptors) on the development and _expression of behavioral sensitization to ethanol (Study I), as well as on the M1 binding, in animals classified as presenting high (AS) or low (BS) sensitization to ethanol (Study II). In Study I, four groups of male Swiss albino mice received one of the following during 21 days: saline+saline (sal/sal); 1.0 mg/kg of scopolamine+saline (escop/sal); salina+2.2 g/kg of ethanol (sal/2.2EtOH) or 1.0 mg/kg scopolamine+2.2 g/kg of ethanol (escop/2.2EtOH). Their locomotor activity was recorded during 20 minutes on the first, 7th, 14th and 21st days of treatment. Acutely, neither ethanol nor scopolamine altered their locomotor activity; however the co-administration of the both drugs induced a significant depressor effect to which tolerance was developed. Only the sal/2.2EtOH group developed sensitization. After the treatment, 3 challenge tests were carried out (on days 28th, 31st and 34th), in which half of the sal/sal group received saline and the other half received the challenge drug (ethanol in challenges 1 and 2 and scopolamine in challenge 3). In challenges 1 and 3 the animals were tested in activity cages and only the sal/2.2EtOH and escop/2.2EtOH groups expressed sensitization, suggesting there is cross-sensitization between ethanol and scopolamine. In challenge 2, which was conducted in a new environment (open-field arena), the _expression of the sensitization was blocked. In Study II, mice were treated during 21 days with saline or 2.2 g/kg ethanol (i.p.) and the ethanol treated mice were classified as AS or BS, according to their locomotor activity on day 21st. The animals were sacrificed and the bindings to M1 sites were examined by auto-radiographic analyses. No significant differences were found among groups (AS, BS and control) in any of the 20 brain regions analyzed. The present results suggest that scopolamine influences the process of sensitization to ethanol and that the cholinergic system participates in this process. However, the neuroadaptation that occurred after chronic ethanol treatment does not seem to change the binding to M1. / TEDE / BV UNIFESP: Teses e dissertações

Au cours des dix dernières années, l’utilisation de l’imagerie par tomographie par émission de positrons (TEP) s’est rapidement développée en oncologie. Certaines tumeurs non visibles en imagerie anatomique conventionnelle sont détectables en mesurant l'activité métabolique dans le corps humain par TEP. L’imagerie TEP est utilisée pour guider la délivrance de traitements locaux tels que par rayonnement ionisants ou ablation thermique. Pour la délivrance de ces traitements, segmenter la zone tumorale avec précision est primordial. Cependant, la faible résolution spatiale des images TEP rend la segmentation difficile. Plusieurs études ont démontré que la segmentation manuelle est sujette à une grande variabilité inter- et intra- individuelle et est fastidieuse. Pour ces raisons, de nombreux algorithmes de segmentation automatiques ont été développés. Cependant, peu de données fiables, avec des résultats histopathologiques existent pour valider ces algorithmes car il est expérimentalement difficile de les produire. Le travail méthodologique mis en place durant cette thèse a eu pour but de développer une méthode permettant de comparer les données histopathologiques aux données obtenue par TEP pour tester et valider des algorithmes de segmentation automatiques. Cette méthode consiste à réaliser des autoradiographies quantitatives de spécimens prélevés lors de biopsies guidées par TEP/tomodensitométrie (TDM); l’autoradiographie permettant d’imager la distribution du radiotraceur dans les échantillons avec une haute résolution spatiale. Les échantillons de tissus sont ensuite finement tranchés pour pouvoir être étudiés à l’aide d’un microscope. L’autoradiographie et les photomicrographes de l’échantillon de tissus sont ensuite recalés à l’image TEP, premièrement en les alignant avec l’aiguille à biopsie visible sur l’image TDM, puis en les transférant sur l’image TEP. Nous avons ensuite cherché à utiliser ces données pour tester deux algorithmes de segmentation automatique d'images TEP, le Fuzzy Locally Adaptive Bayesian (FLAB) développé au Laboratoire de Traitement de l'Information Médicale (LaTIM) à Brest, ainsi qu’une méthode de segmentation par seuillage. Cependant, la qualité de ces données repose sur la précision du recalage des images TEP, autoradiographiques et des micrographes. La principale source d’erreur dans le recalage de ces images venant de la fusion des images TEP/TDM, une méthode a été développée afin de quantifier la précision du recalage. Les résultats obtenus pour les patients inclus dans cette étude montrent que la précision de la fusion varie de 1.1 à 10.9 mm. En se basant sur ces résultats, les données ont été triées, pour finalement sélectionner les données acquises sur 4 patients jugées satisfaisantes pour tester les algorithmes de segmentation. Les résultats montrent qu’au point de la biopsie, les contours obtenus avec FLAB concordent davantage avec le bord de la lésion observé sur les micrographes. Cependant les deux méthodes de segmentation donnent des contours similaires, les lésions étant peu hétérogènes. / During the last decade, positron emission tomography (PET) has been finding broader application in oncology. Some tumors that are non-visible in standard anatomic imaging like computerized tomography (CT) or ultrasounds, can be detected by measuring in 3D the metabolic activity of the body, using PET imaging. PET images can also be used to deliver localized therapy like radiation therapy or ablation. In order to deliver localized therapy, the tumor border has to be delineated with very high accuracy. However, the poor spatial resolution of PET images makes the segmentation challenging. Studies have shown that manual segmentation introduces a large inter- and intra- variability, and is very time consuming. For these reasons, many automatic segmentation algorithms have been developed. However, few datasets with histopathological information are available to test and validate these algorithms since it is experimentally difficult to produce them. The aim of the method developed was to evaluate PET segmentation algorithms against the underlying histopathology. This method consists in acquiring quantitative autoradiography of biopsy specimen extracted under PET/CT guidance. The autoradiography allows imaging the radiotracer distribution in the biopsy specimen with a very high spatial accuracy. Histopathological sections of the specimen can then obtained and observed under the microscope. The autoradiography and the micrograph of the histological sections can then be registered with the PET image, by aligning them first with the biopsy needle seen on the CT image and then transferring them onto the PET image. The next step was to use this dataset to test two PET automatic segmentation algorithms: the Fuzzy Locally Adaptive Bayesian (FLAB) developed at the Laboratory of Medical Information Processing (LaTIM) in Brest, France, as well as a fix threshold segmentation method. However, the reliability of the dataset produced depends on the accuracy of the registration of the PET, autoradiography and micrograph images. The main source of uncertainty in the registration of these images comes from the registration between the CT and the PET. In order to evaluate the accuracy of the registration, a method was developed. The results obtained with this method showed that the registration error ranges from 1.1 to 10.9mm. Based on those results, the dataset obtained from 4 patients was judged satisfying to test the segmentation algorithms. The comparison of the contours obtained with FLAB and with the fixed threshold method shows that at the point of biopsy, the FLAB contour is closer than that to the histopathology contour. However, the two segmentation methods give similar contours, because the lesions were homogeneous.

No description available.

Previous studies have used new angiotensin II (AII) receptor subtype selective compounds to localize AII receptor subtypes within discrete rat brain nuclei. The purpose of this autoradiographic study was to extend these preliminary findings and provide a comprehensive analysis of AII binding sites in 22 rat brain nuclei and the anterior pituitary, to include estimates of the binding affinity for 125I sar1 ile8 AII (125I SIAII) at each nucleus, and determine the fractional distribution of each subtype at each nucleus. Estimates of K(D), in separate experiments revealed that AT1 nuclei had a consistently higher affinity for 125I SIAII than AT2 nuclei (0.66 vs. 2.55 nM). Displacement of subsaturating concentrations of 125I SIAII by 10-8-10-4 M DuP753 (selective for the AT1 subtype) or PD123177 (selective for the AT2 subtype) indicated that approximately half of the brain regions surveyed contained predominantly AT1 sites and half contained predominantly AT2 sites. Binding was partially displaced by both compounds in several regions and two site analyses were performed to estimate the distribution of subtypes within each nucleus. The data were then corrected for differential occupancy by 125I SIAII. Brain nuclei associated with cardiovascular or dipsogenic actions of AII, e.g., subfornical organ, organum vasculosum of the lamina terminalis, median preoptic nucleus, nucleus of the solitary tract and area postrema, contained pure, or almost pure, populations of AT1 receptors. The functions of AII in brain regions containing predominantly AT2 binding sites, e.g., thalamus, colliculi, inferior olive and locus ceruleus, remain undefined. Thus, AII binding sites in the rat brain have been differentiated into two subtypes with similar characteristics to those reported in peripheral tissues. However, the unexpected finding that they can be differentiated on the basis of their affinity for 125I SIAII raises questions concerning their coidentity with peripheral receptor subtypes.

No description available.

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.

Growth hormone (GH) stimulates growth and metabolism but also displays profound effects on the central nervous system (CNS). GH affects neurogenesis and neuroprotection, and has been shown to counteract drug-induced apoptosis in the brain. Anabolic androgenic steroids (AAS), mainly abused for their anabolic and performance-enhancing properties, can cause several adverse effects, such as cardiovascular complications, sterility, depression, and aggression. GH and AAS are both believed to interact with several signaling systems in the CNS. The aim of this thesis was to further investigate the impact of GH and AAS on neurochemistry and cognitive functions. Recombinant human GH (rhGH) and the steroid nandrolone decanoate (ND) were administered, separately and in combination with each other, to male rats. The results demonstrated that administration of GH improved spatial memory, assessed in a water maze test. Furthermore, GH induced alterations of the GABAB receptor mRNA expression, density, and functionality in the brain, for example in regions associated with cognition. GH also altered the mu opioid peptide (MOP) receptor, but not the delta opioid peptide (DOP) receptor functionality in the brain. Thus, some of the GH effects on cognition may involve effects on the GABAB receptors and MOP receptors. ND, on the contrary, seemed to induce impairments of memory and also altered the GABAB receptor mRNA expression in the brain. Furthermore, ND lowered the IGF-1 plasma concentrations and attenuated the IGF-1, IGF-2, and GHR mRNA expression in the pituitary. In addition, significant effects of GH and ND were found on plasma steroid concentrations, organ weight, as well as body weight. In conclusion, this thesis contributes with further knowledge on the cognitive and neurochemical consequences of GH and ND use. The findings regarding ND are worrying considering the common use of AAS among adolescents. GH improves memory functions and affects signaling systems in the brain associated with cognition, hence the hypothesis that GH can reverse drug-induced impairments is further strengthened.

The retina is a thin layer of tissue lining the back of the eye and is primarily responsible for sight in vertebrates. The neural retina has a distinct layered structure with three dense nuclear layers, separated by plexiform layers comprising of axons and dendrites, and a layer of photoreceptor segments. The retinal and choroidal vasculatures nourish the retina from either side, with an avascular layer comprised largely of photoreceptor cells. Diseases that directly affect the neural retina like retinal degeneration as well as those of vascular origin like diabetic retinopathy can lead to partial or total blindness. Early detection of these diseases can potentially pave the way for a timely intervention and improve patient prognosis. Current techniques of retinal imaging rely mainly on optical techniques, which have limited depth resolution and depend mainly on the clarity of visual pathway. Magnetic resonance imaging is a versatile tool that has long been used for anatomical and functional imaging in humans and animals, and can potentially be used for retinal imaging without the limitations of optical methods. The work reported in this thesis involves the development of high resolution magnetic resonance imaging techniques for anatomical and functional imaging of the retina in rats. The rats were anesthetized using isoflurane, mechanically ventilated and paralyzed using pancuronium bromide to reduce eye motion during retinal MRI. The retina was imaged using a small, single-turn surface coil placed directly over the eye. The several physiological parameters, like rectal temperature, fraction of inspired oxygen, end-tidal CO2, were continuously monitored in all rats. MRI parameters like T1, T2, and the apparent diffusion coefficient of water molecules were determined from the rat retina at high spatial resolution and found to be similar to those obtained from the brain at the same field strength. High-resolution MRI of the retina detected the three layers in wild-type rats, which were identified as the retinal vasculature, the avascular layer and the choroidal vasculature. Anatomical MRI performed 24 hours post intravitreal injection of MnCl2, an MRI contrast agent, revealed seven distinct layers within the retina. These layers were identified as the various nuclear and plexiform layers, the photoreceptor segment layer and the choroidal vasculature using Mn54Cl2 emulsion autoradiography. Blood-oxygenlevel dependent (BOLD) functional MRI (fMRI) revealed layer-specific vascular responses to hyperoxic and hypercapnic challenges. Relative blood volume of the retina calculated by using microcrystalline iron oxide nano-colloid, an intravascular contrast agent, revealed high blood-volume in the choroidal vasculature. Fractional changes to blood volume during systemic challenges revealed a higher degree of autoregulation in the retinal vasculature compared to the choroidal vasculature, corroborating the BOLD fMRI data. Finally, the retinal MRI techniques developed were applied to detect structural and vascular changes in a rat model of retinal dystrophy. We conclude that retinal MRI is a powerful investigative tool to resolve layer-specific structure and function in the retina and to probe for changes in retinal diseases. We expect the anatomical and functional retinal MRI techniques developed herein to contribute towards the early detection of diseases and longitudinal evaluation of treatment options without interference from overlying tissue or opacity of the visual pathway.