L’acide docosahexaénoïque (DHA, 22:6n-3) est le principal acide gras oméga-3 des tissus cérébraux et est essentiel au développement et aux fonctions du cerveau. Une diminution de la concentration cérébrale du DHA est observée chez les patients souffrant de maladies neurodégénératives telles que les maladies d'Alzheimer et de Parkinson. Un apport ciblé du DHA au cerveau pourrait compenser ces carences. Le DHA sanguin est transporté à travers la barrière hémato-encéphalique (BHE) plus efficacement lorsqu’il est estérifié en position sn-2 de la lysophosphatidylcholine (lysoPC). Nous produisons au laboratoire une phosphatidylcholine structurée pour mimer la 2-docosahexaénoyl-lysoPC (lysoPC-DHA), nommée AceDoPC (1-acétyl,2-docosahexaénoyl-glycérophosphocholine), qui peut être considérée comme une forme stabilisée de la lysoPC-DHA physiologique et qui est neuroprotectrice dans l’accident ischémique cérébral. Le premier objectif de ce travail a été de comparer le passage du DHA marqué non estérifié ou estérifié dans l’AceDoPC ou dans une phosphatidylcholine (PC-DHA), lié au plasma, à travers un modèle in vitro de la BHE. Nous montrons un passage préférentiel à travers la monocouche endothéliale et une captation préférentielle par les cellules gliales de l’AceDoPC comparativement au DHA non estérifié et à la PC-DHA. Le deuxième objectif de ce travail a été de confirmer si cette préférence pour la forme AceDoPC était également observée in vivo. Nous avons donc étudié, chez des rats âgés de 20 jours, la captation cérébrale des différentes formes d’apport du DHA précédemment utilisées (DHA, AceDoPC, PC-DHA). Nous démontrons que l’AceDoPC apporte le DHA au cerveau plus efficacement que les autres formes d’apport de DHA et que cette préférence pour l’AceDoPC est spécifique au cerveau puisqu’elle n’est pas observée pour les autres organes étudiés. L’AceDoPC est trouvée, en partie, sous forme intacte dans le cerveau. L’autoradiographie ex vivo du cerveau de rat révèle que le DHA provenant de l’AceDoPC est localisé dans des régions cérébrales spécifiques jouant un rôle important dans la mémoire et les fonctions cognitives. Enfin, en utilisant des approches de modélisation moléculaire, nous démontrons que les potentiels électrostatiques et hydrophobes sont distribués de manière très similaire au niveau des surfaces de l’AceDoPC et de la lysoPC-DHA. En conclusion, nos études montrent que l’AceDoPC est un transporteur privilégié et spécifique du DHA au cerveau. En considérant les rôles essentiels du DHA pour le cerveau, cette nouvelle approche de ciblage cérébral du DHA offre des perspectives prometteuses dans le développement de stratégies préventives et thérapeutiques pour les maladies neurologiques. / Docosahexaenoic acid (DHA, 22:6n-3) is the main essential omega-3 fatty acid in brain tissues required for normal brain development and function. A decrease in the cerebral concentration of DHA is observed in patients suffering from neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Targeted intake of DHA to the brain could compensate for these deficiencies. Blood DHA is transported across the blood-brain barrier (BBB) more efficiently when esterified at the sn-2 position of lysophosphatidylcholine (lysoPC). We produce in the laboratory a structured phosphatidylcholine to mimic 2-docosahexaenoyl-lysoPC (lysoPC-DHA), named AceDoPC (1-acetyl,2-docosahexaenoyl-glycerophosphocholine), that may be considered as a stabilized form of the physiological lysoPC-DHA and that is neuroprotective in experimental ischemic stroke. The first objective of this work was to compare the passage of either labeled unesterified DHA or DHA esterified in AceDoPC or in phosphatidylcholine (PC-DHA), bound to plasma, through an in vitro model of the BBB. This model is constituted of a co-culture of bovine brain capillary endothelial cells and glial cells from newborn rats. We show a preferential passage through the endothelial monolayer and a preferential uptake by glial cells of AceDoPC compared to unesterified DHA and PC-DHA. We also show that AceDoPC is hydrolyzed, partly, into lysoPC-DHA and that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most labeled lipid classes in endothelial cells and glial cells. AceDoPC is found, partly, as a whole molecule in the cells. The second objective of this work was to confirm whether this preference for AceDoPC was also observed in vivo. We studied, in 20 days old rats, the brain uptake of different forms of DHA previously used (DHA, AceDoPC, PC-DHA). We demonstrate that AceDoPC provided the brain with DHA more efficiently than the other forms of DHA and that this preference for AceDoPC is specific for the brain because it is not observed for other studied organs. AceDoPC is found, partly, intact in the brain. Ex vivo autoradiography of rat brain reveals that DHA provided from AceDoPC is localized in specific brain regions playing key roles in memory and cognitive functions. Finally, by using molecular modelling approaches, we demonstrate that electrostatic and hydrophobic potentials are distributed very similarly at the surfaces of AceDoPC and lysoPC-DHA. In conclusion, our studies demonstrate that AceDoPC is a privileged and specific carrier of DHA to the brain. Considering the essential roles of DHA for the brain, this new approach to target the brain with DHA offers promising perspectives in the development of preventive and therapeutic strategies for neurological diseases.

Au cours de cette thèse, réalisée dans le cadre du projet Iseult/INUMAC, nous avons réalisé un travail de développements méthodologiques et technologiques, dans l'optique de permettre à l'IRM de devenir un outil quantitatif pour l'imagerie moléculaire de pathologies cérébrales sur des modèles rongeurs. Pour cela, nous avons développé une stratégie de quantification, utilisant des séquences de cartographie T1 et T2, pour acquérir des cartes de concentration en agents de contraste paramagnétiques et superparamagnétiques avec une excellente sensibilité, une résolution spatiale élevée, ainsi qu'une résolution temporelle compatible avec l'imagerie in vivo. La méthodologie générale que nous avons mise en place lors de ces travaux de thèse nous a permis d'aborder un certain nombre de problématiques propres à l'imagerie moléculaire de pathologies cérébrales par IRM. Dans un premier temps, nous nous sommes intéressés à l'imagerie d'un biomarqueur endovasculaire de l'angiogenèse tumorale sur un modèle de glioblastome cérébral induit chez des souris immuno-déprimées. Nous avons étudié la fixation d'une émulsion paramagnétique, fonctionnalisée par l'ajout de peptides RGD, sur l'intégrine alpha-nu-beta-3 surexprimée à la surface des cellules endothéliales de capillaires tumoraux. Nous nous sommes ensuite intéressés à la délivrance des agents de contraste aux tissus cérébraux. À l'aide d'un protocole optimisé d'ouverture de la barrière hématoencéphalique (BHE) par ultrasons focalisés sous IRM, nous avons étudié les caractéristiques de cette ouverture, ainsi que la dynamique de refermeture sur des modèles de rats et souris sains. Dans une autre étude, la mesure du coefficient de diffusion apparent d'agents de contraste dans les tissus cérébraux de rats sains nous a permis d'évaluer le temps nécessaire à ces agents de tailles différentes pour atteindre leurs cibles, une fois la BHE franchie. Ces caractéristiques représentent des informations capitales dans le cadre de la délivrance d'agents de contraste aux tissus cérébraux. Ils sont en effet susceptibles d'intéresser les industriels pharmaceutiques pour optimiser la conception d'agents diagnostiques et thérapeutiques dédiés aux pathologies cérébrales. / In this thesis, which was part of the Iseult/INUMAC project, we propose several methodological and technological developments aiming to allow MRI to become a quantitative tool for molecular imaging of brain pathologies. To do so, we developed a quantification strategy based on T1 and T2 mapping sequences in order to acquire quantitative concentration maps of paramagnetic and superparamagnetic contrast agents with excellent sensitivity, high spatial resolution and temporal resolution compatible with in vivo imaging. This general methodology allowed us to address several issues specific to molecular imaging of cerebral pathologies using MRI. First, we focused on the imaging of a vascular biomarker of tumor angiogenesis on a glioblastoma mouse model. We studied the binding of a paramagnetic emulsion, functionalized using RGD peptides, on alpha-nu-beta-3 integrin over-expressed at the surface of freshly formed endothelial cells. Then, we focused on the delivery of contrast agents to brain parenchyma. A system was developed and optimized to open transiently and non-invasively rodents blood brain barrier (BBB) using focalized ultrasound monitored by MRI. The BBB opening features and closure dynamics induced by this protocol were extensively characterized. In another study, we measured the apparent diffusion coefficient of contrast agents with different sizes in cerebral tissues of healthy rats. From these measures we could estimate the time necessary for these particles to reach their targets once the BBB is crossed. These parameters are highly valuable in the context of drug delivery to the brain. They might indeed be used by pharmaceutical industries to optimize the design of diagnostic and therapeutic agents dedicated to cerebral diseases.

INTRODUÇÃO: As imagens de lesões hiperdensas encontradas em exames de tomografia (TC) de crânio após o tratamento endovascular do acidente vascular cerebral isquêmico (AVCi) agudo têm sido correlacionadas ao risco de transformação hemorrágica após o AVC. Entretanto, a correlação entre as lesões hiperdensas e a área cerebral infartada é desconhecida. O objetivo deste estudo é determinar a correlação entre as lesões hiperdensas encontradas em TC de crânio realizadas logo após tratamento endovascular do AVCi agudo e a área de AVC isquêmico. MATERIAIS E MÉTODOS: Foram coletados retrospectivamente dados radiológicos de pacientes com AVCi agudo por oclusão de grandes vasos da circulação anterior submetidos ao tratamento endovascular. Foram analisadas imagens de TC de crânio nas primeiras 24 horas e até 21 dias após o tratamento. As áreas hiperdensas foram classificadas utilizando o escore ASPECTS e comparadas com as áreas de AVC isquêmico final pelo mesmo escore. As imagens foram analisadas independentemente por dois avaliadores, sendo que um terceiro avaliador analisou os casos discordantes. A concordância entre avaliadores (CCI) e os valores de sensibilidade, especificidade, preditivos positivo e negativos e acurácia foram calculados. RESULTADOS: Lesões hiperdensas foram encontradas em 71 dos 93 (76,34%) pacientes com AVC isquêmico de circulação anterior. As áreas captantes de contraste corresponderam às áreas de AVC final segundo o escore ASPECTS (CCI=0,58 [0,40 0,71]). Os valores para cada região individual foram avaliados e a sensibilidade variou de 58,3% a 96,9%, a especificidade de 42,9% a 95,6%, os valores preditivos positivos de 71,4% a 97,7%, os valores preditivos negativos de 53,8% a 79,5% e os valores de acurácia de 0,68 a 0,91. Os maiores valores de sensibilidade foram encontrados para os núcleos lentiforme (96,9%) e caudado (80,4%) e para a cápsula interna (87,5%) e os menores para os córtices M1 (58,3%) e M6 (66,7%). CONCLUSÕES: A aplicação do escore ASPECTS para avaliação das imagens de tomografia de crânio após o tratamento endovascular do AVCi agudo que apresentam captação de contraste, demonstrou ser uma ferramenta útil para a predição da área final de infarto cerebral. A predição foi maior na região profunda e menor nos córtices cerebrais, provavelmente devido maior circulação colateral cortical. Além disso, o método se mostrou reprodutível e de fácil utilização. / INTRODUCTION: The hyperdense lesions images found in head computed tomography (CT) scan after endovascular treatment have been correlated to risk of hemorrhagic transformation after stroke. However, the correlation between hyperdense lesions and the infarcted brain area is unknown. The aim of this study is to determine the correlation between the hyperdense lesions found on CT scan performed after endovacular treatment of acute stroke and final ischemic stroke area. MATERIALS AND METHODS: It was collected radiological data of patients with acute ischemic stroke by occlusion of large vessels in the anterior circulation were treated with endovascular treatment. Head CT scan were evaluated in the first 24 hours and by 21 days after treatment. The hyperdense areas were rated using the ASPECTS score and compared with final ischemic stroke by the same score. The images were analyzed independently by two reviewers, and a third evaluator examined the discordant cases. The interrater agreement (ICC) and the sensitivity, specificity, positive and negative predictive values and accuracy were calculated. RESULTS: hyperdense lesions were found in 71 of 93 (76.34%) patients with ischemic stroke of anterior circulation. The contrast iodineaccumulating areas corresponded to the final stroke areas (ICC = 0.58 [0.40 to 0.71]) as the ASPECTS score. The values for each individual region were evaluated and the sensitivity ranged from 58.3% to 96.9%, specificity of 42.9% to 95.6%, the positive predictive value of 71.4% to 97, 7%, the negative predictive value of 53.8% to 79.5% and the accuracy of values from 0.68 to 0.91. The higher sensitivity found for lenticular nuclei (96.9%) and caudate (80.4%) and the internal capsule (87.5%) and lower for M1 (58.3%) and M6 (66.7%) cortices. CONCLUSIONS: The use of the ASPECTS score for evaluation of CT head scan after endovascular treatment of acute ischemic stroke images that exhibit contrast enhancement proved to be a useful tool for predicting the final ischemic stroke area. The prediction was higher in the deep region and lower in the cerebral cortex, probably because the cortical collateral circulation. Futhermore, these method was reproducible and easy to use.

Hypertension is the chronic elevation in blood pressure that is regulated in part through the retention and regulation of sodium retention and excretion in the kidneys. Hence the kidney has been considered the organ that regulates blood pressure. There are a cohort of patients that suffer with high blood pressure due to lack of 11β-hydroxysteroid dehydrogenase-type 2 (11β-HSD2) expression (which inactivates glucocorticoids (GCs), allowing selective activation of mineralocorticoid receptors (MR) by aldosterone) that results in hypertensive and increased salt appetite phenotypes - a condition known as syndrome of apparent mineralocorticoid excess (SAME). This disorder can be recapitulated in the mouse through the global deletion of 11β-HSD2, which results in over activation of the MR driving an elevation in blood pressure. However, the distinction between blood pressure elevation because of kidney dysfunction with loss of 11β-HSD2 or increased salt appetite due to loss of brain 11β-HSD2 expression is not clear from the global 11β-HSD2 knockout model. Salt appetite is regulated by regions of the brain out-with the blood-brain barrier, known as circumventricular organs. In the mouse, salt appetite is controlled by aldosterone-sensitive cells in the nucleus of the solitary tract (NTS) in the brain stem, where 11β-HSD2 is expressed to provide mineralocorticoid selectivity. However, in the fetal brain, 11β-HSD2 is widely expressed, protecting against adverse GC action that alters brain development and increases susceptibility to psychiatric disorders as adults. 11β-HSD2 deletion solely in the brain from embryonic day 12 resulting in GC fetal programming (HSD2BKO) causes effects on both behaviour and salt appetite. To determine the role of developmental versus adult expression of brain 11β- HSD2, mice with deletion of brain 11β-HSD2 from mid gestation (HSD2BKO) and mice with adult deletion of 11β-HSD2 in the NTS using lentivirus (HSD2.v- BKD) were compared. The phenotypes (salt appetite, blood pressure (BP), baroreceptor response (BRR) and cognition), can be categorised as either due to GC fetal programming (as indicated by HSD2BKO groups), or increased activation of MR in adult 11β-HSD2 expressing neurons (recapitulated in the HSD2.v-Cre groups). Salt appetite increased in both HSD2BKO and HSD2.v-BKD cohorts (mean percentage increase 65% n=8 and 46% n=6, compared to their respective controls), leading to an increased BP in both groups (+12% and +8%, respectively) as well as an impaired BRR, indicating all phenotypes are mediated by adult NTS neurons. However, spatial recognition memory (Object-in-Place task) is abolished in HSD2BKO mice, whereas, HSD2.v-BKD mice still retain short-term memory. Our data suggest that neural 11β-HSD2 protects against inappropriate activation of MR by corticosterone to regulate salt appetite and salt-induced rises in blood pressure. However, spatial recognition memory is not influenced by deletion of 11β-HSD2 in the adult brain, confirmation that this phenotype is underpinned by developmental programming by GCs, which is observed in the 11β-HSD2 brain KO. Salt appetite has been shown to be centrally regulated through the adult deletion of 11β-HSD2. From this, our data suggest that an increased salt appetite is due to adult loss of function of 11β-HSD2 rather than GC programming during development. Highlighting the NTS as a region for drug delivery to try and control salt appetite in salt sensitive individuals who struggle with administering a recommended change in diet. To develop this further, minimally invasive modes of delivery of viruses and drugs into the brain were investigated. In so doing, a non-invasive and reversible method to temporarily disrupt the blood brain barrier (BBB) was optimised. The technique required acoustic insonation of ultrasonic contrast agents (CAs) (gas microbubbles) adjacent to the BBB. These microbubbles (SonoVueTM, Bracco) were delivered via tail vein injection into the vasculature. To target the BBB, an ultrasonic transducer was suspended and focused through coupling gel onto the area of interest in the brain with skull the intact. The optimisation of this technique required determination of the focal position of the 3.5MHz transducer that was utilised, in addition to optimisation of the pulse length, pulse repetition frequency and power output of the ultrasound beam to enable the BBB to be disrupted. In addition, measurement of the attenuation of the ultrasound beam through ex vivo mouse skulls were measured. These results showed a 50% reduction in pressure amplitude from the baseline of 335.2mV (Baseline mean = 100% +/-SEM 0 n=3 (No skull), five regions across the skull averaged 47.79% +/-SEM 1.913 n=25 (using 5 different animals). In in vivo mice, after co-injection of the microbubbles with Evans Blue and insonation of the brain, disruption of the BBB was confirmed by the presence of Evans Blue dye in the brain, with no measurable damage occurring in the brain. This was confirmed by cell and nuclear morphology with no red blood cell extravasation into the surrounding tissue. The parameters used to open the BBB used a peak negative pressure of 2.1MPa (single pulse), transducer frequency 3.5MHz, 35,000 cycles over a 10ms burst at a pulse repetition frequency of 10Hz. The technique when applied in vivo in recovery animals is speculated to work by the focused ultrasound causing the microbubbles to oscillate within the vasculature adjacent to the BBB, resulting in high-shear stresses being generated on the tight junctions within the BBB. The resultant gaps in the BBB allow free circulating compounds (e.g. large dye molecules (Evans Blue - 960.8g/mol molecular weight) and adeno-associated-viruses (25nm with a packing capacity of 4.5kb) within the blood to pass into the brain, but there is no penetration of red blood cells (7μm). Longitudinal mouse experiments demonstrated that within 12-hours these gaps close with no long-term damage observed. Currently, utilising this technique, successful passage of an adeno-associated virus expressing GFP (as a marker) has been shown to pass into the brain (n=6 for each cohort including control) - indicating that the virus requires the ultrasound and microbubbles to facilitate its movement into the brain. Further technique optimisation is being explored looking at the role of CAs used in the opening and disruption of the BBB, comparing composition and size of the CAs. Microbubbles (2-3μm) and nanobubbles (200nm) were compared as well as lipid and non-ionic surfactant surface compositions, using volume of drug delivery and degree of disruption as outputs. Using this technique, the hydrophilic drug mimic calcein was delivered into the brain (n=5 non-ionic surfactant nanobubble, n=5 lipid nanobubble). Results have indicated that the delivery of calcein is most efficient when using non-ionic surfactant nanobubbles as opposed to lipid nanobubbles - with a greater volume of the drug being delivered into the cerebral tissue. Furthermore, the concentration and surface composition of the nanobubble have an effect as to the size and potential damage to the brain when opening the BBB. In conclusion, it has been shown that it is possible to non-invasively open the BBB and deliver viruses and dye into the brain. In addition, this thesis has investigated the use of nanobubbles as both facilitators to opening the BBB and delivery vectors for potentially therapeutic drugs. Finally, a non-invasive opening of the BBB has been achieved using focused ultrasound. Ultimately this non-invasive opening of the BBB can be used to achieve delivery of larger molecules (such as antibodies and viruses) into the brain to target treatments. Focused ultrasound brain targeting can be applied to the potential treatment of salt appetite regulation in the NTS. For the individuals who suffer from salt sensitive hypertension, the NTS can be targeted to reduce the drive to ingest high salt diets. Furthermore, the continuation of research into the central control of BP, salt appetite and baroreceptor reflex control can become better understood, using less invasive delivery techniques to the brain.

Breast cancer metastasizes to the brain in 15-30% of all breast cancer cases, and metastasis is the predominant cause of breast cancer-related deaths. Patients with HER2-enriched and triple-negative breast cancers (TNBCs) are more likely to develop brain metastases. While targeted therapies exist for HER2-enriched breast cancers, there are no effective treatments for TNBCs. Thus, a greater understanding of how these cancers spread to the brain is critical. In order to spread to the brain, disseminated breast cancer cells must overcome 2 major steps—crossing the blood-brain barrier (BBB) and survival and successful colonization of the distinctive and mostly cellular brain environment. Here, we report a novel role for breast cancer cell surface receptor, Syndecan-1 (Sdc1), a heparan sulfate proteoglycan, in promoting breast cancer cell transmigration across the BBB. We found that when we silenced Sdc1 expression in a highly metastatic TNBC cell line, MDA-MB-231, these cells exhibited reduced migration across an in vitro BBB model system. Further, in an in vivo experimental model of metastasis, mice injected with MDA-MB-231 Sdc1 KD (knock-down) cells developed less brain metastases than mice injected with control non-silencing (NS1) cells. Conversely, we found that overexpression of Sdc1 in a metastatic triple-negative mouse mammary carcinoma cell line, 4T1, led to an increase in brain metastases compared to empty vector control-treated mice. We predicted that a secreted factor(s) facilitated BBB disruption that allowed for Sdc1-mediated BBB transmigration, and found that silencing Sdc1 led to decreases in the production and/or release of various cytokines and chemokines implicated in BBB permeability and transmigration. In addition to supporting BBB transmigration, through an in vitro tissue section adhesion assay, we found that Sdc1 also facilitates adhesion of breast cancer cells to the brain, and not to the liver or lungs, revealing specificity for the brain. Further, we report that Sdc1 is expressed in 81% of breast cancer patient brain metastases in our tissue microarray study and that patients with TNBC and high Sdc1 expression have shorter disease-free survival based on a study performed using data from The Cancer Genome Atlas. Taken together, we predict that breast cancer cell Sdc1-regulated cytokines and chemokines promote BBB permeability and/or support transmigration to facilitate breast cancer metastasis to the brain. We also provide evidence for breast cancer-secreted extracellular vesicles, namely exosomes, in supporting the formation of a pro-metastatic brain environment. We compared exosomes derived from the metastatic 4T1 mouse mammary carcinoma cell line to a non-metastatic counterpart, the 67NR cell line, to assess their microRNA and protein composition and their effect(s) on recipient astrocytes, known mediators of brain metastasis. We found that there are inherent differences in both the microRNA and protein cargo from the metastatic 4T1 cells compared to the non-metastatic 67NR cells, whereby the metastatic 4T1 cells contained various tumor-promoting microRNAs and proteins, and also contained 4.5-fold more protein than the non-metastatic 67NR cells. Mouse astrocytes treated with the metastatic 4T1 exosomes exhibited a shift towards a pro-metastatic phenotype, characterized by upregulation of pro-inflammatory genes, and genes associated with astrocyte reactivity and cancer, whereby 67NR exosome-treated astrocytes exhibited a response profile that overlapped with untreated controls. Overall, these findings reveal an important role for exosomes in driving changes in the brain microenvironment to create a site conducive for cancer growth. Together, both studies help to elucidate how breast cancer cells can invade and colonize the unique brain environment.

No description available.

Although survival rates are increasing, brain injury continues to be a leading cause of death after cardiac arrest (CA). Permanent brain damage after CA is determined by limited tolerance to ischemia from CA and cardiopulmonary resuscitation (CPR), as well as the unique cerebral response to reperfusion after return of spontaneous circulation (ROSC). A major pathway leading to neurotoxic cascade and neuronal injury after CA involves the increased presence of reactive oxygen and nitrogen species generated during ischemia and reperfusion. The magnitude of cerebral oxidative injury induced by free radicals increased with the duration of CA (Paper I). Nitric oxide (NO), a free radical responsible for the formation of reactive nitrogen species, is increased during global ischemia from CA and reperfusion (Paper IV). Hypothetically, the administration of a drug that counteracts the overproduction of NO and also acts as a scavenger of oxygen free radicals might be warranted in order to reduce the damage caused by nitrosative and oxidative stress. For these purposes we used methylene blue (MB), an old dye that has been used in medicine for almost half a century, and an experimental pig model of 20 min of ventricular fibrillation (VF) to reflect a clinical scenario of ischemia/reperfusion injury. Administration of MB added to a hypertonic-hyperoncotic solution (MBHSD) that was started during CPR and continued for 50 min after ROSC increased short-term survival by decreasing myocardial damage, as well as cerebral peroxidation and inflammatory injury (Paper II). Immunostaining of cerebral tissue collected at different time points after CA and ROSC (Paper IV) provided experimental evidence that cortical blood-brain barrier (BBB) disruption begins as early as  during the initial phase of untreated as well as treated CA. The results indicated that MB administration reduced the neurologic injury and BBB disruption considerably, but did not reverse the ongoing detrimental processes. The demonstrated positive effects of MB were related to a decrease of nitrite/nitrate tissue content, and thus to a decrease of excess NO due to the MB inhibitory effects on NOS isoforms. A mixture of MB in hypertonic sodium lactate (MBL) was investigated to facilitate administration of MB in “the field.” Based on findings that MBL cardio- and neuroprotective properties were similar to those of MBHSD, there is reason to believe that the use of MBL might be extended during ongoing CPR and after ROSC (Paper III). It would therefore make sense to try using MB as a pharmacological neuroprotectant during or after clinical CPR in order to expand the temporal therapeutic window before other measures for neuroprotection such as hypothermia are available.

<p>Mice have become the preferred model system for studying brain function and disease. With the powerful genetic tools available, mouse models can be created to study the underlying molecular basis of neurobiology in vivo. Just as magnetic resonance imaging is the dominant tool for evaluating the human brain, high-resolution MRI--magnetic resonance microscopy (MRM)--is a useful tool for studying the brain of mouse models. However, the need for high spatial resolution limits the signal-to-noise ratio (SNR) of the MRM images. To address this problem, T1-shortening contrast agents can be used, which not only improve the tissue contrast-to-noise ratio (CNR) but also increase SNR by allowing the MR signal to recover faster between pulses. By "actively staining" the tissue with these T1-shortening agents, MRM can be performed with higher resolution, greater contrast, and shorter scan times. In this work, active staining with T1-shortening agents was used to enhance three types of in vivo mouse brain MRM: (1) angiographic imaging of the neurovasculature, (2) anatomical imaging of the brain parenchyma, and (3) functional imaging of neuronal activity.</p> <p></p> <p>For magnetic resonance angiography (MRA) of the mouse, typical contrast agents are not useful because they are quickly cleared by the body and/or extravasate from the blood pool before a high-resolution image can be acquired. To address these limitations, a novel contrast agent--SC-Gd liposomes--has been developed, which is cleared slowly by the body and is too large to extravasate from the blood pool. In this work, MRA protocols were optimized for both the standard technique (time-of-flight contrast) and SC-Gd liposomes. When the blood was stained with SC-Gd liposomes, small vessel CNR improved to 250% that of time-of-flight. The SC-Gd liposomes could also be used to reduce scan time by 75% while still improving CNR by 32%.</p> <p>For MRM of the mouse brain parenchyma, active staining has been used to make dramatic improvements in the imaging of ex vivo specimens. However for in vivo imaging, the blood-brain barrier (BBB) prevents T1-shortening agents from entering the brain parenchyma. In this work, a noninvasive technique was developed for BBB opening with microbubbles and ultrasound (BOMUS). Using BOMUS, the parenchyma of the brain could be actively stained with the T1-shortening contrast agent, Gd-DTPA, and MRM images could be acquired in vivo with unprecedented resolution (52 x 52 x 100 micrometers3) in less than 1 hour.</p> <p>Functional MRI (fMRI), which uses blood oxygen level dependant (BOLD) contrast to detect neuronal activity, has been a revolutionary technique for studying brain function in humans. However, in mice, BOLD contrast has been difficult to detect and thus routine fMRI in mice has not been feasible. An alternative approach for detecting neuronal activity uses manganese (Mn2+). Mn2+ is a T1-shortening agent that can enter depolarized neurons via calcium channels. Thus, Mn2+ is a functional contrast agent with affinity for active neurons. In this work, Mn2+ (administered with the BOMUS technique) was used to map the neuronal response to stimulation of the vibrissae. The resultant activation map showed close agreement to published maps of the posterior-lateral and anterior-medial barrel field of the primary sensory cortex.</p> <p>The use of T1-shortening agents to actively stain tissues of interest--blood, brain parenchyma, or active neurons--will facilitate the use of MRM for studying mouse models of brain development, function, and disease.</p> / Dissertation

Η ηπατική εγκεφαλοπάθεια είναι ένα σύνθετο νευροψυχιατρικό σύνδρομο το οποίο εκδηλώνεται κυρίως σε συνθήκες ηπατικής κίρρωσης. Διάφορες παθολογικές και τοξικές καταστάσεις μπορεί να επηρεάσουν την ηπατική λειτουργία σε τέτοια βαθμό ώστε να προκληθεί ηπατική εγκεφαλοπάθεια. Το οξειδωτικό στρές έχει ενεπλακεί σε διάφορες μελέτες στην παθογένεση της ηπατικής εγκεφαλοπάθειας. Επιπλέον, η ανάπτυξη αυξημένου οξειδωτικού στρές υπό την επίδραση αποφρακτικού ικτέρου έχει μετρηθεί σε διάφορα όργανα πειραματοζώων, συμπεριλαμβανομένου και του εγκεφάλου. Ο σκοπός της παρούσας μελέτης ήταν να ανιχνεύσει της αλλαγές στις αποφρακτικές συνδέσεις των τριχοειδικών αγγείων του εγκεφάλου που συσχετίζονται με την οκκλουδίνη. Για να το επιτύχουμε αυτό χρησιμοποιήσαμε ένα μοντέλο απολίνωσης του κοινού χοληδόχου πόρου (BDL) σε πειραματόζωα (αρουραίους). Στο πείραμα 1 η έκφραση της οκκλουδίνης εκτιμήθηκε μέσω της μεθόδου ποσοτικοποίησης κατά Westernblot. Σε αυτό το πείραμα χρησιμοποιήθηκαν πέντε (BDL) και πέντε ψευδώς χειρουργηθέντα πειραματόζωα (sham). Τα πειραματόζωα θανατώθηκαν δέκα ημέρες μετα το χειρουργείο. Στην κατά Westernblot ποσοτικοποίηση παρατηρήθηκε μεγάλη μείωση της ποσότητας της οκκλουδίνης στα BDL πειραματόζωα σε σχέση με τα sham. Στο πείραμα 2 χρησιμοποιήθηκαν εννέα BDL και εννέα sham πειραματόζωα. Τρία πειραματόζωα από τις δύο παραπάνω ομάδες θανατώθηκαν την πρώτη, την πέμπτη και τη δέκατη μετεγχειρητική ημέρα. Τα επίπεδα οκκουδίνης σε αυτά τα πειραματόζωα συσχετιστήκαν με τις τιμές τηςinvivo μέτρησης των ελευθέρων ριζών οξυγόνο. Τα αποτελέσματα ανέδειξαν ότι η τιμή της οκκλουδίνης στα BDL ζώα ήταν σημαντικά μειωμένη σε σχέση με τα sham όλες τις χρονικές στιγμές που έγιναν οι μετρήσεις, ενώ οι χαμηλότερες τιμές καταγράφηκαν στα πειραματόζωα που παρέμειναν υπό αποφρακτικό ίκτερο για δέκα ημέρες. Επιπλέον καταδείχθη ότι η χρονικά συσχετιζόμενη μείωση των επιπέδων της οκκλουδίνης στα ενδοθηλιακά κύτταρα του εγκεφάλου συσχετίζεται με τα αυξανόμενα επίπεδα ελευθέρων ριζών οξυγόνου της ημέρες μετά το χειρουργείο, φανερώνοντας τη σχέση μεταξύ αυτών των δύο φαινομένων. Συμπερασματικά, η παρούσα μελέτη πρώτη παραθέτει στοιχεία που προτείνουν την εμπλοκή της οκκλουδίνης στην παθοφυσιολογία της ηπατικής εγκεφαλοπάθειας σε συνθήκες εξωηπατικής χολόστασης. Αυτό γίνεται μέσω της μείωσης των επιπέδων τις οκκλουδίνης στις αποφρακτικές συνδέσεις των ενδοθηλιακών κυττάρων του εγκεφάλου υπο την επίδραση του χολοστατικού ικτέρου, η οποία οδηγεί σε άρση του αιματο-εγκεφαλικού φραγμού. / Hepatic encephalopathy in a complicate neuro-psychiatric syndrome which is common under hepatic cirrhosis. Various pathological and toxic lesions can deteriorate liver function in such way, so hepatic encephalopathy can be inflicted. Oxidative stress is involved in pathogenesis of hepatic encephalopathy in several protocols. Moreover the development of increased oxidative stress in the context of obstructive cholestasis has been proven in various rats' organs including the brain. The present study aimed to detect alterations of tight junction-associated occludin in rat brain capillaries. To accomplish that we have used a rats bile duct ligation experimental model (BDL). In experiment 1, occludin expression was evaluated by Western blot analysis. In this experiment were used five BDL and five sham rats. The experimental animals were sacrificed ten days after the operation. Western blot analysis revealed significant decrease of occlidins amount in BDL rats compared to the sham rats. In experiment 2, nine BDL and nine sham animals were used. Three animals from each group were sacrificed during the first, fifth and tenth post-operate day. The results of occludin level to these animals were associated with the in vivo superoxide radical production. The results indicated that occludin level in BDL animals, as opposed to sham-operated, was significantly reduced at every time point studied, being lowest in the rats remaining on BDL condition for 10 days. Moreover, it was demonstrated that the time-dependent reduction of occludin level in the brain endothelial was significantly correlated with the time dependent increase of brain superoxide radical level, implying a relationship between these two abnormalities. In conclusion, the evidence presented herein suggests for first time the implication of occludin in pathophysiology of hepatic encephalopathy under extra-hepatic cholestasis. This phenomenon occur due to the reduce of occludin level to cerebral endothelial cells’ tight junctions under cholestatic jaundice, which drives to lift of brain-blood barrier.

Ο αιματο-εγκεφαλικός φραγμός (ΑΕΦ) παίζει έναν καθοριστικό ρόλο στην ομοιόσταση του Κεντρικού Νευρικού Συστήματος. Οι στενοσύνδεσμοι (TJ) των ενδοθηλιακών κυττάρων των εγκεφαλικών τριχοειδών συνεισφέρουν σημαντικά στη λειτουργία του ΑΕΦ περιορίζοντας την παρακυτταρική διάχυση. Η οκλουδίνη, μια διαμεμβρανική πρωτεΐνη, αποτελεί μείζον συστατικό των TJ και παίζει σημαντικό ρόλο στη ρύθμιση της λειτουργίας τους. Το οξειδωτικό στρες αποτελεί κοινό χαρακτηριστικό πολλών νευροεκφυλιστικών και νευροφλεγμονωδών παθήσεων και η δυσλειτουργία των TJ με τη συνοδό διαταραχή του ΑΕΦ, συνδέονται με αυτό. Το αυξημένο οξειδωτικό φορτίο στα πλαίσια της αποφρακτικής χολόστασης έχει αποδειχθεί σε διάφορα όργανα αρουραίων μεταξύ των οποίων και ο εγκέφαλος. Στην παρούσα μελέτη χρησιμοποιήθηκε ένα πειραματικό μοντέλο αρουραίων με απολίνωση του χοληδόχου πόρου (BDL), για να εξεταστεί η επίδραση της χολόστασης στην εντόπιση της οκλουδίνης στο ενδοθήλιο εγκεφαλικών τριχοειδών με τη χρήση ηλεκτρονικού μικροσκοπίου. Αρσενικοί αρουραίοι Wistar χωρίστηκαν τυχαίως σε δύο ομάδες. Ομάδα Ι ψευδώς χειρουργημένοι αρουραίοι και ομάδα ΙΙ αρουραίοι που υπεβλήθησαν σε απολίνωση χοληδόχου πόρου (BDL) και οι δύο την ίδια ημέρα 0. Την 10η μετεγχειρητική ημέρα, όλα τα επιζήσαντα ζώα θυσιάστηκαν δια αποκεφαλισμού. Μετά από κατάλληλη προετοιμασία για σήμανση με ανοσοχρυσό της οκλουδίνης, δείγματα από το μετωπιαίο λοβό, το μεσεγκέφαλο και την παρεγκεφαλίδα από κάθε ομάδα παρατηρήθηκαν με ηλεκτρονικό μικροσκόπιο για διαφορές στην εντόπιση της οκλουδίνης σε σχέση με τη διενδοθηλιακή σχισμή. Τα αποτελέσματα ανέδειξαν μετακίνηση της οκλουδίνης μακριά από την περιοχή των στενοσυνδέσμων του τριχοειδικού ενδοθηλίου. Σημαντική αύξηση της απόστασης της οκλουδίνης από τη διενδοθηλιακή σχισμή παρατηρήθηκε στο μεσεγκέφαλο και στην παρεγκεφαλίδα και όχι στο μετωπιαίο λοβό, σε σχέση με την ομάδα ελέγχου (control). Τα συγκεκριμένα αποτελέσματα υπαινίσσονται μια εκλεκτική ως προς την περιοχή αποδιοργάνωση της οκλουδίνης σε απάντηση στην ηπατική δυσλειτουργία και ένα σημάδι δυσλειτουργίας των TJ με λογικό συνεπακόλουθο τη δυσλειτουργία και του ΑΕΦ. Προκαταρκτικά δεδομένα της χρήσης αντιοξειδωτικών παραγόντων, όπως η αλλοπουρινόλη, αφήνουν να διαφανεί ένας προστατευτικός ρόλος όσον αφορά τη μετατόπιση της οκλουδίνης σε BDL αρουραίους. Εν συντομία, η παρούσα πειραματική μελέτη παρουσιάζει την επίδραση του οξειδωτικού στρες, στην εντόπιση της πρωτεΐνης των στενοσυνδέσμων οκλουδίνη στο ενδοθήλιο των εγκεφαλικών τριχοειδών αγγείων σε αρουραίους με απολίνωση χοληδόχου πόρου. Για τη μελέτη χρησιμοποιήθηκαν τεχνικές ανοσοσήμανσης συνδυαζόμενες με ηλεκτρονική μικροσκοπία και παρουσιάστηκαν δεδομένα εκλεκτικής ως προς την περιοχή εγκεφαλικής δυσλειτουργίας στην ηπατική πάθηση με δυνητική συσχέτιση με τις κλινικές εκδηλώσεις της ηπατικής εγκεφαλοπάθειας. / The blood–brain barrier (BBB) plays a critical role in central nervous system homeostasis. Interendothelial tight junction (TJ) protein complexes of the brain microvasculature have a major contribution to the BBB function by limiting paracellular diffusion Occludin, a transmembrane protein, is a major component of the TJ which plays a significant role in its regulation. Oxidative stress is a major underlying cause of neurodegenerative and neuroinflammatory disorders while TJ dysfunction leading to BBB disruption is associated with it. The development of increased oxidative stress in the context of obstructive cholestasis has been proven in various rats' organs including the brain. The present study used a rat model with bile duct ligation, to examine the effect of cholestasis, to the localization of occludin in brain capillary endothelium by means of electronic microscopy. Male Wistar rats were randomly divided into Group I, rats subjected to sham operation, and Group II, rats subjected to bile duct ligation (BDL) on day 0 and on post-operative day 10 all surviving animals were sacrificed by instant decapitation. After specific treatment for immunogold labeling of occludin, samples from frontal cortex, midbrain and cerebellum from each group were observed for differences in occludin location in relation to the interendothelial cleft. The results demonstrated a dislocation of occludin away from the tight junction sites of brain endothelial cells. A significant increase of the occludin-interendothelial cleft distance was demonstrated in the midbrain and the cerebellum samples but not in the frontal cortex, compared to the control group samples. These findings imply a brain region selective derangement of occludin in response to liver disease and a sign of TJ impairment and thus, a speculated BBB dysfunction. Preliminary data of use of antioxidant agents, as allopurinol, imply a protective effect concerning the dislocation of occludin in BDL rats. In brief, this experimental study demonstrates the effect of oxidative stress, in the location of TJ protein occludin in brain capillary endothelium of BDL rats. The study used immunolabeling techniques combined with electron microscopy and presented data of region-specific brain abnormalities in liver disease with potential correlation with clinical manifestations of hepatic encephalopathy.