Tesi sul tema "Cerebral ischemia – Animal models"

Stroke is a serious cerebral vascular event and a leading cause of death and disability worldwide, and ischemic stroke is the most common type. Evidence from animal research in acute cerebral ischemia shows that a combination of neuroprotectants might be more efficacious than the single agent given individually. Both melatonin and electro-acupuncture (EA) have been suggested to be effective treatments against cerebral ischemia. However, it is unknown whether a combination of these two therapies could be beneficial against focal cerebral ischemia. In the first study, the effect of post-treatment with a combination of melatonin and EA on regional cerebral blood flow (rCBF), neurological deficit score and infarct volume was investigated in both permanent and transient middle cerebral artery occlusion (MCAO) models in rats. When compared with the single treatment of melatonin or EA, the combination therapy resulted in a significant improvement of neurological function and a dramatic reduction of infarct volume at 72 hr after transient MCAO. A significant upregulatory effect on rCBF has been exerted by the combined treatment. The effect of a combination of melatonin and EA on inflammatory reaction was investigated in the second study. Post-treatment of the combination therapy effectively inhibited neutrophil infiltration as well as the expression of some pro-inflammatory mediators, and increased the anti-inflammatory protein expression at 72 hr after transient MCAO. This beneficial effect may be due to the respective anti-inflammatory effects of melatonin and EA. In the third study, the effect of a combination of melatonin and EA on apoptosis was examined. When compared with the EA treatment alone, post-treatment of the combination therapy exerted a greater inhibitory effect on tissue apoptosis and expression of the pro-apoptotic proteins as well as an upregulatory effect on the anti-apoptotic protein expression. In the fourth study, the effect of continuous post-treatment of a combination of melatonin and EA on transient MCAO was investigated. The combination treatment significantly improved neurological function and decreased infarct volume at 7 days after transient MCAO. Cell proliferation and expression of the neurotrophic factor were increased by the combined treatment. The effect of pretreatment with a combination of melatonin and EA was examined in the fifth study. Neurological function was improved and infarct volume was reduced by the combination pretreatment at 24 hr after transient MCAO. The inflammatory and apoptotic reaction were inhibited by the combined pretreatment through the modulatory effect of the related proteins. In summary, our results show that, when compared with the single treatment of either melatonin or EA, post-treatment with a combination of melatonin and EA induced a complementary neuroprotective effect on improvement of neurological function and a dramatic reduction of infarct volume after transient MCAO. The complementary protection may be partially mediated via anti-inflammation and anti-apoptosis after transient cerebral ischemia. Pretreatment with a combination of melatonin and EA may be more effective in preventing ischemic brain injury after transient focal cerebral ischemia.
published_or_final_version
Medicine
Doctoral
Doctor of Philosophy

Cerebral ischemia is defined as a decrease in blood flow to the brain. It is most often caused by obstruction of a cerebral blood vessel, and is recognized by the World Health Organization as the leading cause of serious adult disability and one of the top three causes of adult death worldwide. Most survivors demonstrate partial restitution of function over time, but the underlying recovery mechanism(s) remain unclear especially in a subset of patients with persistent neurological morbidities despite normal-appearing brain on neuroimaging. The optimal way to understand any human disease state is via clinical studies. Unfortunately, well-controlled experiments in humans are difficult due to small patient populations, the presence of numerous confounding variables, and ethical issues associated with invasive or discomforting experimental procedures. Anesthetized animal models of cerebral ischemia afford a means of avoiding the above difficulties. However, anesthesia and physiological perturbations that occasionally follow brain ischemia may affect the reliability of certain tools used to study this disease, such as functional magnetic resonance imaging (fMRI). Therefore, the central goals of this thesis were: 1) to evaluate the feasibility of performing fMRI in anesthetized and awake animals, 2) to assess fMRI responses under various perturbations of cerebral perfusion and tissue oxygenation in order to identify key factors that may modulate functional signal changes following ischemia, and 3) to utilize fMRI, behavioral tests and histology in an anesthetized animal model of transient focal cerebral ischemia to explore postischemic changes in brain pathology/function and how they relate to changes in behavior. In the first study of this dissertation, I report the evaluation of fMRI responses in anesthetized and awake animals. Anesthesia is frequently used in animal models of cerebral ischemia, but is known to alter brain perfusion and metabolism which may, in turn, affect fMRI responsivity. Perfusion-based fMRI was used to evaluate cerebral blood flow (CBF) and blood oxygenation level-dependent (BOLD) responses to hypercapnia in awake and isoflurane-anesthetized rats. Hypercapnia produced significant CBF and BOLD fMRI signal changes throughout the cerebrum in awake and isoflurane-anesthetized groups. These results show that perfusion-based fMRI can successfully detect stimulus-evoked hemodynamic changes in the brains of both conscious and isoflurane-anesthetized animals. The second study of this dissertation: 1) investigates the effects of alterations in cerebral perfusion and oxygenation on fMRI signal changes, and 2) examines the self-consistency of an imaging-based formalism for the calculation of the cerebral metabolic rate of oxygen (CMRO2). Functional MRI responses to a stimulus can be described in terms of relative or absolute signal change. A relative fMRI response is defined as a percent-change relative to its own respective baseline value. An absolute fMRI response is defined as a quantitative change relative to a single fixed baseline value that serves as a control. Thus, an absolute fMRI signal change is largely independent of the baseline state and may more accurately index brain activity when baseline fMRI signals change significantly over time due to, for example, hemodynamic-metabolic disturbances that occur during and/or after brain ischemia. To address these issues, the effects of inspired hypoxic, normoxic, hyperoxic, and hypercapnic gases on baseline and forepaw stimulation-evoked changes in BOLD and CBF fMRI signals were examined in isoflurane-anesthetized rats. Relative fMRI responses to forepaw stimulation varied-whereas. absolute responses were similar--across gas conditions. These results demonstrate that absolute measurements of fMRI signal change may lend a more accurate measure of brain activity during states of altered basal physiology as well as support the self-consistency of the imaging-based CMRO2 formalism under these conditions. The third and last study of this dissertation utilized multimodal MRI, behavioral tests, and histology at acute to chronic periods following transient middle cerebral artery occlusion (tMCAO) in the rat to examine the evolution of pathological, functional, and behavioral parameters following transient focal cerebral ischemia. MRI was used to track the evolution of brain pathology and function following cerebral ischemia, and it was found that the cerebral sensorimotor network, critical for sensory and motor behavioral functions, showed profoundly abnormal signal changes that required up to one day to normalize. Adhesive removal, forepaw placement and beam-walk behavioral tests demonstrated sensorimotor dysfunctions that gradually improved but remained long after the recovery of MRI parameters. Postmortem histology confirmed the presence of selective neural cell death within the sensorimotor network at time points when behavior was abnormal. These results suggest that subtle postischemic pathological changes in the brain undetectable by MRI may be responsible for persistent behavioral deficits-a finding which may be relevant to a clinical subset of patients with persistent neurological morbidities despite negative MRI results following cerebral ischemia.

Cerebral ischaemia is a leading cause of disability and death world-wide. The only effective treatments are thrombolytic therapy (plasminogen activator; tPA) and hypothermia (33?C). However, tPA has limited clinical application due to its short therapeutic time window and its specific application in thrombo-embolic stroke. Moderate hypothermia (33?C) is only being used following cardiac arrest in comatose survivors. Hence more treatments are urgently required. The first step in developing new treatments is the identification and characterisation of a potential therapeutic target. Since brain damage following cerebral ischaemia is associated with disturbances in intracellular calcium homeostasis, the sodium-calcium exchanger (NCX) is a potential therapeutic target due to its ability to regulate intracellular calcium. Currently, however there is uncertainty as to whether the plasma membrane NCX has a neuroprotective or neurodamaging role following cerebral ischemia. To address this issue I compared hippocampal neuronal injury in NCX3 knockout mice (Ncx3-/-) and wild-type mice (Ncx3+/+) following global cerebral ischaemia. In order to perform this study I first established a bilateral common carotid occlusion (BCCAO) model of global ischaemia in wild-type C57/BlHsnD mice using controlled ventilation. After trials of several ischaemic time points, 17 minutes was established as the optimum duration of ischaemia to produce selective hippocampal CA1 neuronal loss in the wild-type mice. I then subjected NCX3 knockout and wild-type mice to 17 minutes of ischaemia. Following the 17 minute period of ischaemia, wild-type mice exhibited 80% CA1 neuronal loss and 40% CA2 neuronal loss. In contrast, NCX3 knockout mice displayed > 95% CA1 neuronal loss and 95% CA2 neuronal loss. Following experiments using a 17 minute duration of global ischaemia, a 15 minute duration of ischaemia was also evaluated. Wild-type mice exposed to a 15 minute period of ischaemia, did not exhibit any significant hippocampal neuronal loss. In contrast, NCX3 knockout mice displayed 45% CA1 neuronal loss and 25% CA2 neuronal loss. The results clearly demonstrate that mice deficient for the NCX3 protein are more susceptible to global cerebral ischaemia than wild-type mice. My findings showing a neuroprotective role for NCX3 following ischaemia, suggest that the exchanger has a positive role in maintaining neuronal intracellular calcium homeostasis. When this function is disrupted, neurons are more susceptible to calcium deregulation, with resultant cell death via calcium mediated pathways. Therefore, improving NCX activity following cerebral ischaemia may provide a therapeutic strategy to reduce neuronal death.

Les défauts de translation des études précliniques vers les essais cliniques dans le domaine des AVC ischémiques et l'échec des développements thérapeutiques pourraient être expliqués par trois aspects : (1) le manque de compréhension des mécanismes des deux formes de rtPA, le traitement pharmacologique de l'AVC; (2) le manque d'outils adaptés d'imagerie de perfusion chez le petit animal et (3) l'influence de l'anesthésie sur l’effet des traitements testés dans les modèles animaux.Le tPA utilisé en clinique (Actilyse®) est un mélange de deux formes de tPA: une forme chaîne simple (sc-rtPA) et une double chaîne (tc-rtPA). Malgré des activités fibrinolytiques similaires, ces deux formes de tPA exercent des fonctions cérébrales distinctes influençant différemment la récupération des patients. Ainsi, nous avons décidé d'étudier en détail dans un modèle murin d'AVC thromboembolique les mécanismes pouvant expliquer ces effets divergents. Nous avons confirmé ces observations à savoir que sc-rtPA est bénéfique lorsqu'il est perfusé tôt après le début de l'AVC, alors que le tc-rtPA est délétère en raison d'une altération de la barrière hémato-encéphalique.L'imagerie en temps réel de la perfusion de l'ensemble du cerveau est un atout pour les études cliniques et précliniques. L'émergence des ultrasons ultra-rapides a conduit au développement du Doppler ultra-rapide et de la Microscopie de Localisation à Ultrasons (ULM), deux méthodes avec différents profils de résolutions spatio-temporelles et une excellente sensibilité aux petits flux sanguins. Nous avons combiné ces deux méthodes pour fournir un suivi longitudinal en 3D de la perfusion cérébrale dans un modèle murin d’AVC thromboembolique avec fibrinolyse par rtPA. Nos données montrent que le FUS et l’ULM présentent un intérêt majeur pour le pronostic précoce de l'AVC ischémique et de la réponse au traitement, avec une corrélation étroite entre la reperfusion précoce à 2h et la récupération tissulaire à 24h.Enfin, l’anesthésie utilisée en laboratoire interfère sur la lésion ischémique et les effets des molécules thérapeutiques testées. Nous nous sommes affranchis de ces effets en développant un nouveau modèle d’AVC ischémique chez des souris totalement éveillées. Le débit sanguin cérébral régional a été suivi par laser Doppler avant, pendant et 45min après le début de l’AVC. Le traitement par rtPA (à 20 min) est bénéfique dans les modèles d’AVC vigile et anesthésié, mais l'anesthésie est associée à un manque de corrélation entre la recanalisation et les volumes de lésion post-ischémie. Nous testons actuellement une molécule neuroprotectrice, qui était prometteuse avant d’échouer lors des essais cliniques (NXY-059), afin d’évaluer la pertinence de ce modèle novateur d’AVC pour les futures études pharmacologiques. Dans l’ensemble, ce travail fournit un panel de données précliniques innovantes pour améliorer nos chances de translation en clinique, incluant un modèle pertinent d'AVC thromboembolique chez des animaux vigiles et une méthode d'imagerie du pronostic précoce de réponse aux traitements vasculaires
The lack of translation between preclinical studies and clinical trials in the field of ischemic stroke and the failure of therapeutic developments could be explained by three aspects: (1) the lack of understanding the mechanism of the two forms of tPA, the pharmacological treatment in stroke; (2) the lack of optimized perfusion imaging tools for small animal and (3) the influence of anesthesia on treatment tested in animal models.tPA used in the clinical setting (Actilyse®) is a mix of two forms of tPA: single chain form (sc-rtPA) and two chains form (tc-rtPA). Despite similar fibrinolytic activities, these two forms exert distinct brain functions therefore influencing differentially the outcome patients. We then decided to further investigate in a relevant model of thromboembolic stroke in rodents, the mechanisms that can explain these differential effects. Here, we have confirmed differential outcomes of the two forms: whereas sc-rtPA is clearly beneficial when infused shortly after stroke onset, tc-rtPA is deleterious due to an increased alteration of the blood brain barrier integrity.Live imaging of cerebral perfusion of the whole brain is an asset for both clinical and preclinical studies. The emergence of ultrafast ultrasound led to the development of ultrafast Doppler (fUS) and Ultrasound Localization Microscopy (ULM), two methods with different sets of spatio-temporal resolutions and excellent sensitivity to small blood flows. We combined these two methods to provide a longitudinal monitoring of whole brain perfusion using the thromboembolic stroke model in mice with rtPA-induced reperfusion. Our data show that fUS and ULM are of major interest for early prognosis of ischemic stroke and response to treatment, with a tight correlation between early reperfusion at 2h and tissue recovery at 24h. Finally, we develop a relevant awake ischemic stroke model to test new therapies, avoiding interferences due to anesthesia commonly used during in vivo studies mice. The patern of the MCA was followed using Laser Doppler monitoring before, during and 45 min after the stroke onset. Although rtPA treatment is beneficial in both awake and anesthetized stroke models, anesthesia is associated with a lack of correlation between recanalization and stroke outcome. We are now testing a neuroprotective molecule, which was promising before failing in clinical trials (NXY-059), to assess the relevance of this innovative stroke model for future pharmacological studies. Altogether, we provide here a set of innovative pre-clinical data to improve our chance of translation to clinic, including a relevant model of thromboembolic stroke in awake animals and an early prognosis imaging method of response to vascular treatments

Neuroprotection with anesthetics has been studied for many decades; important advances in this field have modified the way Anesthesiologists treat patients in the operating room. Animal models have played an important role in the study of ischemia in the operating room. Recent studies have demonstrated that the effect of anesthetics seems to be different in different animal models. We decided to evaluate anesthetics in a well-known model of cerebral ischemia and also in hypotensive models designed by us. We used a model of cerebral ischemia (MCAO) to test anesthetics neuroprotective effect in a two-week period. Then, we used a model of hypotension to characterize the damage caused by this type of insult. Finally we characterized a model of hypotension plus hypoxia that can mimic real situations in the OR. We found that anesthetics alone do not have a neuroprotective effect after two weeks in the MCAO model; but the combination of anesthetics with caspase inhibitors can decrease the damage caused by ischemia. The caspase inhibitor by itself did not show a significant neuroprotective effect. We also found that repetitive periods of profound hypotension can cause important damage in the hippocampus but no memory or neurological changes were seen. The induction of only one episode of hypotension plus hypoxia did not alter the morphology of the hippocampus although induced memory changes that were reverted by the use of anesthetics.

An interruption of the blood supply to the brain, as occurs during ischemic stroke, results in a rapid decline of ATP levels and a subsequent loss of neuronal function and viability. Under physiological conditions the brain reuses ATP degradation metabolites, such as hypoxanthine, via the purine salvage pathway, to restore its ATP pool. However, the massive degradation of ATP during ischemia results in the accumulation and loss of diffusible purine metabolites and thereby leads to a reduction in the post-ischemic ATP pool size, leaving the brain more vulnerable to secondary ischemic insults (recurrent strokes) and less able to deploy reparative mechanisms. The aim of this study was to improve the recovery of post-ischemic ATP levels by enhancing the purine salvage pathway, with substances that are already known to be tolerated in humans. Using acute hippocampal rat brain slices, I found that 1 mM Ribose (Rib) and 50 μM Adenine (Ade), two main metabolites of the purine salvage pathway, significantly increased the tissue ATP levels under basal conditions. Rib/Ade pre-treatment results in accelerated decline of synaptic transmission after onset of oxygen/glucose deprivation (OGD), due to increased adenosine release. However, this intervention does not delay the onset of anoxic depolarisation, or improve the recovery of synaptic transmission after prolonged ischemic periods. Pre-treatment of brain slices with 1 mM creatine, which increases phosphocreatine levels and thereby buffers the rapid decline of ATP levels upon energy shortage, significantly delays the onset of AD and helps to improve the recovery of synaptic transmission. By using cultured cerebellar granule cells, for more protracted studies on cell viability after OGD, I show that addition of Rib/Ade after ischemia helps to improves cell viability. Therefore my results suggest that both, delaying the decline of ATP upon onset of OGD (pre-treatment with creatine), or enhancing the post-ischemic recovery of ATP (post-treatment with Rib/Ade) are useful strategies to improve cell survival and function after in vitro ischemia.

Ziele der hier vorliegenden Arbeit waren eine immunhistochemische Analyse von GFAP (‚glial fibrillary acidic protein’) und GLUT-1 (Glukosetransporter-1) nach fokaler zerebraler Ischämie sowie deren mögliche Beeinflussung durch eine intravenöse Transplantation autologer mononukleärer Knochenmarkzellen (mKMZ) im Schafmodell. Eine differenzierte Analyse der Zielstrukturen in grauer und weißer Substanz (GS bzw. WS) sollte Aufschluss über eventuell unterschiedliche Reaktionsmuster liefern. Das Gehirnmaterial von zehn Tieren der bereits 2006/2007 stattgefundenen Studie, welche mit PET und MRT-Untersuchungen sowie der Durchführung von Verhaltenstests einherging, wurde retrospektiv im Rahmen der vorliegenden Arbeit untersucht. Je fünf gehörten zu einer Kontroll- bzw. Therapiegruppe (KG bzw. TG). Bei allen Versuchstieren wurde durch die permanente Okklusion der linken mittleren Zerebralarterie (pMCAO) eine fokale zerebrale Ischämie im Bereich des Neokortex hervorgerufen. Die Tiere der Therapiegruppe erhielten 24 Stunden nach dem Eingriff eine Transplantation autologer mKMZ (4x106/kg KGew). Nach sieben Wochen wurden die Versuchstiere getötet, ihre Schädel perfundiert und ihre Gehirne fixiert. Eine Lamelle der Gehirne wurde für die anschließende histologische Untersuchung in 30% Saccharose konserviert. Nach der Etablierung der Antikörper GFAP und GLUT-1 wurden vier Regionen der Gehirn-lamellen immunhistochemisch markiert und abschließend qualitativ und quantitativ analysiert. Die Regionen I (infarktnah) und III (infarktfern) lagen in der ipsilateralen Hemisphäre, die Regionen II (korrespondierend zu Region I) und IV (korrespondierend zu Region III) in der kontralateralen Hemisphäre. Durch den höheren Substanzverlust an Gehirnmasse in der ipsi-lateralen Hemisphäre der KG, wurden in dieser Tiergruppe die Regionen III und IV nicht ausgewertet. Vor der Analyse sind die physiologischen Markierungsmuster der vier Regionen in grauer und weißer Substanz an zwei gesunden Tieren (Prozesskontrolle) aufgezeigt worden. Durch die elektronenmikroskopische Untersuchung von Präparaten und anhand von GFAP/GLUT-1 doppelmarkierten Präparaten konnte festgestellt werden, dass die Astrozytenendfüßchen durch den hier verwendeten GLUT-1 Antikörper nicht markiert wur-den, sondern dass alleinig die gefäßständige, 55 kDa schwere Isoform detektiert worden ist. Die fokale zerebrale Ischämie führte in beiden Gruppen zu einer hochgradigen reaktiven Astrogliose mit Ausprägung einer Glianarbe in Region I. Protoplasmatische Astrozyten der grauen und fibrilläre Astrozyten der weißen Substanz zeigten hypertrophe Veränderungen. Die reaktive Astrogliose von Region I spiegelte sich in einer erhöhten GFAP-Dichte wider (p<0,05 in der Therapiegruppe). Region III hatte die gleiche GFAP-Dichte wie die Regionen II und IV. Der direkte Vergleich zwischen den Regionen I der beiden Gruppen zeigte Veränderungen der GFAP-Dichte durch die Zelltherapie auf: In der GS der Therapiegruppe lag eine geringere GFAP-Dichte vor, in der WS eine höhere (≠ p<0,05; GS und WS). Die Ergebnisse der GLUT-1-Analyse sind denen der GFAP-Analyse sehr ähnlich. Durch den Schlaganfall ist es zu einer erhöhten GLUT-1-Expression in GS und WS (p<0,05 WS) von Region I der Kontrollgruppe gekommen. Auch in Region I der Therapiegruppe konnten er-höhte GLUT-1-Dichten in GS und WS (p<0,05 WS) detektiert werden, zusätzlich dazu lag in der GS von Region III der Therapiegruppe eine erhöhte GLUT-1-Dichte vor (p<0,05). Der Vergleich zwischen beiden Gruppen zeigte Veränderungen durch die Therapie für die Regio-nen I und II auf. Die GLUT-1-Dichte der WS war in beiden Regionen in der TG erhöht (p<0,05), die GS von Region I zeigte in der Therapiegruppe eine geringere GLUT-1-Dichte. Ein Schlaganfall führt zu einer Erhöhung der GFAP sowie GLUT-1-Dichten in WS und GS im infarktnahen Gebiet. Durch die Transplantation von 4x106 autologen mononukleären Knochenmarkzellen pro kg KGew 24 Stunden nach dem Schlaganfall können diese Strukturen in ihren Expressionsmustern beeinflusst werden, dabei reagieren graue und weiße Substanz unterschiedlich: Die GS mit einer Verringerung, die WS mit einer Erhöhung der GFAP- bzw. GLUT-1-Dichte (p<0,05 WS, GLUT-1). Die Funktionskreisläufe in infarktfernen Regionen sind sieben Wochen nach dem Schlaganfall auf Astrozytenebene normalisiert (vgl. Region III). Die erhöhte GLUT-1-Dichte (p<0,05) in der GS der infarktfernen Region ist möglicherweise mit einem erhöhten Glukosemetabolismus in Verbindung zu setzen. Dies kann jedoch erst durch die Auswertung der FDG-PET-Daten beantwortet werden. Ob die durch Transplantation autologer mKMZ festgestellten Veränderungen der GFAP- und GLUT-1-Dichte in der Therapiegruppe zusätzlich mit einer verbesserten motorischen Leistung der Tiere einhergingen, wird erst durch die Analyse der Daten aus den Verhaltenstests festgestellt werden können.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2002.
Includes bibliographical references.
Diffusion (DWI) and perfusion weighted (PWI) magnetic resonance imaging (MRI) provide significant insight into acute stroke and can potentially be useful for clinical decision-making. In particular, current therapeutic decisions for acute human cerebral ischemia are typically based on time of symptom onset, limiting the number of patients treated. Imaging, however, offers insight into the physiologic integrity of brain tissue that is not attainable with time of symptom onset alone. This thesis extends existing imaging techniques for acute human stroke in order to improve identification of tissue at risk of infarction, thereby assisting clinical decision-making at the stage when intervention may be most effective. DWI and PWI have both been shown to identify infarcted tissue earlier than conventional stroke imaging. However, these techniques are limited in their existing implementations. DWI in most acute stroke settings has been restricted to isotropic imaging, measuring only mean diffusivity. In this thesis, DWI is extended to diffusion tensor imaging (DTI) with results demonstrating that DTI can detect ultrastructural changes in acute human stroke. PWI measures perfusion status by tracking the first pass of a bolus of contrast agent. In this dissertation, using numerical simulations, delay in contrast agent arrival is found to result in biased estimates of perfusion indices. A deconvolution technique using a block-circulant matrix is therefore proposed to compensate for delayed arrival, and its performance is compared to non-block circulant techniques in simulations as well as in clinically acquired human data sets.
(cont.) The results show that decoupling delay-associated effects reduces bias in tissue perfusion estimates. Algorithms combining DWI and PWI information are also evaluated to determine whether they predict tissue outcome in acute stroke better than models using only subsets of these parameters. Results show that algorithms combining DWI and PWI on a voxel-by-voxel basis predict tissue that infarct with higher specificity and sensitivity than algorithms using DWI or PWI individually. These combination algorithms are then used to investigate the efficacy of a novel therapeutic agent by evaluating the performance of the model as a function of treatment dose. Findings suggest that predictive models allow evaluation of novel therapies using smaller sample sizes than traditional endpoints. The results of this dissertation demonstrate that imaging can be used to identify tissue at risk of infarction, which may aid diagnosis and prognosis by providing clinicians unique insight into the underlying pathophysiology of stroke.
by Ona Wu.
Ph.D.

This thesis concerns the possible existence of central nervous system (CNS) chemosensitive mechanisms influencing sympathetic activity. The thesis is based on observations of sympathetic neuron and cardiovascular responses to CNS ischemia, systemic hypoxia and systemic hypercapnia. Investigation of the pressor response to cerebral ischemia in the cat indicates that it is mediated by superficial regions of the ventral medulla also involved in the pressor response to central hypercapnia. Experiments concerning the sympathetic response to systemic hypoxia in the CNS-intact sino-aortic denervated cat revealed a two-component response of the firing rates of single sympathetic preganglionic neurons (SPN), the mass activity of the cervical sympathetic trunk, and the neurogenic component of hindlink vascular resistance (N-HLVR). The response consisted of: (i) an increase of all three variables during extreme hypoxia, and (ii) a decrease during moderate hypoxia. The hypoxic sympatho-depression resulted from loss of central respiratory input to SPNs as well as of respiration-independent input. The hypoxic sympatho-excitation involved only the latter input. Investigation of the sympathetic response to systemic hypercapnia in the acute C$ sb1$ spinal cat demonstrated a direct relationship between SPN firing rate or N-HLVR and arterial PCO$ sb2$ between normocapnia and severe hypercapnia. N-HLVR also increased in this preparation during systemic hypoxia.

Cette thèse porte sur l’étude de la séquence d’imagerie IRM IVIM (« Intravoxel incoherent motion »). Cette séquence permet l’étude des microvaisseaux sanguins tels que les capillaires, artérioles et veinules. Pour être sensible seulement aux groupes de spins non statiques dans les tissus, des gradients de diffusion sont ajoutés avant et après l’impulsion 180° d’une séquence d’écho de spin. La composante du signal correspondant aux spins qui diffusent dans le tissu peut être séparée de celle des spins en mouvement dans les vaisseaux sanguins qui est appelée signal IVIM. Ces deux composantes sont pondérées par f IVIM qui représente la fraction volumique du sang à l’intérieur du tissu. Le signal IVIM est en général modélisé par une fonction mono-exponentielle (ME) caractérisée par un coefficient de pseudo-diffusion D*. Nous proposons un modèle IVIM bi-exponentiel formé d’une composante lente caractérisée F slow et D* slow qui correspondrait aux capillaires comme dans le modèle ME, et d’une composante rapide caractérisée par F fast et D* fast qui correspondrait à des vaisseaux plus gros comme des artérioles et veinules. Ce modèle a été validé expérimentalement et des informations supplémentaires ont été obtenues en comparant les signaux expérimentaux avec un dictionnaire de signaux IVIM simulés numériquement. L’influence de la séquence d’impulsions, du temps de répétition et du temps d’encodage de diffusion a également été étudiée. Enfin, la séquence IVIM a été appliquée à l’étude d’un modèle animal de la maladie d’Alzheimer
This PhD thesis is centered on the study of the IVIM (“Intravoxel Incoherent Motion”) MRI sequence. This sequence allows for the study of the blood microvasculature such as the capillaries, arterioles and venules. To be sensitive only to moving groups of spins, diffusion gradients are added before and after the 180° pulse of a spin echo (SE) sequence. The signal component corresponding to spins diffusing in the tissue can be separated from the one related to spins travelling in the blood vessels which is called the IVIM signal. These two components are weighted by f IVIM which represents the volume fraction of blood inside the tissue. The IVIM signal is usually modelled by a mono-exponential (ME) function and characterized by a pseudo-diffusion coefficient, D*. We propose instead a bi-exponential IVIM model consisting of a slow pool, characterized by F slow and D* slow corresponding to the capillaries as in the ME model, and a fast pool, characterized by F fast and D* fast, related to larger vessels such as medium-size arterioles and venules. This model was validated experimentally and more information was retrieved by comparing the experimental signals to a dictionary of simulated IVIM signals. The influence of the pulse sequence, the repetition time and the diffusion encoding time was also studied. Finally, the IVIM sequence was applied to the study of an animal model of Alzheimer’s disease

Background. Over the past decades metabolic profiling has become a valuable tool for the investigation of metabolic changes specific to particular diseases. As this concept considers participants of several metabolic networks and processes within one analyt- ical step, it has been considered suitable for the investigation of cellular mechanisms underlying brain diseases whose pathological backgrounds are assumed to be inher- ently complex. One main technical platform of metabolic profiling is based on in vitro nuclear magnetic resonance (NMR) spectroscopy: a technique that is also available for the non-invasive application in vivo. Hence, it provides a promising mean for the translation from in vitro research to the eventual clinical use. Aim. The studies presented in this thesis applied in vitro NMR spectroscopy based metabolic profiling to extracts of cerebral tissue samples of rodent models of mental and neurodegenerative disorders to evaluate its ability as search tool for biomarkers in these conditions. Method Validation. To verify the comparability between the performance of the in vitro and in vivo application of nuclear magnetic resonance spectroscopy, tissue ex- traction methods were firstly tested for metabolic content, precision and suitability for metabolic profiling studies. Secondly, the in vitro results of the study of socially isolated rats were compared to in vivo results of the same model. This study demonstrated that the two modalities produced similar outcomes with respect to the (patho)physiological research questions.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May, 2005" Includes bibliographical references.

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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
The aim was to determine the effect of ethanolic extract of pequi mesocarp on induced brain damage and ERK1/2 and AMPKα active forms in rats subjected to a hypercaloric diet. 48 rats were sorted into two groups of 24 according to the diet used, hypercaloric or commercial, provided daily for 60 days. The animals were separated into two subgroups of 12, treated or not with the extract, given daily for 30 days after diet start. Quantification of triglycerides, induction of global cerebral ischemia followed by reperfusion was performed. Later, euthanasia, brains harvest and visceral fat weighing were performed. Histopathological evaluation of brain lesions, quantification of the number of viable and non-viable cells in the cerebral cortex and hippocampus, and of cells marked by the anti-pKR-ERK1/2 and p-AMPKα antibodies in the cerebral cortex were performed. Hypertriglyceridemia and a significant increase in the amount of visceral fat were observed in the group that received hypercaloric diet (p <0.05). Histopathological evaluations showed that the group that received hypercaloric diet and was treated with the extract had fewer brain lesions of ischemia and reperfusion. The extract did not influence the number of viable and nonviable cells in the cerebral cortex and hippocampus, but significantly reduced p-ERK1 / 2 and p-AMPKα cell labelling in the hypercaloric diet group (p <0.05). In conclusion, ethanolic extract of pequi peel reduces induced brain lesions in rats fed a hypercaloric diet and has a modulatory effect on the expression of ERK1 / 2 and AMPKα in the cerebral cortex.
O objetivo com esse estudo foi determinar o efeito do extrato etanólico da casca de pequi sobre dano cerebral induzido em ratas submetidas a dieta hipercalórica e as expressões das formas ativas da ERK1/2 e AMPKα no córtex cerebral. Utilizaram-se 48 ratas alocadas em dois grupos de 24, de acordo com a dieta utilizada, hipercalórica ou comercial, fornecidas diariamente por 60 dias. Os animais foram distribuídos em dois subgrupos de 12, tratados ou não com o extrato, administrado diariamente 30 dias após o início das dietas. Foi realizada quantificação dos triglicerídeos e indução de isquemia cerebral global seguida de reperfusão. Seguido à eutanásia, colheram-se os encéfalos e pesou-se a gordura visceral. Foram feitas avaliação histopatológica das lesões no encéfalo, quantificação do número de células viáveis e inviáveis no córtex cerebral e hipocampo e células marcadas pelos anticorpos anti p-ERK1/2 e p-AMPKα no córtex cerebral. Havia hipertrigliceridemia e aumento significativo na quantidade de gordura visceral no grupo que recebeu dieta hipercalórica (p < 0,05). O grupo que recebeu dieta hipercalórica e foi tratado com o extrato apresentou menos lesões cerebrais de isquemia e reperfusão. O extrato não influenciou o número de células viáveis e inviáveis no córtex cerebral e hipocampo, porém, reduziu significativamente a marcação pela pERK1/2 e p-AMPKα no grupo da dieta hipercalórica (p < 0,05). Concluiu-se que o extrato etanólico da casca de pequi reduz lesões cerebrais induzidas em ratas alimentadas com dieta hipercalórica e apresenta efeito modulatório sobre a expressão da ERK1/2 e da AMPKα no córtex cerebral.

This thesis investigates the behavioural and anatomical correlates of recovery from motor cortex damage in rats. The effectiveness of behavioural, pharmacological, and regenerative treatments was investigated using models of focal stroke. Chronic bilateral motor deficits were found after motor cortex damage induced by various methods. These behavioural deficits were similar in severity and duration although they were correlated with different patterns of reorganization seen in Golgi-stained tissue. Animals with motor cortex injury benefited from postinjury olfactory stimulation, chronic administration of nicotine, and infusions of epidermal growth factor followed by erythroprotein. Different mechanisms of plasticity in remaining cortical circuits are discussed as possible candidates responsible for the behavioural improvement. The current thesis expands the current knowledge of the effects of adult cortical damage to ares critical to motor control. It may also stimulate research on therapies and possible mechanisms that might enhance recovery after stroke.
xviii, 299 leaves : ill. (some col.) ; 29 cm.

Stroke is a leading cause of neurological disability, often resulting in long-term motor impairments due to damage to the striatum and/or motor cortex. While both humans and animals show spontaneous recovery following stroke, little is known about how the injury location affects recovery and what causes recovery to plateau. This information is essential in order to improve current rehabilitation practice and develop new therapies to enhance recovery. In this thesis, we used endothelin-1 (ET-1), a potent vasoconstrictor, to produce focal infarcts in the forelimb motor cortex (FMC), the dorsolateral striatum (DLS) or both the FMC and DLS in male Sprague-Dawley rats. In the first experiment, the spontaneous recovery profile of animals was followed over an 8-week period using multiple behavioural tasks assessing motor function and limb preference to identify how recovery varies depending on injury location. Infarct volumes were measured to determine the association between injury and behavioural outcome. All three groups had significant functional impairments on the Montoya staircase, beam traversal, and cylinder tests following stroke, with the combined group having the largest and most persistent impairments. Importantly, spontaneous recovery was not simply dependent on lesion volume but on the lesion location and the behavioural test employed. In the second experiment, we focused on a potential cellular mechanism thought to underlie post-stroke plasticity and functional recovery. In a separate cohort of animals, we assessed how plasticity-limiting perineuronal nets (PNNs) and associated parvalbumin-positive (PV) GABAergic interneurons change following similar ET-1 strokes as in the prior experiment. A significant reduction in the density of PNNs was observed in the perilesional cortex of animals that received a cortical-only or combined stroke but not a striatal-only injury. Although there were no significant differences in the density of PV interneurons between sham and stroked groups, a significant negative correlation existed between cortical infarct volume and the density of PV interneurons in the perilesional cortex. Taken together these results demonstrate that lesion location influences motor recovery and neuroplastic changes following stroke. This supports the idea that a “one size fits all” approach for stroke rehabilitation may not be effective and treatment needs to be individualized to the patient.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
A obstruÃÃo das artÃrias uterinas promove isquemia e/ou necrose no Ãtero, no entanto nÃo se conhece com que intensidade essas lesÃes ocorrem. Os objetivos deste estudo sÃo: descrever e padronizar uma tÃcnica de ligadura da artÃria uterina (LAU) direita em ratas nÃo grÃvidas e avaliar os efeitos deste modelo em Ãteros e ovÃrios de ratas. Estudo experimental, utilizando 64 ratos, 48 fÃmeas e 16 machos (Rattus norvergicus, variedade albina) maduros, de fertilidade comprovada. As ratas foram alocadas aleatoriamente em 8 grupos, de seis indivÃduos. Quatro grupos foram submetidos à tÃcnica de LAU direita e sacrificados nos dias 1, 7, 14 e 21 apÃs o procedimento. Outros 3 grupos foram acasalados nos dias 1, 7 e 14 apÃs a LAU, e comparados com o grupo controle quanto à fertilidade. ApÃs o sacrifÃcio, eram retirados, para anÃlise histopatolÃgica, os ovÃrios, e os hemi-Ãteros. Realizou-se anÃlise histolÃgica, que avaliou o Ãtero quanto à congestÃo, hemorragia, edema intersticial e perda de coesÃo celular atravÃs de um escore que varia de 0 a 3. Os ovÃrios foram avaliados de acordo com o nÃmero de folÃculos em desenvolvimento e corpos lÃteos. Para a anÃlise estatÃstica foi utilizado o software SPSS (Statistical Package for Social Sciences) versÃo 13.0, p < 0,05 foi considerado estatisticamente significativo. O projeto de pesquisa foi enviado para a ComissÃo de Ãtica em Pesquisa Animal (CEPA) da Universidade Federal do CearÃ, com protocolo de nÃmero 90/08. O modelo da tÃcnica foi realizado em ratas nÃo-grÃvidas utilizando a ligadura na porÃÃo inferior da artÃria uterina direita, mantendo o hemi-Ãtero esquerdo (HUE) como controle. Em nenhum momento dos dias de sacrifÃcio, apÃs a LAU (1, 7, 14 e 21 dias), houve diferenÃa entre os escores histolÃgicos de isquemia dos hemi-Ãteros direitos (HUD) distais e proximais (p > 0,05). Os escores histolÃgicos de isquemia dos hemi-Ãteros direitos no decorrer do tempo aumentaram substancialmente a partir do 7 dia (p=0,003). Da mesma maneira ocorreu com os hemi-Ãteros esquerdos (p=0,001). A Ãnica diferenÃa significativa observada na comparaÃÃo dos escores dos hemi-Ãteros direito e esquerdo ocorreu no 1 dia apÃs LAU (p=0,026), à custa de congestÃo. Os ovÃrios esquerdos nÃo apresentaram alteraÃÃes no nÃmero de folÃculos e de corpos lÃteos apÃs LAU e, os ovÃrios direitos apresentaram nÃmero de folÃculos e corpos lÃteos semelhante ao controle a partir do 21 dia da LAU. A percentagem de ratas grÃvidas que foram submetidas a LAU foi de 44,4%, comparado com 100% das ratas controle que engravidaram (p=0,024). Observou-se ainda uma reduÃÃo na mÃdia do nÃmero de fetos por rata (p=0,029). Pode-se concluir que o modelo estabelecido foi efetivo e de fÃcil reprodutibilidade, bem como as alteraÃÃes histolÃgicas encontradas no Ãtero ocorrem de forma discreta. Em relaÃÃo a funÃÃo ovulatÃria, pode-se dizer que o nÃmero de folÃculos e corpos lÃteos dos ovÃrios direitos, a partir do 21 dia, permaneceram semelhantes aos dos esquerdos (controle). A fertilidade, porÃm, mostrou-se reduzida apÃs o estabelecimento desta tÃcnica.
The obstruction of the uterine arteries promotes ischemia or necrosis in the uterus. However it is not known how strongly these injuries occur. The objectives of this study are to describe and standardize a technique of right uterine artery ligation (UAL) in non-pregnant rats and to evaluate the effects of this model in the uteri and ovaries of female rats. An experimental study using 64 rats, 48 females and 16 males (Rattus norvegicus, albino variety) mature, with proven fertility. The rats were randomly allocated into 8 groups of six individuals. Four groups were subjected to the technique of right UAL and sacrificed on days 1, 7, 14 and 21 after the procedure. Other 3 groups were mated on days 1, 7 and 14 after the UAL, and compared with the control group regarding fertility. After sacrifice, ovaries and the hemi-uteri, were removed for histological analysis wich was carried out histological analysis, which evaluated the uteri and the congestion, hemorrhage, interstitial edema and loss of cell cohesion through a score ranging from 0 to 3. Ovaries were evaluated according to the number of follicles and corpora lutea. For statistical analysis we used SPSS software (Statistical Package for Social Sciences) version 13.0, p <0.05 was considered statistically significant. The research project was submitted to the Ethics Committee on Animal Research (ECAR) Federal University of CearÃ, under the number 90/08. The model of the technique was performed in non-pregnant rats using ligation in the lower portion of the right uterine artery, keeping the left hemi-uteri (LHU) as control. At no time during periods of ischemia established (1, 7, 14 and 21 days) there was any difference between the histological scores of ischemia of the right hemi-uteri (RHU) distal and proximal (p> 0.05). The histological scores of ischemia of the right hemi-uteri over time increased significantly from day 7 (p = 0.003), just as occurred with the left hemi-uteri (p = 0.001). The only significant difference observed when comparing the scores of the right hemi-uteri and left occurred on day 1 after UAL (p = 0.026), due to congestion. The left ovary showed no changes in the number of follicles and corpora lutea after UAL, and the right ovary showed a number of follicles and corpora lutea similar to control from the 21th day of UAL. The percentage of pregnant rats that were subjected to ischemia was 44.4%, compared with 100% of control rats that became pregnant (p = 0.024). There was also a reduction in the average number of fetuses per mother (p = 0.029). It can be concluded that the model established was effective and highly reproducible, and histological changes found in the uteri are mild. As to ovulatory function, one can say that the number of follicles and corpora lutea of the right ovaries, after 21 days, remained similar to the left (control). Fertility, however, was reduced after the establishment of this technique.

Middle cerebral artery (MCA) stroke can produce chronic incapacitating motor impairments. Understanding the neural basis of the motor syndromes is complicated by the diversity of neural structures damaged but the problem can be addressed in laboratory rats by inducing selective infarcts. Nevertheless, the motor syndromes that ensue from stroke in rats remain poorly understood and undermine its potential as a model for clinical stroke. The objective of the present thesis was to document the skilled reaching impairments from neocortical and subcortical MCA infarcts in rats. In addition, the integrity of the motor system components spared by the infarct was assessed neurophysiologically and neuroanatomically. Characteristic reaching impairments emerged from each infarct but there were also some overlapping features that might be explained by neural dysfunction extending beyond the boundaries of the infarct. The present studies showed that the laboratory rat is an ideal animal model for studying stroke, which should be of interest to both clinical and research scientists studying stroke.
xiii, 345 leaves : ill. ; 29 cm. + 1 CD-ROM

Orientadores: Ilka de Fátima Santana Ferreira Boin, Artur Udelsmann
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-27T00:05:18Z (GMT). No. of bitstreams: 1 Jorge_GracindadeLourdes_D.pdf: 6636878 bytes, checksum: 9167ec95670521d52d8c88a19fc3ceb9 (MD5) Previous issue date: 2015
Resumo: A oclusão vascular temporária do fluxo hepático é um dos procedimentos essenciais nas cirurgias hepáticas. Muitas pesquisas relacionadas à isquemia e reperfusão (I/R) em ratos Wistar, seja através de interrupção por pouco tempo, ou período maior, têm demonstrado sérias complicações causadas por lesões de I/R. Pesquisas demonstram grandes alterações metabólicas e fisiológicas quando analisadas durante, ou nas primeiras horas após o experimento. O objetivo deste trabalho foi avaliar as alterações hepáticas morfológicas e bioquímicas tardias, ocorridas após pinçamento total ou parcial do hilo hepático em ratos Wistar. Um total de 26 ratos Wistar, machos, peso médio 315,5 ± 61,5g foram utilizados em dois estudos. No primeiro estudo, 12 ratos foram divididos em dois grupos: Grupo Pinçamento do Ducto Biliar Comum (PDBC; n=6) foram submetidos à anestesia com tiopental sódico iv, à incisão abdominal até 2cm, tendo o ducto biliar isolado, dissecado e pinçado por 10 minutos. Após este tempo, a pinça foi retirada e a incisão fechada. Grupo Operação Simulada I (OSI; n=6) em condições de normalidade, os animais foram submetidos unicamente à anestesia e laparotomia e, posteriormente, a exames de controle. Nos dois grupos após o 28ºdia foram realizadas biópsias hepáticas e exames bioquímicos seguidos de eutanásia dos animais. Observamos que 83% dos animas do grupo PDBC apresentaram dilatação do colédoco, com alterações histológicas hepáticas: proliferação ductular, formação de septos, focos de necrose do parênquima, com formação de micro abcessos e alterações dos exames bioquímicos, quando comparados aos animais do grupo OSI (p < 0,05). No segundo estudo, 14 animais foram anestesiados com ketamina 5% (30mg/Kg) e xylazina 2% (30mg/Kg) via intraperitoneal. No grupo clampeamento intermitente do pedículo hepático (CIPH; n=7) os animais foram submetidos à incisão em U no abdome; o pedículo hepático foi isolado, dissecado e submetido a pinçamento, com micro pinça, intermitente por 4 ciclos de 5 minutos de isquemia, seguidos de 5 minutos de reperfusão, e a incisão foi fechada. No Grupo Operação Simulada II (OSII; n=7) os animais foram submetidos à anestesia, laparotomia e manipulação do pedículo hepático e,posteriormente, ao controle dos exames. Em todos os animais no 35o dia, após jejum de 12 horas, foi realizada nova anestesia para coleta da biópsia hepática e sangue para dosagem de alanina amino transferase (ALT) e de aspartato amino transferase (AST). A análise estatística foi realizada pelo teste Mann-Whitney para comparação de médias com significância de 5%. Observamos que todos os animais do grupo CIPH apresentaram dilatação do colédoco e aumento significativo nas enzimas hepáticas (p<0,05). Na avaliação histológica constatamos proliferação ductular (100% dos casos), septos porta-porta (42,8%), formação de nódulos (42,8%), focos de necrose (14,2%) e rolhas de bile (14,2%). No grupo OSII estas alterações não foram encontradas. Concluímos que o pinçamento do ducto biliar (10 minutos) foi suficiente para gerar importantes alterações morfológicas hepáticas e do colédoco, confirmadas através de análise enzimática e histológica, podendo, portanto, ser utilizado como modelo de obstrução biliar, visando estudos semelhantes. Constatamos, também, que o pinçamento intermitente do pedículo hepático provocou lesões semelhantes na árvore biliar e no parênquima hepático
Abstract: The temporary vascular occlusion of the hepatic blood flow is one of the essential procedures in hepatic surgery. Many researches are related to ischemia and reperfusion (I/R) in rats, either through interruption for a short time period or higher and all has shown serious complications caused by I/R injury. Researches have demonstrated, in the early analysis severe metabolic and physiological disorders but there are not reports about hepatic injuries after late analysis. The objective was to assess the morphological and biochemical hepatic late alterations occurring after total or partial hepatic pedicle clamping, in Wistar rats. A total of 26 male Wistar rats, weighting 315.6 ±61.9g were used in two studies. In the first, 12 rats were distributed into two groups: bile duct clamping group (BDCG; n = 6); anesthetized with thiopental sodium; with bile duct dissection, isolation and clamped for 10 minutes. After this time, the clamp was removed and the incision was closed. In simulated operation group I (SOGI; n = 6) the animals were submitted solely to anesthesia and laparotomy and subsequently control exams.On the 28th day, liver biopsies and biochemical exams were performed. All animals were sacrificed while still under anesthesia. We observed that 83% of the animals of BDCG group showed dilation of the common bile duct with hepatic histological changes such as ductular proliferation, septa formation, parenchymal foci of necrosis with formation of micro abscesses and changes of biochemical tests when compared to SOGI (p<0.05).In the second study, 14 animals were anesthetized with ketamine 5% (30mg/kg) and xylazine 2% (30mg/kg)intraperitoneally. In intermittent Hepatic Pedicle Group (IHPC; n = 7) the animals had the hepatic pedicle isolated, dissected and submitted to clamping applying 4 cycles of 5 minutes of ischemia followed by 5 minutes of reperfusion, and the incision was closed.In group operation simulated II (SOGII; n = 7), the animals were anesthetized, laparotomy and manipulation of the hepatic pedicle were performed. On the 35th day, after 12 hours fasting, anesthesia wasinduced for liver biopsy and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) collection. The Mann-Whitney test for comparison of means with significance of 5% was used. In result all the animals of IHPC group had a dilated common bile duct and the significant increase in hepatic enzymes (p <0.05). In histological evaluation we found ductular proliferation (100% of cases), septa port-portal (42.8%), nodules formation (42.8%), foci of necrosis (14.2%) and bile plugs (14.2%). In the group SOGII these changes were not found. We conclude that the short clamping time of the bile duct (for 10 minutes) was enough to cause major hepatic and bile duct morphological changes confirmed by enzymatic and histological analysis, and may therefore be used as biliary obstruction model in order to similar studies. We also noted that the intermittent clamping of the hepatic pedicle caused similar injuries in the biliary tree and in the hepatic parenchyma
Doutorado
Fisiopatologia Cirúrgica
Doutora em Ciências

Introdução: O aumento da pressão intracraniana (PIC) é um problema comum na prática neurocirúrgica, e a monitoração invasiva deste parâmetro faz parte da rotina de unidades de terapia intensiva. O Doppler transcraniano vem sendo testado na avaliação da hemodinâmica cerebral como parâmetro de avaliação não invasiva da PIC, porém há controvérsias na literatura sobre seu real benefício e utilidade nesta situação. Este estudo objetivou correlacionar os dados de avaliação do fluxo sanguíneo cerebral através da técnica de Doppler com as variações da monitoração invasiva da PIC na fase aguda de hipertensão intracraniana em um modelo animal. Métodos: Trata-se de um estudo experimental realizado em suínos. O experimento constou de dois grupos de animais (A e B) com hipertensão intracraniana gerada por insuflação com soro fisiológico de um balão no parênquima cerebral, sendo o grupo A com 4 mL e o grupo B com 7 mL. Nos dois grupos houve uma intervenção clínica com infusão de solução salina a 3% e uma simulação de intervenção cirúrgica (desinsuflação do balão). Em todos os momentos de insuflação do balão e das intervenções foram registrados os valores dos monitores de PIC e do Doppler: velocidades sistólica (VS), diastólica (VD), média (VM) do fluxo sanguíneo cerebral e índice de pulsatilidade (IP). Foram realizadas comparações do comportamento dos parâmetros avaliados pela ultrassonografia Doppler craniana (VS, VD, VM e IP) em relação às variações da PIC intraparenquimatosa. Resultados: Foram estudados 20 suínos sendo 10 no grupo A e 10 no grupo B. Um animal do grupo B foi excluído do estudo, pois foi a óbito antes do término do experimento. Após a insuflação do balão, como era de se esperar, a PIC no grupo B foi superior à do grupo A em todos os momentos, até a desinsuflação do mesmo. Realizada a correlação de Spearman observou-se correlação significativa entre IP e PIC, principalmente logo após insuflação do balão, ou seja, na elevação abrupta da PIC. Não houve correlação entre a PIC e os parâmetros VS, VD e VM. Também não houve variação significativa da PIC após infusão endovenosa de solução salina hipertônica. Conclusão: Este resultado demonstra o potencial do IP como bom parâmetro de avaliação de pacientes com suspeita de elevação hiperaguda e recente da PIC. Não se conseguiu demonstrar os mesmos resultados de correlação entre a PIC e as demais variáveis VS, VD e VM. Diante destes achados, adicionados aos dados conflitantes da literatura disponível até o momento, não se recomenda, por enquanto, a utilização desses parâmetros isoladamente como substitutos da monitoração invasiva da PIC, evidenciando a necessidade de mais estudos clínicos e experimentais
Introduction: Increased intracranial pressure (ICP) is a common problem in neurosurgical practice. Invasive monitoring of ICP in these cases is part of the intensive care unit routine. Transcranial Doppler has been tested in the evaluation of cerebral hemodynamics as a non-invasive evaluation of ICP, but there are controversies in the literature about its real benefit and usefulness in this situation. Thus, this study aimed to correlate the data of cerebral blood flow assessment using the Doppler technique and the invasive monitoring of ICP in the acute phase of intracranial hypertension in an animal model. Methods: This is an experimental study in pigs. During the experiment, an intracerebral expansive mass with an inflatable balloon was simulated. The experiment consisted of two groups (A and B) of animals with intracranial hypertension generated by a ballon inflation inside the cerebral parenchima, group A with 4 mL and group B with 7 mL. In both groups there was a clinical intervention with infusion of 3% saline solution and a simulation of surgical intervention (balloon drain out). The values of ICP and Doppler parameters (systolic (FVs), diastolic (FVd), and mean (FVm) cerebral blood flow velocities) were collected at all moments of balloon inflation and interventions, as well as the pulsatility index (PI). Comparisons of the behavior of the parameters evaluated by Doppler ultrasound (FVs, FVd, FVm and PI) were performed in relation to intraparenchymal ICP. Results: Twenty pigs were studied, 10 in group A and 10 in group B. One pig died in group B and it was excluded. After balloon inflation, as expected, ICP in group B was higher than in group A at all times, until the ballon was empty again. Significant correlation between PI and ICP was obtained when Spearman correlation was performed, mainly shortly after balloon inflation, that is, in the abrupt elevation of ICP. There was no correlation between ICP and FVs, FVd or FVm. There was also no significant change in ICP after intravenous infusion of hypertonic saline solution. Conclusion: These results demonstrate the potential of PI as a good parameter for the evaluation of patients with suspected ICP elevation. It was not possible to demonstrate the same correlation results between the ICP and FVs, FVd or FVm. Due to these results and also to the literature conflicting data to date, the use of these parameters alone as substitutes for the invasive monitoring of ICP is not recommended until now, which shows the need for further clinical and experimental studies

INTRODUÇÃO: O transplante de intestino (TI) estabeleceu-se como tratamento para pacientes com falência intestinal e complicações da nutrição parenteral. Entretanto, sepse continua sendo a principal causa de mortalidade. A lesão causada pela isquemia seguida de reperfusão (LIR) é apontada como um dos fatores de ruptura da barreira mucosa intestinal, com consequente translocação bacteriana e sepse, seja precocemente, por lesão epitelial direta, seja mais tardiamente pela sua associação com o desenvolvimento da rejeição celular aguda. Criou-se um modelo de TI em porcos jovens com a finalidade de estudar a LIR e seus efeitos no epitélio intestinal. MÉTODOS: Para a padronização do modelo, foram realizados 25 procedimentos, tendo sido testados os tamanhos dos animais, as soluções de preservação, o tipo de drenagem venosa, o tipo de reconstrução intestinal e o tempo de duração do experimento. Na pesquisa propriamente dita, 20 porcos jovens foram submetidos a TI ortotópico. Dois grupos foram determinados conforme o tempo de isquemia fria a que foi submetido o intestino: grupo 1 (n=12) 90 minutos (min) e grupo 2 (n=8) 180 min. O procedimento foi realizado sob técnica asséptica e as anastomoses vasculares realizadas entre a aorta do doador e a aorta infra-renal do receptor e a veia porta do doador e a veia cava inferior do receptor. O trânsito intestinal foi reconstruído através de anastomoses entre o jejuno proximal do doador e do receptor e o íleo terminal do doador e do receptor. A solução de preservação utilizada foi Euro Collins. Não foi administrada medicação imunossupressora, exceto pela metilprednisolona (20mg/kg) no momento da reperfusão. Fragmentos de intestino foram obtidos: 1 no momento da laparotomia do doador, o fragmento basal, considerado controle, 2 30 min após a reperfusão e 3 3 dias após o transplante. Os fragmentos assim obtidos foram submetidos a: 1 análise histológica com coloração de hematoxilina-eosina (HE), 2 análise imunoistoquímica para a detecção de infiltração da mucosa por neutrófilos (marcados pelos grânulos ricos em mieloperoxidase MPO), 3 análise histoquímica para quantificação de células epiteliais em apoptose pelo método TUNEL, 4 análise da expressão dos genes da endotelina-1 (ET-1) e da interleucina-6 (IL-6), do gene antiapoptótico Bcl-XL e do gene pró-apoptótico Bak. A análise estatística foi realizada utilizando-se o teste de Mann-Whitney na comparação entre os grupos e, na análise da evolução temporal da LIR em cada grupo, o teste de Friedman seguido do teste post hoc de Dunn quando detectada diferença estatisticamente significante. RESULTADOS: Não foram encontradas diferenças entre os grupos quanto às alterações histológicas estudadas. O grau de infiltração da mucosa por neutrófilos elevou-se significantemente nos dois grupos 30 min após perfusão, tendo persistido elevado 3 dias após o TI somente no grupo 2. O número de células epiteliais em apoptose detectadas pelo método TUNEL sofreu incremento significante apenas no grupo 1, 3 dias após o procedimento. As duas citocinas estudadas, IL-6 e ET-1 mostraram elevação significante 30 minutos após a reperfusão, tendo retornado aos níveis basais 3 dias após a cirurgia em ambos os grupos. Detectou-se redução significante da expressão do Bcl-XL somente no grupo 1, 3 dias após o TI. CONCLUSÕES: As citocinas estudadas estão envolvidas no processo de LIR nas fases iniciais do TI. Ocorre diminuição da expressão de gene anti-apoptótico e aumento do número de células em processo de morte celular de maneira mais intensa no grupo submetido a menor tempo de isquemia
INTRODUCTION: Intestinal transplantation (ITx) has become an accepted mode of treatment of intestinal failure patients who develop parenteral nutrition-related complications. Overall outcomes have dramatically improved but sepsis remains the leading cause of mortality. Ischemia-reperfusion injury (IRI) has been related to the development of sepsis due either to direct mucosal damage or to increased risk of acute cellular rejection. An experimental ITx model has been idealized in order to better characterize IRI-associated mucosal damage. METHOD: 25 procedures involving 75 outbred pigs were necessary to standardize the procedure. Weight of the animals, venous drainage, intestinal transit reconstruction as well as the time period the animals should be maintained alive were evaluated. Orthotopic ITx was performed in 20 hybrid pigs. Two groups were assigned according to cold ischemia time (CI): group 1 (n=12) 90 minutes (min), group 2 (n=8) 180 min. The procedure was performed under aseptic technique and portal drainage was adopted as standard. Intestinal transit reconstruction involved the performance of termino-terminal anastomosis between donor and recipient jejunum and donor and recipient terminal ileum. Euro-Collins was used as preservation solution. 20mg/kg of metilprednisolone was administered at reperfusion and no other immunosuppressive drug has been employed. Specimens were collected from the donor at laparotomy, and from the receptor, 30 min, and 3 days after reperfusion. Mucosal damage was assigned by histological evaluation with hematoxylin-eosin dye. Neutrophilic infiltration was quantified using myeloperoxidase (MPO) immunohistochemical assay and epithelial cell apoptosis was also assigned by means of TUNEL assay. Molecular biology involved the quantification of the expression of the IL-6, ET-1, Bak, and Bcl-XL genes. RESULTS: No statistical difference was detected between the groups as far as plain histological evaluation is regarded. Neutrophilic infiltration increased in a similar fashion in both groups, but lasted longer in group 2. Apoptosis detected by TUNEL showed significant increase in group 1, 3 days after surgery. Anti-apoptotic gene Bcl-XL had its expression decreased in group 1, in 3 days as well. Endothelin-1 and IL-6 genes expression increased 30 min after the procedure and had already returned to baseline 3 d after surgery. CONCLUSION: IL-6 and ET-1 are involved precociously in the development of intestinal IRI. Neutrophilic infiltration lasted longer in the group submitted to longer CI. Although there were no significant differences between the groups, significant increase in the number of apoptotic epithelial cells 3 days after reperfusion could be detected in animals

A lo largo de esta tesis se han utilizado las oscilaciones lentas (SO), que emergen durante el sueño de onda lenta y bajo el efecto de ciertos tipos de anestesia, como paradigma para detectar alteraciones funcionales en la corteza de varios modelos murinos de la Enfermedad de Alzheimer (AD) y el Síndrome X Frágil (FXS), para monitorizar la progresión de su enfermedad, y para evaluar la efectividad de dos intervenciones terapéuticas. La caracterización detallada de los parámetros de las SO en varias áreas corticales de los modelos de la AD 3xTg-AD y SAMP8 mostró que dichos parámetros se alteran de manera muy similar en ambos modelos, de un modo que es consistente con una red cortical menos excitable en estos animales. Los parámetros de las SO registradas cerca de las placas de β-amiloide en el modelo APP/PS1 también se alteraron de un modo que sugiere una reducción de la excitabilidad de la neuronas que se encuentran a su alrededor. La realización de ocho semanas de ejercicio físico voluntario en los animales SAMP8 de 7 meses de edad no consiguió cancelar las diferencias que presentaban respecto a los animales control, pero atenuó los efectos de la edad sobre algunos parámetros de la oscilación lenta en ambos grupos, que después de hacer ejercicio presentaron valores más parecidos a los de los 5 meses de edad. Los cambios detectados en el modelo del FXS Fmr1KO fueron contrarios a los detectados en los modelos de la AD, y consistentes con una red cortical hiperexcitable, cambios que fueron revertidos por la subexpresión del receptor cannabinoide CB1, que restauró las diferencias que se habían encontrado entre los animales control y Fmr1KO no tratados. El trabajo realizado en esta tesis debe ser relevante para obtener un fenotipo de red en estos modelos animales, que puede ser contrastado con el que está presente en humanos, pero también puede aportar indicios sobre los mecanismos subyacentes a la actividad de red alterada, que podría estar contribuyendo a los déficits cognitivos característicos de la Enfermedad de Alzheimer y el Síndrome X Frágil.
The slow oscillation (SO) is a rhythmic cortical pattern that emerges during slow-wave sleep and under the effect of certain types of anesthesia. In this Thesis, we used this pattern as a paradigm to 1) identify functional alterations in the cerebral cortex of several mouse models of Alzheimer’s disease (AD) and the Fragile X Syndrome (FXS), 2) to monitor the progression of these diseases, and 3) to evaluate the effectiveness of two therapeutic interventions. Slow oscillations were recorded in different cortical areas of these mouse models, and several parameters of this rhythmic pattern were quantified and compared between them and control groups. The parameters of the SO were similarly altered in 3xTg-AD and SAMP8 mouse models of AD, in a way that suggests a reduced excitability of the cortical network. In addition, these parameters were also altered in the vicinity of amyloid- β plaques in the APP/PS1 mouse model of AD, suggesting a reduced excitability of the neurons located around them. The realization of eight weeks of voluntary physical exercise on 7-month-old SAMP8 animals failed to cancel the differences between them and control animals, but attenuated the effects of aging on some parameters of the SO in both groups, showing similar values to those observed at 5 months of age. Changes in the SO parameters detected in Fmr1KO mouse model of FXS occurred in the opposite way to those detected in the AD mouse models, and were suggestive of a hyperexcitable cortical network. Those changes in the Fmr1KO mouse model were reverted by genetic partial blockade of CB1 cannabinoid receptors, which restored the differences between control and untreated Fmr1KO animals. The work conducted in this Thesis may be relevant to obtain a network phenotype in these animal models, which can be contrasted with that present in humans. Also, it provides clues about the mechanisms underlying the altered network activity, which could be contributing to the cognitive deficits present in the AD and FXS pathological conditions.

Les avancées technologiques endovasculaires des dernières décennies ont été nombreuses ; la diversion de flux en fait partie. Lorsqu’une nouvelle approche permet de traiter de façon efficace et sûre un certain nombre de patients présentant des défis jusque-là difficiles à surmonter, son adoption en pratique clinique peut-être précoce, voire prématurée. Nous avons dans un premier travail réalisé une revue systématique sur les stents dits «Flow Diverters» (FD) et les modèles animaux. Puis nous avons mené quatre expérimentations animales évaluant l’efficacité des FDs dans différents modèles d’anévrismes canins adaptés à l’hypothèse de travail par l’application d’une méthodologie rigoureuse. Nous avons été en mesure de montrer que la technique de diversion de flux est plus à même d’occlure les anévrismes avec de petits collets, des anévrismes dont la branche couverte par le FD est occluse, ou encore quand la porosité du FD en regard de l’anévrisme est diminuée par l’opérateur. Dans le sixième travail, nous avons expérimenté les résultats de la mise en place d’un clip chirurgical sur ces FDs avant d’en déconseiller la pratique. Puis nous avons étudié la variabilité dans la décision des opérateurs d’implanter un FD pour le traitement d’un anévrisme à l’aide d’un questionnaire et ainsi montré l’importante variabilité présente. Enfin nous rapportons le design de l’étude randomisée, pragmatique, multicentrique FIAT (Flow diversion In Aneurysm Treatment) ainsi que ces résultats
Flow Diversion is one of the relevant technical improvements of the past decade in the endovascular treatment of cerebral aneurysms. When the efficacy and safety of a new tool allow treating challenging aneurysms, this adoption in daily practice can be fast even if the benefit of use is not clearly, scientifically show. We performed a systematic review of studies of these stents called “Flow Diverters” (FD) in animal models. Then we performed 4 animal studies in models we create in order to isolate the propriety of the FD we wanted to study. By using this methodology, we have been able to show that Flow Diversion is more likely to occlude small neck aneurysms, aneurysms in which the jailed branch has been occluded, or when the operator compact the FD in order to decrease the porosity of the device. In a 6th study, we test the result of the use of a clip to occlude a FD. Regarding the results of the test, we recommand to avoid clipping FDs.Then by using a questionaire; we showed the poor agreement of using FD in daily practice by using clinical vignettes. Then we presented the design and the result of the first randomized clinical study on flow diverters FIAT (Flow diversion In Aneurysm Treatment)

INTRODUÇÃO: A lesão de isquemia-reperfusão continua sendo considerada a maior causa de mortalidade relacionada ao transplante de pulmão e sua gravidade é influenciada por diversos fatores, dentre eles, a preservação pulmonar. OBJETIVO: Comparar duas soluções de preservação pulmonar, Perfadex® e Celsior®, quanto a capacidade de preservação de tecido pulmonar isquêmico. MÉTODOS: Sessenta pulmões de ratos preservados com Perfadex®, Celsior® ou solução salina após períodos de isquemia hipotérmica de 6 ou 12 horas, foram reperfundidos com sangue homólogo em modelo experimental ex-vivo durante 60 minutos consecutivos. A cada 10 minutos os dados de gasometria, hematócrito, mecânica ventilatória, hemodinâmica e peso do bloco cardiopulmonar foram registrados. Ao final da reperfusão o pulmão esquerdo foi pesado e acondicionado por 48h a 70oC para obtenção da razão peso úmido/peso seco, bem como amostras de tecido pulmonar foram retiradas para histopatologia, microscopia eletrônica e TUNEL. A análise estatística incluiu a comparação entre as soluções e os tempos de isquemia, utilizando ANOVA e Kruskall-Wallis. O nível de significância foi de 5%. RESULTADOS: A comparação entre as complacências de pulmões preservados com Celsior® e Perfadex® nos tempos de isquemia de 6 e 12 horas não apresentou significância estatística (p=0,161 e p=0,316, respectivamente). Os pulmões submetidos a 6 horas de isquemia apresentaram complacência pulmonar superior aos de 12 horas (Perfadex® p=0,02; Celsior® p=0,019; Salina p=0,016). Os valores de pressão arterial pulmonar foram semelhantes entre as três soluções nos dois tempos de isquemia, bem como na comparação entre os tempos de 6 e 12 horas, independente da solução. A Capacidade Relativa de Oxigenação não demonstrou diferença estatística entre as três soluções, independentemente do tempo de isquemia. Na comparação entre os dois tempos de isquemia, o desempenho da oxigenação foi significativamente pior nos pulmões preservados com salina por 12 horas (p=0,001). A razão peso úmido/peso seco não apresentou diferença estatística significante entre as três soluções nos dois tempos de isquemia, porém na comparação entre os tempos de isquemia, os pulmões preservados com Perfadex® apresentaram uma relação peso úmido/peso seco maior no tempo de isquemia mais longo (p=0,001). À microscopia óptica, pulmões preservados com salina apresentaram mais edema que os demais, independentemente do tempo de isquemia. A avaliação da apoptose celular através do método de TUNEL não mostrou diferença estatisticamente significativa na comparação entre os grupos. CONCLUSÃO: Os pulmões preservados com Perfadex® e Celsior® apresentaram desempenho similar em relação às trocas gasosas e parâmetros hemodinâmicos e de mecânica ventilatória. Os pulmões preservados com Perfadex® por 12 horas apresentaram mais edema. Os achados histopatológicos não diferiram entre os grupos estudados
INTRODUCTION: Ischemia-reperfusion injury remaisn the leading cause of mortality related to lung transplantation. Its severity is influenced by several factors including lung preservation. OBJECTIVE: To compare two lung preservation solutions, Perfadex® and Celsior® and its ability to preserve ischemic lung tissue. METHODS: Sixty rat lungs were preserved with Perfadex®, Celsior® or saline after a cold ischemic period of 6 or 12 hours and were then reperfused with homologous blood in an ex vivo experimental model for 60 consecutive minutes. At 10-minute intervals during reperfusion of the heart-lung blocks, data were collected for blood gases, hematocrit, mechanical ventilation, hemodynamic and the heart-lung block weight was recorded. At the end of reperfusion, the left lung was weighed and packaged kept at 70oC for 48h to obtain the wet-to-dry weight ratio. Lung tissue samples were processed for histology, electron microscopy and TUNEL. Statistical analysis included a comparison of the solutions and ischemic times, using ANOVA and Kruskal-Wallis. The significance level was set at 5%. RESULTS: The comparison between the compliance of lungs preserved with Celsior® and Perfadex® in ischemic times of 6 and 12 hours was not statistically significant (p=0.161 and p=0.316, respectively). The lungs subjected to 6 hours of ischemia showed higher lung compliance compared to 12 hours (p=0.02 Perfadex®; Celsior® p=0.019; saline p=0.016). The pulmonary artery pressure values were similar between the three solutions in two stages of ischemia and comparing the times of 6 and 12 hours, regardless of the solution. The Relative Oxygenation Capacity showed no significant difference between the three solutions tested, regardless of the ischemic time. The comparison between the two ischemic times showed that oxygenation capacity was significantly worse in lungs preserved with saline for 12 hours (p=0.001). The wet-to-dry weight ratio showed no statistically significant difference between the three solutions in both ischemic times. However, when ischemic times were compared, Perfadex® showed greater wet-to-dry weight ratio in lungs submitted to 12 hours of ischemia (p=0.001). Light microscopy showed that lungs preserved with saline had more edema than the others, regardless of the ischemic time. Assessment of apoptosis by the TUNEL assay showed no statistically significant difference in the comparison between the groups. CONCLUSIONS: The lungs preserved with Celsior® and Perfadex® performed evenly in regards to gas exchange, hemodynamics and ventilatory mechanics. The lungs preserved with Perfadex® for 12 hours were more edematous. Histopathology findings did not differ between the groups

INTRODUÇÃO: O transplante hepático se consolidou como terapêutica para crianças com doença hepática em estágio terminal. A principal indicação nessa faixa etária é a atresia das vias biliares, doença colestática que leva à cirrose hepática na maioria dos casos nos primeiros anos de vida mesmo nas crianças submetidas a portoenterostomia. Essas crianças evoluem entre outras complicações com desnutrição grave e necessitam do transplante precocemente. A relação ideal do peso do enxerto hepático e o peso do receptor varia de 1 a 3%. No entanto, quando o transplante é realizado em crianças em idade inferior a 2 anos, essa relação na maioria das vezes é maior do que 5%. Essa situação é conhecida como large-for-size, em que a perfusão sanguínea do enxerto pode ser insuficiente, o que leva a disfunção do órgão. Os mecanismos de lesão neste cenário ainda não estão bem esclarecidos. A lesão de isquemia-reperfusão (IR) é apontada como um dos fatores para o mal funcionamento do enxerto bem como de outras complicações. Nessa lesão estão envolvidos elementos da cascata inflamatória culminando em morte celular por apoptose bem como mecanismos de regeneração celular. Um fenômeno conhecido que atenua a lesão de IR é o pré-condicionamento isquêmico (PCI). Nenhum trabalho experimental foi realizado com o objetivo de avaliar a lesão hepática na situação large-for-size e o efeito do PCI nessa situação. MÉTODOS: Para a padronização do modelo foram realizados 8 transplantes, tendo sido testados os pesos dos animais, relação entre o peso do enxerto hepático e peso do receptor e técnica cirúrgica. Na fase experimental, foram realizados 21 transplantes e os animais foram divididos em 3 grupos. No primeiro grupo (controle) o transplante era realizado com doadores e receptores com pesos similares. Para caracterização da situação large-for-size, no segundo grupo os doadores tinham aproximadamente o dobro do peso dos receptores (LFS). No terceiro grupo a distribuição de peso foi semelhante a do segundo, porém era realizado o PCI no doador (LFS+PCI). Os procedimentos foram realizados sob anestesia geral e técnica asséptica e os animais foram mantidos vivos por 3 horas após a reperfusão. Foram obtidas amostras de sangue em 3 períodos (logo após abertura da cavidade abdominal, 1 e 3 horas após reperfusão) e fragmentos de fígado 1 e 3 horas após reperfusão. As amostras de sangue foram submetidas a gasometria arterial, dosagem de sódio, potássio e enzimas hepatocelulares. Os fragmentos de tecido hepático foram submetidos a análise histológica pela coloração da hematoxilina-eosina (HE) e a análise de quantificação da expressão dos genes pró-apoptótico (Bax), anti-apoptótico (Bcl- XL), da óxido nítrico-sintase endotelial (eNOS) e da interleucina-6 (IL-6). RESULTADOS: A mortalidade observada foi de 28%. A relação peso do enxerto e peso do paciente foi de 2,9% no grupo controle, 6,3% no grupo LFS e 6,4% no grupo LFS+PCI. A natremia 1 hora após a reperfusão foi mais elevada no grupo controle do que nos demais, já com relação ao potássio, a dosagem foi mais baixa nesse grupo no mesmo intervalo. A dosagem da aspartato aminotransferase (AST) foi mais elevada nos grupos LFS e LFS+PCI do que no controle. A análise histológica não Resumo demonstrou diferença entre os grupos. A expressão do gene Bax foi mais elevada no grupo LFS do que nos demais, enquanto que a do gene eNOS foi mais acentuada no grupo LFS+PCI do que nos demais nos dois intervalos estudados. Após 3 horas de reperfusão a expressão do gene IL-6 foi maior no grupo LFS+PCI. CONCLUSÕES: O modelo de transplante hepático large-for-size em suínos foi exequível e adequado para a pesquisa. A lesão IR foi mais acentuada no grupo LFS do que no grupo controle e apesar de não ter alterado a elevação de enzimas hepatocelulares, o PCI teve papel de aumentar a expressão dos genes IL-6 e eNOS e diminuir a do gene Bax
INTRODUCTION: Liver transplantation has been established as therapy for children with end-stage liver disease. The main indication is biliary atresia, cholestatic disease that leads to cirrhosis in most cases even in children undergoing portoenterostomy. These children develop other complications as severely malnutrition and need early transplantation. The ideal graft to body weight ratio (GBWR) for transplantation varies from 1 to 3%. However, when transplantation is performed in children younger than 2 years, this ratio in most cases is higher than 5%. This situation, known as large-for-size, may be related to insufficient graft perfusion and liver disfunction. The mechanisms of injury involved in this scenario are not well established. Ischemia-reperfusion injury (IRI) has been related as a factor for poor graft function and other complications. Inflammatory cascade culminating in cell death by apoptosis and cellular mechanisms of regeneration are the elements involved in IRI. Ischemic preconditioning (IPC) is a phenomenon that attenuates IR injury. There are no experimental studies conducted to evaluate the hepatic injury in large-for-size situation and the effect of IPC in this situation. METHODS: 8 procedures were required for standardize the model. Weight of the animals, GBWR and surgical technique were evaluated. In the experimental phase, 21 transplantations were performed. The animals were divided in three groups. In the first group (control), liver transplantation was performed with donors and recipients with similar weights. To characterize large-for-size situation, donors of the second group had approximately twice the weight receptor (LFS). In the third group the weight distribution was similar to the second but IPC was performed in donor (LFS+IPC). Procedures were performed under general anesthesia and aseptic technique and the animals were kept alive for 3 hours after reperfusion. Blood samples were obtained at three times (immediately after opening the abdominal cavity, 1 and 3 hours after reperfusion) and fragments of liver 1 and 3 hours after reperfusion. Blood samples were analyzed for arterial blood gases, sodium, potassium and hepatocellular enzymes. The fragments of liver tissues were subjected to histological analysis by hematoxylin-eosin staining (HE). Molecular biology involved quantification of the expression of Bax, Bcl-XL, endothelial nitric oxide synthase (eNOS) and interleukin- 6 (IL-6) genes. RESULTS: The mortality rate was 28%. The GBWR was 2.9% in the control group, 6.3% in the LFS group and 6.4% in the LFS+ICP group. The natremia 1 hour after reperfusion was higher in the control group and potassium dosage was lower in this group in the same period. The dosage of aspartate aminotransferase (AST) was higher in LFS and LFS+IPC groups than control. Histological analysis showed no difference between groups. The Bax gene expression was higher in the LFS, while the eNOS gene was more expressed in LFS + PCI group. After 3 hours of reperfusion the expression of IL-6 gene was higher in the PCI + LFS. CONCLUSIONS: The model of large-for-size liver transplantation in swine was feasible and appropriate for the research. IR injury was more pronounced in the Summary group LFS than in the control group and despite not having changed the elevation of hepatocellular enzymes, the PCI was responsible for increasing the expression of IL- 6 and eNOS and decreasing the Bax gene expression

Le present travail qui s'inscrit dans le cadre general des etudes consacrees a la protection pharmacologique des tissus cardiaque et vasculaire au cours de la reperfusion post-ischemique, comprend deux parties principales. La premiere a ete realisee sur un modele d'ischemie/reperfusion myocardique sur un modele experimental de coeur isole de rat. Les resultats sont exprimes en termes de donnees fonctionnelles, metaboliques et ultrastructurales. La seconde partie est une etude pharmacologique menee sur un modele d'anneaux d'aorte isolee de rat et comporte essentiellement des mesures de contractilite vasculaire. Dans la premiere partie, nous avons etudie l'effet protecteur du chlorure de manganese sur la recuperation post-ischemique du myocarde. Nous avons demontre que le manganese, administre durant la phase precoce de la reperfusion, ameliore la recuperation metabolique et fonctionnelle, vraisemblablement par le biais d'une protection antioxydante de la membrane des cardiomyocytes. Nous avons egalement mis en evidence un effet protecteur du manganese administre avant la periode d'ischemie sur la fonction et l'ultrastructure du myocarde post-ischemique. Dans la seconde partie de notre travail, nous avons montre qu'euk8, un compose de type salen-manganese presentant de fortes activites sod et catalase, exerce in vitro un effet vasorelaxant dose-dependant, non medie par une simple protection antioxydante de no, mais essentiellement du a une activation de l'adenylate cyclase et de la guanylate cyclase soluble des cellules musculaires lisses vasculaires. Enfin, une etude des effets vasomoteurs du manganese nous a amenes a conclure qu'il induit une relaxation par le biais de mecanismes plus complexes qu'une simple protection antioxydante de no et qu'un simple effet antagoniste calcique direct sur les cellules musculaires lisses vasculaires.

O infarto do miocárdio (IM) é a doença cardiovascular que mais causa morte e invalidez em todo o mundo. O uso de animais experimentais tem auxiliado a compreender melhor a fisiopatologia e as formas de tratamento do IM. Sabendo que as dislipidemias estão associadas com o IM e que o treinamento físico pode ser prescrito para prevenção e tratamento de doenças cardiovasculares, no presente trabalho, investigamos os efeitos de dois tipos de treinamentos físicos em um modelo experimental de dislipidemia e isquemia miocárdica. Camundongos selvagens (WT) e knockout para o receptor LDL (LDL-/-) foram divididos em oito grupos: a) LDLr-/- sedentário (LDL-S); b) LDLr-/- infartado sedentário (LDL-IM-S); c) LDLr-/- infartado submetido a treinamento contínuo (LDL-IM-C); d) LDLr-/- infartado submetido a treinamento intervalado (LDL-IM-I); e) WT sedentário (WT-S); f) WT infartado sedentário (WT-IM-S); g) WT infartado submetido a treinamento contínuo (WT-IM-C); h) WT infartado submetido a treinamento intervalado (WT-IM-I). Após 60 dias da ligadura da artéria coronária descendente, o treino contínuo constou de corrida a 60% do máximo e o intervalado de 8 tiros de 4min a 80% do máximo e recuperação de 4min a 40% do máximo. Nos animais WT infartados, ambos os treinamentos aumentaram a tolerância ao esforço e provocaram diminuição do balanço simpatovagal e aumento do índice alfa em magnitudes semelhantes. O treinamento intervalado reduziu o número de fibras do tipo II em relação aos grupos WT-S e WT-IM-C, bem como reduziu a quantidade de fibras do tipo II-X em relação aos WT-S. A área de secção transversa das fibras do tipo I foi maior no grupo WT-IM-I do que no WT-IM-S e WT-S. A razão capilar/fibra foi maior nos animais do grupo WT-I do que no WT-S. A fração de ejeção e a fração de encurtamento foi menor no grupo LDL-IM-I em relação aos demais, mas sem diferenças entre os grupos WT-S, WT-IM-C e WT-IM-I. Nos animais LDL-/-, o LDL foi maior e o VLDL menor no grupo LDL-IM-C em relação aos demais. O HDLtg(%) foi superior no LDL-C em relação ao LDL-S. O HDLc (mg e %) do LDL-IM-I foi maior que o do grupo LDL-IM-C, sendo que o HDLc (mg) do LDL-IM-I foi, ainda maior do que o grupo LDL-S. O triglicérides total foi menor no grupo LDL-IM-C do que no LDL-S. Somente o grupo LDL-IM-I diminuiu a FC de repouso em relação ao grupo LDL-IM-S. A PA diastólica foi menor no grupo LDL-IM-S em relação ao LDL-S, enquanto que o grupo LDL-IM-I apresentou PA diastólica maior do que o grupo LDL-IM-C. A variância do intervalo de pulso foi maior no grupo LDL-S somente em relação ao grupo LDL-IM-I. Em conjunto nossos resultados demonstraram que os animais LDL possuem diferenças funcionais e fisiológicas importantes em relação ao WT, especialmente na morfologia muscular, na hemodinâmica e no controle autonômico. Que o IM acarretou prejuízos em ambas as linhagens investigadas e que os dois tipos de TF atenuaram semelhantemente esses prejuízos em grande parte das variáveis analisadas
Myocardial infarction (MI) is a major cause of death and disability worldwide. The use of experimental animals has supported to better understand the pathophysiology and treatment forms of myocardial infarction (MI). Knowing that the dyslipidemia associated with IM and that physical training can be prescribed for prevention and treatment of cardiovascular diseases, the present study investigated the effects of two types of physical training on an experimental model of dyslipidemia and myocardial ischemia. Wild mice (WT) and LDL receptor knockout (LDL-/- ) were divided into eight groups: a) LDLr-/- sedentary (LDL-S), b) LDLr-/- myocardium infarction sedentary (LDL-MI-S), c) LDLr-/- myocardium infarction submitted to continuous training (LDL-MI-C), d) LDLr-/- myocardium infarction submitted to interval training (LDLMI- I), e) sedentary WT (WT-S); f) WT myocardium infarction sedentary (WT-MI-S); g) WT myocardium infarction submitted to continuous training (WT-MI-C), h) WT myocardium infarction submitted to interval training (WT-IM-I). After 60 days of descending coronary artery ligation, the continuous training consisted of running at 60% of maximum, while the interval training consisted of eight sprints of 4 min at 80% of maximum and a 4 min recovery at 40% of maximum. In infarcted WT animals, both training programs increased exercise tolerance and promoted decrease of sympathetic-vagal balance and increase of alpha index in similar magnitudes. Nevertheless, the interval training reduced the number of type II fibers in infarcted WT animals compared to WT-S and WT-MI-C groups, as well as reduced the amount of fiber type II-X compared to WT-S. The cross-sectional area of the fiber type I was higher in the WTMI- I animals than in WT-MI-S and S-WT groups. The reason capillary/fiber was higher in group WT-I than in the WT-S. Ejection fraction and shortening fraction were lower in LDL-MII compared to the others, but with no differences among the WT-S, WT-IMI-C and WT-MI-I groups. About the LDL-/- animals, the LDL was higher and VLDL was lower in the group LDL-MI-C in relation to the others. The HDLtg (%) was higher in LDL-C compared to LDL-S. The HDLc (mg and %) of LDL-MI-I was higher than the LDL-MI-C group, and the HDLc (mg) of LDL-MI-I was even higher than LDL-S group. The total triglycerides was lower in LDL- MIC than in LDL-S animals. Only in LDL-MI-I group the resting HR was decreased in comparison to LDL-MI-S. The diastolic blood pressure was lower in LDL-MI-S in relation to LDL-S, while the LDL-MI-I group presented a higher diastolic BP than the LDL-MI-C group. The pulse interval variance was greater in LDL-S than in LDL-MI-I only. In conclusion, our results demonstrate that LDL animals have important functional and physiological differences compared to WT, especially in relation to muscle morphology, hemodynamic and autonomic cardiovascular control. Furthermore, MI leads to damage in both investigated strains and the two types of physical training attenuate similarly the impairment of most of the analyzed variables

In a mouse model of ischemic stroke, infarct volume is highly variable and strain dependent, but the natural genetic determinants responsible for this difference remain unknown. To identify genetic determinants regulating ischemic neuronal damage and to dissect apart the role of individual genes and physiological mechanisms in infarction in mice, we performed forward genetic mapping analyses of surgically induced cerebral infarct volume. We have identified multiple quantitative trait loci (QTL) that modulate infarct volume, with a major locus (Civq1 ) on chromosome 7 accounting for over 50% of the variation, with a combined LOD score of 21.7. Measurement of infarct volume in chromosome substitution strains (CSS) and two additional intercrosses validate that Civq1 on chromosome 7 is present in multiple inbred strains. Interval-specific ancestral SNP haplotype analysis for Civq1 results in 5 candidate genes. A causative gene underlying Civq1 may regulate collateral artery formation and genetic variations in the gene may result in the differential outcome of cerebral infarction. Additionally, we have identified a locus of large effect, Civq4, modulating infarct volume through a mechanism different from collateral circulation. In conclusion, the extent of ischemic tissue damage after distal middle cerebral artery occlusion (MCAO) in inbred strains of mice is regulated by genetic variation mapping to at least 4 different loci. A single locus on chromosome 7 determines the majority of the observed variation in the trait in multiple mouse strains. Civq1 appears to be identical to Lsq1, a locus conferring limb salvage and reperfusion in hindlimb ischemia. The identification of the genes underlying these loci may uncover novel genetic and physiological pathways that modulate cerebral infarction and provide new targets for therapeutic intervention in ischemic stroke, and possibly other human vascular occlusive diseases.

Dissertation

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in the embryonic neural development and adult neurogenesis. But the effects of 5-HT on stem cells are not fully known. In this study, the effects and underlying signal pathways of 5- HT on proliferation and neural differentiation of mouse embryonic stem (ES) cells, neural progenitor (NP) cell line C 17.2 and embryonic neural stem (NS) cells were explored. Molecular analysis, immunostaining and western blotting revealed that NP/NB cells expressed the rate-limiting enzyme tryptophan hydroxylase (TPH) and produced endogenous 5-HT. While mouse ES cells showed no expression of TPH. Quantitative PCR demonstrated that ES cells and NPINS cells expressed majority of 5-HT receptor sUbtypes. In serum free propagation culture, WST1, BrdU incorporation and neural colony forming cell assay demonstrated that 5-HT enhanced proliferation of ES cells and NPINS cells in a dose-dependent manner. Tryptophan hydroxylase (TPH) inhibitor para-chlorophenylalanine (PCPA) which can inhibit biosynthesis of endogenous 5-HT decreased viability of mouse NP/NS cells. Mouse ES cells derived embryoid bodies (EB) and NS/NP cells were subjected to neural induction in serum-free medium with and without 5-HT or PCPA. On day 8 of EB cultures, immunofluorescence staining displayed a less percentage of SSEA-1+ cells derived from cultures supplemented with 5-HT. Nestin positivity are comparable. Quantitative PCR analysis suggested that supplement of 5-HT in EB culture inhibit neural differentiation of ES cells and induce mesodermal commitment. On day 21 of ES cells neural induction, compared to cultures without 5-HT treatment, a significantly less number of ß-tubulin III+ neurons, GEAP+ astrocytes and GaIC+ oligodendrocytes were noted in 5-HT -supplemented cultures. For NS/NP cells, the inhibitory effects of 5-HT on neuronal and oligodendrocytic commitment were also observed. And the application of PCPA exerted a promoting effect on neural differentiation of NS cells. Manipulating 5-HT level can affect the expression level of key genes which involved in 5-HT metabolism. ES and NS/NP cells treated with 5-HT showed decreased production of endogenous reactive oxygen species (ROS). 5-HT demonstrated a significant anti-apoptotic effect on NP cells and this antiapoptotic effect may be mediated by up-regulated expression of anti-apoptotic gene Bel- 2. Whole genome cDNA microarray analysis and quantitative RT-PCR revealed that notch signal pathway was involved in mediating the biological effects of 5-HT. Western blotting further confirmed that 5-HT treatment up-regulated the protein level of NICD and notch downstream effectors Hes-l and Hes-5. Finally, the therapeutic effects of ES cell-derived neural cells were testified in a mouse model of global ischemia. Two weeks post-transplantation, BrdU labeled ES cell-derived neural cells survived and migrated throughout brain parenchyma. A majority of transplanted cells remained nestin positive. The cognitive functions of cell transplanted groups showed significant recovery compared with untransplanted arms, but no significant difference was observed between transplanted groups treated with and without 5-HT. Taken together, data of this study indicated 5-HT play an important role in neural development and ES cell-derived neural cells might be applicable in the treatment of stroke.
Li, Jin.
"November 2011."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 195-241).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
Abstracts in English.
ACKNOWLEDGEMENTS --- p.i
LIST OF PUBLICATIONS --- p.ii
ABSTRACT --- p.iii
ABSTRACT [in Chinese] --- p.v
TABLE OF CONTENT --- p.vi
LISTS OF FLOWCHARTS --- p.xii
LISTS OF FIGURES --- p.xiii
LIST OF TABLES --- p.xvi
LIST OF EQUIPMENTS --- p.xvii
LIST OF ABBREVATIONS --- p.xvii
Chapter Chapter1 --- Introduction --- p.1
Chapter 1.1 --- Central nervous system disorder --- p.1
Chapter 1.1.1 --- Stroke --- p.1
Chapter 1.1.2 --- Spinal cord injuries --- p.4
Chapter 1.1.3 --- Parkinson's disease --- p.6
Chapter 1.1.4 --- Amyotrophic Lateral Sclerosis --- p.8
Chapter 1.2 --- Stem cell therapy --- p.10
Chapter 1.2.1 --- General considerations in stem cell therapy --- p.11
Chapter 1.2.2 --- Stem cell therapy for stroke --- p.11
Chapter 1.2.3 --- Stem cell therapy for spinal cord injury --- p.15
Chapter 1.2.4 --- Stem cell therapy for Parkinson's disease --- p.16
Chapter 1.2.5 --- Stem cell therapy for ALS --- p.18
Chapter 1.3 --- Stem cells --- p.20
Chapter 1.3.1 --- Embryonic stem cells --- p.21
Chapter 1.3.1.1 --- Derivation and characterization --- p.21
Chapter 1.3.1.2 --- Biology of ES cells --- p.21
Chapter 1.3.1.2.1 --- Pluripotency of ES cells --- p.21
Chapter 1.3.1.2.2 --- Differentiation of ES cells to multiple lineages --- p.24
Chapter 1.3.1.2.2.1 --- Ectodermal differentiation --- p.25
Chapter 1.3.1.2.2.2 --- Mesodermal differentiation --- p.27
Chapter 1.3.1.2.2.3 --- Endodermal differentiation --- p.28
Chapter 1.3.2 --- Neural stem cells --- p.30
Chapter 1.3.2.1 --- Derivation and characterization --- p.30
Chapter 1.3.2.2 --- Biology of NS cells --- p.32
Chapter 1.3.3 --- Induced pluripotent stem cells --- p.34
Chapter 1.3.4 --- Mesenchymal stem cells --- p.35
Chapter 1.4 --- Serotonin (5-HT) --- p.36
Chapter 1.4.1 --- Distribution --- p.37
Chapter 1.4.2 --- Metabolism --- p.37
Chapter 1.4.3 --- Biological effects of 5-HT --- p.38
Chapter 1.4.4 --- Serotonin receptor subtypes and receptor signal transduction pathways --- p.40
Chapter Chapter2 --- Aim --- p.43
Chapter 2.1 --- Hypothesis and study objectives --- p.43
Chapter Chapter3 --- Materials and Methods --- p.49
Chapter 3.1 --- Chemicals and Reagents --- p.49
Chapter 3.1.1 --- Cell culture --- p.49
Chapter 3.1.2 --- Serotonin, serotonin receptor subtypes specific agonists/antagonists and drugs that regulate serotonin metabolism --- p.51
Chapter 3.1.3 --- Cell proliferation assay --- p.52
Chapter 3.1.4 --- Cell apoptosis assay --- p.52
Chapter 3.1.5 --- Immunohistochemistry and staining --- p.52
Chapter 3.1.6 --- Western blotting --- p.55
Chapter 3.1.7 --- Molecular biology --- p.56
Chapter 3.1.8 --- Whole genome cDNA micro array --- p.58
Chapter 3.1.9 --- MAO activity assay --- p.58
Chapter 3.1.10 --- Endogenous ROS production assay --- p.58
Chapter 3.2 --- Consumable --- p.58
Chapter 3.3 --- Cells --- p.60
Chapter 3.3.1 --- Feeder cell --- p.60
Chapter 3.3.1.1 --- Mouse embryonic fibroblasts --- p.60
Chapter 3.3.2 --- ES cells --- p.61
Chapter 3.3.2.1 --- ES cell D3 --- p.61
Chapter 3.3.2.2 --- ES cell-E14TG2a --- p.61
Chapter 3.3.3 --- NS cells --- p.61
Chapter 3.3.3.1 --- Neural progenitor cells line C172 --- p.61
Chapter 3.3.3.2 --- Mouse embryonic neural stem cells --- p.61
Chapter 3.4 --- In-house prepared solutions --- p.62
Chapter 3.4.1 --- Stock solution ofInsulin, Transferrin, Selentine (ITS) Supplement --- p.63
Chapter 3.4.2 --- Gelatin solution 01% --- p.62
Chapter 3.4.3 --- Paraformaldehyde solution 4% (PFA) --- p.62
Chapter 3.4.4 --- Tritox X-lOO solution 03% --- p.63
Chapter 3.4.5 --- Popidium iodide solution 1 ug/ml (PI) --- p.63
Chapter 3.4.6 --- Poly-L-ornithine solution --- p.63
Chapter 3.4.7 --- Laminin solution --- p.64
Chapter 3.4.7 --- MEF Maintenance medium --- p.64
Chapter 3.4.9 --- Cryopreservation Media for MEF and C172 (2X) --- p.64
Chapter 3.4.10 --- Cryopreservation Media for mouse ES cell (2X) --- p.65
Chapter 3.4.11 --- Cryopreservation Media for mouse NS cell (2X) --- p.65
Chapter 3.4.12 --- Serum based maintenance medium for C172 --- p.65
Chapter 3.4.13 --- Serum free maintenance medium for C172 --- p.66
Chapter 3.4.14 --- Serum-based propagation medium for ES cells --- p.66
Chapter 3.4.15 --- Serum-free propagation medium forES cells --- p.67
Chapter 3.4.16 --- Serum-free induction medium for ES cells --- p.67
Chapter 3.4.16.1 --- Serum-free induction medium I --- p.67
Chapter 3.4.16.2 --- Serum-free induction medium II --- p.68
Chapter 3.4.16.3 --- Serum-free induction medium III --- p.68
Chapter 3.4.17 --- Tris-HCl (1 M), pH 74 --- p.68
Chapter 3.4.18 --- Tris-HCl (1 M), pH 87 --- p.69
Chapter 3.4.19 --- Tris-HCI (1 M), pH 69 --- p.69
Chapter 3.4.20 --- APS 10% (wt/vol) --- p.69
Chapter 3.4.21 --- Protease inhibitor (10X) --- p.70
Chapter 3.4.22 --- RIPA --- p.70
Chapter 3.4.23 --- Resolving buffer (8X) --- p.70
Chapter 3.4.24 --- Stacking buffer (4X) --- p.71
Chapter 3.4.25 --- Protein running buffer (lOX) --- p.71
Chapter 3.4.26 --- Transfer buffer (10X) --- p.72
Chapter 3.4.27 --- Transfer buffer (IX) --- p.72
Chapter 3.4.28 --- Blocking buffer (lOX) --- p.72
Chapter 3.4.29 --- TBS (10X) --- p.73
Chapter 3.4.30 --- TBS-T (IX) --- p.73
Chapter 3.4.31 --- Stacking gel --- p.73
Chapter 3.4.32 --- Resolving gel --- p.74
Chapter 3.5 --- Methods --- p.75
Chapter 3.5.1 --- Cell culture --- p.75
Chapter 3.5.1.1 --- Preparation of acid washed cover slips --- p.75
Chapter 3.5.1.2 --- Preparation of gelatinized culture wares --- p.75
Chapter 3.5.1.3 --- Poly-L-omithine and laminin coating --- p.76
Chapter 3.5.1.4 --- Thawing cryopreserved cells --- p.76
Chapter 3.5.1.5 --- Passage of culture --- p.77
Chapter 3.5.1.5 --- 6 Cell count --- p.78
Chapter 3.5.1.7 --- Cytospin --- p.78
Chapter 3.5.1.8 --- Trypan blue dye exclusion test --- p.78
Chapter 3.5.1.9 --- Cryopreservation --- p.79
Chapter 3.5.1.10 --- Derivation and culture of mouse embryonic fibroblasts (MEF) --- p.79
Chapter 3.5.1.11 --- Propagation of ES cells in serum-based/free medium --- p.81
Chapter 3.5.1.12 --- Neural differentiation ofES cells --- p.83
Chapter 3.5.1.13 --- Propagation ofNP cell C172 in serum-based or serum-free medium --- p.84
Chapter 3.5.1.14 --- Neural differentiation ofC172 --- p.85
Chapter 3.5.1.15 --- Derivation and propagation of embryonic NS cells --- p.85
Chapter 3.5.1.13 --- Neural differentiation of embryonic NS cells --- p.86
Chapter 3.5.1.17 --- BrdU labeling of the ES cells derived products --- p.87
Chapter 3.5.2 --- Cell proliferation assay --- p.87
Chapter 3.5.2.1 --- Cell morphology --- p.87
Chapter 3.5.2.2 --- WST-1 assay --- p.88
Chapter 3.5.2.3 --- BrdU incorporation assay --- p.88
Chapter 3.5.2.4 --- NCFC assay --- p.89
Chapter 3.5.3 --- Conventional and quantitative RT-PCR --- p.89
Chapter 3.5.3.1 --- RNA extraction --- p.89
Chapter 3.5.3.2 --- RNA quantitation --- p.90
Chapter 3.5.3.3 --- Reverse Transcription ofthe First Strand complementary DNA --- p.90
Chapter 3.5.3.4 --- Polymerase chain reaction --- p.91
Chapter 3.5.3.5 --- RNA Integrity Check --- p.91
Chapter 3.5.3.6 --- Electrophoresis and visualization of gene products --- p.91
Chapter 3.5.3.7 --- Real-time quantitative PCR --- p.92
Chapter 3.5.4 --- Microarray --- p.94
Chapter 3.5.5 --- Immunofluoresent staining --- p.94
Chapter 3.5.6 --- Western blot --- p.95
Chapter 3.5.6.1 --- Harvesting samples --- p.95
Chapter 3.5.6.2 --- Protein extraction --- p.96
Chapter 3.5.6.3 --- Protein quantification --- p.96
Chapter 3.5.6.4 --- SDS-PAGE --- p.97
Chapter 3.5.6.5 --- Wet transfer of protein to PVDF membrane --- p.97
Chapter 3.5.6.6 --- Blocking the membrane --- p.97
Chapter 3.5.6.7 --- Immunoblotting --- p.97
Chapter 3.5.6.8 --- Signal detection --- p.98
Chapter 3.5.7 --- Cell apoptosis assay --- p.98
Chapter 3.5.7.1 --- ANNEXINV-FITC apoptosis detection --- p.98
Chapter 3.5.7.2 --- TUNEL --- p.99
Chapter 3.5.8 --- Endogenous ROS assay --- p.100
Chapter 3.5.9 --- In vivo studies --- p.101
Chapter 3.5.9.1 --- Induction of cerebral ischemia in mice --- p.101
Chapter 3.5.9.2 --- Transplantation --- p.101
Chapter 3.5.9.3 --- Assessment of learning ability and memory --- p.102
Chapter 3.5.10 --- Histological analysis --- p.103
Chapter 3.5.10.1 --- Animal sacrifice for brain harvest --- p.103
Chapter 3.5.10.2 --- Cryosectioning --- p.103
Chapter 3.5.10.3 --- Haematoxylin and eosin staining --- p.104
Chapter 3.6 --- Data analysis --- p.104
Chapter Chapter4 --- Results --- p.113
Chapter 4.1 --- Expression profile of 5-HT receptors and metablism of endogenous 5-HT --- p.113
Chapter 4.1.1 --- Expression profiles of 5-HT receptors in stem cells --- p.113
Chapter 4.1.2 --- Biosynthesis of endogenous 5-HT --- p.115
Chapter 4.2 --- Effects of 5-HT on proliferation of mouse ES cells and NS cells --- p.115
Chapter 4.2.1 --- Effects of 5-HT on proliferation ofES cells --- p.115
Chapter 4.2.2 --- Effects of 5-HT on proliferation ofNP and NS cells --- p.117
Chapter 4.3 --- Effects of 5-HT on differentiation of mouse ES cells and NS cells --- p.119
Chapter 4.3.1 --- Neural differentiation ofES cells --- p.119
Chapter 4.3.2 --- Effects of 5-HT on differentiation ofES cells --- p.119
Chapter 4.3.3 --- Neural differentiation ofNP and NS cells --- p.120
Chapter 4.3.4 --- Effects of 5-HT on differentiation ofNP and NS cells --- p.121
Chapter 4.4 --- 5-HT metabolism in mouse ES cells and NS cells --- p.122
Chapter 4.4.1 --- Expression of key 5-HT metablic genes in stem cells --- p.122
Chapter 4.4.2 --- Detection ofROS generation in mouse NS cells --- p.123
Chapter 4.4.3 --- Effects of 5-HT on expression level of MAO-A, MAO-B and SERT --- p.123
Chapter 4.5 --- Anti-apoptotic effect of 5-HT on NP and NS cells in neural induction --- p.127
Chapter 4.6 --- Potential signaling pathways mediated by 5-HT --- p.130
Chapter 4.7 --- Therapeutic effects of 5-HT treated mouse ES cell-derived cells in a stoke model --- p.130
Chapter 4.7.1 --- Induction of global ischemia by transient BCCAO --- p.130
Chapter 4.7.1.1 --- HE staining of post ischemic brain --- p.131
Chapter 4.7.1.2 --- TUNEL analysis of cell apoptosis at post ischemia day 3 --- p.132
Chapter 4.7.2 --- Cell labelling --- p.132
Chapter 4.7.3 --- Cognition monitoring post transplantation --- p.133
Chapter 4.7.4 --- Survival, migration and differentiation of transplanted neural cells --- p.135
Chapter Chapter5 --- Discussion --- p.180
Chapter Chapter6 --- Conclusions --- p.192
References --- p.195

Baicalin, which is a flavonoid, was previously shown to exert neuroprotective effects against ischemic injury and oxidative insults. In this study, baicalin was found to induce neuronal differentiation on both C17.2 NSC and primary mouse NSC originated from hippocampuses of E14.5 mouse embryos. The baicalin-mediated differentiation of C17.2 NSC was noted in dose- and time-dependent manners. Baicalin-treated NSC displayed long processes of neurites. The gene expression of neuronal markers, NF-L, TUBB3 and MAP2 was also significantly increased after treated with 20 to 50 muM baicalin on C17.2 NSC. Treating C17.2 NSC with baicalin significantly increased the number of TUBB3 positive cells by 300%. A significant increase in the gene expression of TUBB3 was also observed on primary NSC upon baicalin treatment at 5 to 10 muM. The number of TUBB3 positive cells was increased by 100% after treating with 10 muM baicalin. C17.2 NSC treated with baicalin also increased the gene expression of GABAergic and serotonergic neuronal subtype specific enzymes GAD1 and TPH1.
Nature provides a vast pool of natural compounds with neuroprotection and neurotrophism. A few of these compounds can induce the differentiation of neural stem cells (NSC). There are ample opportunities to discover more natural compounds with differentiation inducing effect on NSC. One of the objectives of this project is to look for novel natural compounds showing neurogenic effect on NSC. This project has established a platform for screening medicinal materials and natural compounds with neural differentiation promoting effect on C17.2 mouse neural stem cell line. Screening results identified total Sanqi saponins, total Renshen saponins, Huangqin extracts and baicalin as potent candidates for inducing this differentiation of NSC.
This project also aims at characterizing the mechanisms involved in the neuronal differentiation effect of baicalin on NSC. Annotation from microarray analysis indicated that baicalin treatment on C17.2 NSC is related to development of tissue and nervous system. qPCR study attested the increased gene expression of nerve growth factor-beta, neurotrophin-3, pro-neural transcriptional factors Ngn1, Ngn2 and NeuroD2. Western blotting showed that baicalin activated ERK1/2 MAP kinase but not JNK and p38 MAP kinases.
This project demonstrated the neurogenic potential of natural resources on NSC. A novel neuronal induction effect of baicalin on NSC was also demonstrated with its mechanisms characterized. This project also revealed that baicalin can be used for promoting functional recovery of post-ischemia animals.
This study showed for the first time that baicalin exerts neuronal differentiation inducing effect on NSC. Another objective of this project is to study whether baicalin can promote functional recovery of animals with ischemia brain injury. Mice having undergone transient occlusion of the bilateral common carotid arteries with blood-reperfusion to induce global cerebral ischemia were treated with baicalin and/or EGFP-NSC. Ischemia animals received implantation of EGFP-NSC into the caudate putamen and/or intravenous injection of baicalin on alternate days for two-week on day seven post-ischemia displayed significant improvement of the cognitive function in terms of the incident of error and escape time in the water T-maze task compared to the control arm of ischemia mice. Data of the study suggested that the therapeutic effect of baicalin would be comparable to that of neural stem cell transplant in improving the cognitive function in a mouse ischemic stroke model.
Li, Ming.
Adviser: P. C. Shaw.
Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 199-232).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

博士
國立成功大學
醫學工程研究所碩博士班
94
The neuroprotective properties of melatonin were evaluated in Sprague-Dawley rats and B6 mice subjected to middle cerebral artery (MCA) occlusion. A series of experiments with delayed treatment of melatonin have been employed to examine whether exogenous melatonin offers neuroprotective action against MCA occlusion which includes ischemic brain damage not only caused by necrotic but also by apoptotic processes. Postmortem infarct volumes will be determined by quantitative image analysis of Nissl-stained brain sections and TTC-staining method. In addition, postischemic electrophysiological recovery was evaluated. The protective efficiency of melatonin against ischemia- and reperfusion-induced neuronal perikarya, axonal, and oligodendrocyte pathology and increases in oxidative stress was also evaluated. The data provides a potential outlook of melatonin to treat ischemic stroke patients. 　　Firstly, neuroprotective properties of melatonin were evaluated in rats subjected to transient middle cerebral artery (MCA) occlusion. The study examined electrophysiological, histological and neurobehavioral outcomes following transient focal cerebral ischemia caused by intraluminal suture occlusion of the middle cerebral artery. Postmortem infarct volumes will be determined by quantitative image analysis of TTC-stained brain sections. The effects of melatonin on cortical blood perfusion were also examined in the model. Our results showed that melatonin could effectively reduce brain infarction and improve electrophysiological and functional outcome. 　　Secondarily, we examined the effect of melatonin on gray and white matter damage after ischemic stroke. The changes in the post-ischemic expression in gray and white matter pathology as well as oxidative stress to cell membrane and DNA by which the neuroprotective effects of melatonin may be mediated were also evaluated. The results showed that melatonin is a good candidate for protecting against gray and white matter pathology after ischemic stroke. 　　Finally, we examined the possible role of melatonin to reduce postischemic damage to late-onset rise in the blood-brain barrier and the t-PA-associated hemorrhagic transformation after ischemic stroke. We have observed that melatonin decreases the late-onset rise in blood-brain barrier and improves the hemorrhagic transformation associated with t-PA therapy after ischemic stroke. Additionally, we examined the possible role of melatonin in neurovascular unit protection and its effects on postischemic neurovascular oxidative and nitrosative damage. We found that melatonin effectively reduces postischemic neurovascular oxidative and nitrosative damage and improves early rise in the blood-brain barrier after stroke. 　　These neuroprotective actions observed with melatonin will further solidify the possible role of the neuroprotective agent for clinical use and provide a potential outlook to treat patients against ischemic stroke. We suggest a need for a pilot clinical trial of melatonin for acute ischemic stroke patients and our data may also benefit neurosurgeons to perform a planned cerebrovascular surgery.

Ischaemic stroke has become one of the leading causes of death and disability in the world. Protease activated receptors (PARs, PAR-1 to PAR-4) belong to G protein coupled receptors that can be self-activated by tethered ligands (TL) revealed through proteolytic cleavage. Based on these TL, many activating peptides (APs) and antagonists have been synthesized to investigate PARs actions.
In the present study, the roles of PARs were examined in two models of ischaemic stroke. For the in vivo model, transient middle cerebral artery occlusion (tMCAO) was performed to establish cerebral ischaemia in rats. For the in vitro model, oxygen and glucose deprivation (OGD) was used to mimic an ischaemia insult in primary cultured rat embryonic cortical neurones.
Western blot studies showed that expressions of PAR-1 and PAR-2 were increased in the rat ischaemic brain cortex, whereas PAR-1 was reduced in the rat cortical neurones subjected to OGD. Pretreatments of PAR-1 AP (SFLLRN-NH₂) and PAR-2 AP (SLIGRL-NH₂) produced significant protection against ischaemia-induced damage. Pretreatment of PAR-3 AP (SFNGGP-NH₂) only improved ischaemic symptoms in in vivo but not in in vitro model. When treated after ischaemia, only PAR-1 AP produced significant reductions on ischaemia-induced damage. Protective actions of PAR-1 and PAR-2 APs were inhibited by PAR-1 antagonist (BMS-200261) and PAR-2 antagonist (ENMD-1068) respectively, but PAR-1 antagonist did not affect posttreatment effects of PAR-1 AP in in vitro model. Pre- and posttreatments of thrombin, and pretreatment of trypsin also protected ischaemia-induced damage in the two models.
PAR-1 AP produced marked increase in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and ratio of bcl-2/bax, but reduced contents of reactive oxygen species (ROS), nitric oxide (NO) and malondialdehyde (MDA) in both ipsilateral ischaemic brain cortices and in rat cortical neurones subjected to OGD. In the in vitro model, PAR-1 AP greatly decreased caspase-3 activity and TUNEL positive cells, while markedly increased mitochondrial membrane potential (MMP). All these protective actions were inhibited by its antagonist, which suggests it was mediated via activation of PAR-1.
In MCA isolated from normal and ischameic rats, PAR-2 AP and trypsin produced vasodilatation while PAR-3 AP elicited vasoconstriction. However, another PAR-3 AP had no effect in the two types of MCA. A high concentration of PAR-1 AP relaxed MCA isolated form ischaemic rats, and it was not inhibited by a PAR-1 antagonist. The vasodilator action of PAR-2 AP was inhibited by one of two PAR-2 antagonists tested. The vasodilator actions induced by PAR-1 and PAR-2 APs involved NO production since L-NAME was effective in inhibiting their actions.
In conclusion, PAR-1 AP was found to be the most efficacious in protecting the brain from ischaemia-induced damage when administered either before or after ischaemia insults. The protective actions were likely to be attributed to its anti-oxidant properties in the ischaemic brain that reduced apoptosis of brain cells. Therefore, PAR-1 was identified as a promising target for development of novel prophylactic and therapeutic treatments of ischaemic brain disease.
缺血性腦中風已經成為全世界導致死亡和殘疾的最主要的疾病之一。蛋白酶激活受體（PARs, PAR-1 to PAR-4）屬於G蛋白偶聯受體並且可以通過蛋白水解生成系鎖配體（TL）從而作用於受體本身而激活信號通路。根據TL的序列已經合成了很多激活肽和拮抗劑，它們可以作為有價值的工具藥進行PAR的作用研究。
當前，PAR的作用在兩個缺血性腦中風模型中進行研究。體內模型是通過大鼠大腦中動脈阻塞手術而建立；體外模型是通過對大鼠胚胎大腦皮層神經元進行氧糖剝奪模擬缺血性損傷。
蛋白質印跡法的實驗表明PAR-1和PAR-2的表達在缺血側大腦皮層中有所增多，而PAR-1在氧糖剝奪的大鼠皮層神經元中表達卻有所降低。預處理PAR-1（SFLLRN-NH₂）和PAR-2（SLIGRL-NH₂）的激活肽顯著改善了缺血導致的損傷。預處理PAR-3激活肽（SFNGGP-NH₂）僅僅改善了體內缺血症狀，卻對體外缺血模型沒有效果。然而，當這些激活肽在缺血后給予的時候，只有PAR-1的激活肽顯著改善了缺血損傷。PAR-1的拮抗劑（BMS-200261）和PAR-2的拮抗劑（ENMD-1068）抑制了PAR-1和PAR-2激活肽的保護作用，但是體外實驗後處理PAR-1激活肽的保護作用卻未收影響。預處理及後處理凝血酶，預處理胰酶都在這兩個模型中顯示出保護缺血性損傷的作用。
PAR-1激活肽在缺血同側大腦皮層以及經受氧糖剝奪的大鼠皮層神經元中，顯著提高了超氧化物歧化酶（SOD）、過氧化氫酶（CAT）、谷胱甘肽過氧化物酶（GSH-Px）的活力以及bcl-2/bax的比例，同時顯著降低了活性氧自由基（ROS）、一氧化氮（NO）以及丙二醛（MDA）的含量。在體外模型中，PAR-1激活肽還顯著降低了caspase-3的活力以及TUNEL陽性細胞的比例，同時顯著提高了線粒體膜電位（MMP）。所有這些作用都可以被拮抗劑抑制，說明PAR-1激活肽的保護作用是通過激活PAR-1介導的。
不管是從正常還是缺血的大鼠中分離出來的大腦中動脈，PAR-2激活肽和胰酶都可以使之舒張，PAR-3激活肽卻對其有收縮作用。然而，另外一種PAR-3激活肽卻未顯現出對血管活性的影響。高劑量的PAR-1激活肽只可以在分離于缺血大鼠的大腦中動脈中引起舒張，但此作用不能被其拮抗劑所抑制。PAR-2激活肽導致的血管舒張只可以被檢測的兩個拮抗劑中的其中一個所抑制。PAR-1和PAR-2激活肽引起的血管舒張與NO的產生有關，因為L-NAME可以有效抑制它們的作用。
總之，不管是預處理還是後處理的給藥方式，PAR-1的激活肽在保護大腦的缺血性損傷中都是最有效果的。保護作用可能可以歸因于其抗氧化以及抗凋亡的特性。所以，PAR-1是研究防治缺血性腦疾病的發展中富有希望的一個靶點。
Zhen, Xia.
Thesis Ph.D. Chinese University of Hong Kong 2014.
Includes bibliographical references (leaves 194-206).
Abstracts also in Chinese.
Title from PDF title page (viewed on 11, October, 2016).
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.

Cerebral radiation necrosis as a consequence of radiation therapy is often observed in patients several months to years after treatment. Complications include painful headaches, seizures, and in the worst-case death. Radiation necrosis is an irreversible condition with the options available to manage it all having noticeable downsides. As such, there is a critical need for better ways of either preventing the onset of necrosis and/or managing its symptoms. As radiation necrosis cannot be induced in humans for ethical reasons, a mouse model that mirrors the features of radiation necrosis observed in patients would allow for new techniques to be tested before being used in human clinical trials. This thesis will explain how our lab designed a murine model of cerebral radiation necrosis that uses a 320 keV cabinet irradiator to produce radiation necrosis and MRI and histology to evaluate the development of radiation necrosis at multiple time points.

Our model required the development of a mouse positioning apparatus that could be used in the cabinet irradiator used as well as the machining of lead shields so that focal semi-hemispheric irradiations could be conducted with other critical structures spared. The MRI scans used as well as the algorithm used to draw radiation necrosis lesions were based off what has been used in previous Gamma Knife models of radiation necrosis. Our initial work showed that since the cabinet irradiator has a relatively flat dose distribution unlike the Gamma Knife, the radiation lesion volumes produced in the former either plateaued or decreased, unlike in the case of the latter where lesion volumes tended to decrease over time. Further work analyzed the effects of fractionation and found minimal sparing using four different fractionation schemes. The effects of strain and sex on the development of radiation necrosis were also analyzed, with strain being found to be a statistically significant parameter while sex was not. Future research should focus on testing the effects of new drugs and techniques for better dealing with radiation necrosis.