Dissertations / Theses on the topic 'Ex injuria'

La thèse de doctorat est consacrée au principe ex injuria jus non oritur qui signifie littéralement que le droit ne naît pas du fait illicite. Alors qu’il est incidemment évoqué par la doctrine ou la jurisprudence comme un principe général du droit international public, il n’a jamais fait l’objet d’une étude systématique destinée à en vérifier l’existence, à en mesurer la portée, à en interroger les limites et à en expliciter les soubassements théoriques. C’est cette lacune que la thèse entend combler, dans une perspective relevant de la théorie du droit international.

Dans une première partie, il s’agit de se demander si ce principe a été reconnu en droit international public, et dans l’affirmative d’en déterminer la portée juridique. La maxime ex injuria jus non oritur ne pouvant être réduite à une règle juridique particulière, la question qui se pose est plutôt de savoir si on peut la qualifier de principe du droit international public qui, sans constituer une source formelle de l’ordre juridique, permet d’expliquer la logique sous-jacente à certaines règles du droit international. Les expressions de ce principe visent d’abord des situations dans lesquelles on remet en cause la validité d’un acte juridique issu de la violation du droit international (invalidité du titre de souveraineté relatif à un territoire acquis ou occupé illégalement, invalidité de l’acte juridique adopté par une autorité illégale, nullité d’un traité dont la conclusion a été obtenue par une contrainte illicite, inadmissibilité comme preuve d’une déclaration obtenue sous la torture, invalidité d’une saisie ou d’une arrestation illégale, invalidité d’un ordre illégal émis par un supérieur hiérarchique). Dans une perspective parallèle, on retrouve le principe dans la règle selon laquelle la violation du droit international ne remet pas en cause sa validité, règle valable dans le domaine du droit des traités, de la coutume ou de la responsabilité internationale. A côté de cette dimension « objective » (dans la mesure où elle recouvre un problème de validité), une dimension plus « subjective » apparaît dans les relations entre sujets du droit international. Ainsi, l’auteur d’une violation du droit international ne peut s’en prévaloir pour revendiquer des droits, et doit plutôt en effacer les conséquences. De même, les Etats tiers ne peuvent reconnaître comme licite une situation résultant de la violation grave d’une norme impérative de droit international, ni ne peuvent prêter aide ou assistance au maintien de cette situation. A l’issue de la première partie de la thèse, on peut établir un constat nuancé :le principe ex injuria jus non oritur constitue un principe général, qui peut être induit de diverses règles de droit international positif, règles qu’il permet d’interpréter en en explicitant l’objet et le but. En même temps, cette existence ne peut être comprise que moyennant une définition stricte et limitée de ce principe, lequel ne prescrit pas, comme on aurait pu s’y attendre, qu’aucun droit ne puisse jamais résulter d’une violation du droit. En premier lieu, et au travers des différents exemples qui viennent d’être mentionnés, on peut remarquer que seules des violations graves —et non des irrégularités mineures— sont de nature à empêcher la création de droits (ainsi, par exemple, dans le domaine de la récolte de preuve). En second lieu, on remarque que seuls les droits qui découleraient directement (dans le sens où ils en consacreraient juridiquement les effets) d’une violation grave du droit ne peuvent être valablement reconnus (ainsi, par exemple, des actes quotidiens d’administration posés par un occupant illégal peuvent être reconnus, ces actes n’étant pas intrinsèquement liés à ce statut d’occupant illégal). Ce n’est que dans cette double mesure que l’on peut affirmer que, en droit international positif, il existe un principe général exprimé par la maxime ex injuria jus non oritur.

Dans la seconde partie de la thèse, le principe est confronté, d’une part (volet empirique) à des précédents dans lesquels il semble avoir été mis à mal (certaines situations semblant avoir résulté de violations graves du principe impératif de l’interdiction du recours à la force) et, d’autre part (volet théorique), à des théories du droit international susceptibles d’en expliquer à la fois le fonctionnement et les limites. Le volet empirique s’appuie sur une étude de cas :la reconnaissance du Bangladesh à la suite d’une intervention militaire de l’Inde au Pakistan, la reconnaissance des gouvernements installés au Cambodge à la suite de l’intervention militaire du Vietnam, la validité des accords conclus par la Yougoslavie à la suite de l’intervention militaire de l’OTAN, la reconnaissance du Kosovo en 2008, et l’administration de l’Irak après l’intervention militaire de 2003. Si le principe ex injuria jus non oritur est sans doute malmené dans les faits, il ne l’est pas dans le discours officiel des Etats, lesquels n’assument pas une remise en cause d’un principe dont ils reconnaissent par ailleurs (comme montré dans la première partie de la thèse) la validité. On peut se demander si cette tension entre un discours légaliste et une réalité parfois caractérisée par la force des effectivités, est susceptible d’être comprise au regard de certaines doctrines qui traitent des relations entre le fait et le droit. Ce volet spécifiquement théorique de la recherche consiste à examiner deux approches, par hypothèses opposées. La première pourrait suggérer une consécration du principe par le biais de la théorie normativiste élaborée par Hans Kelsen. Selon cette théorie, le droit (international) se définirait comme un ensemble cohérent de normes, chaque norme juridique tirant sa validité d’une autre norme juridique valide, ce qui semble exclure qu’une norme puisse s’appuyer sur une violation du droit. A l’analyse, le normativisme paraît néanmoins réfractaire à une reconnaissance du principe ex injuria jus non oritur, la validité du droit ne pouvant être détachée de toute considération fondée sur l’effectivité, et celle-ci pouvant même le cas échéant aboutir à la consécration d’une situation résultant d’une violation du droit. A l’opposé, on pourrait s’attendre à ce que l’approche critique, définie par référence aux travaux de l’ « école de Reims » qui se sont développés autour de Charles Chaumont, rejette ex injuria jus non oritur comme une maxime formaliste et fictive, la force du fait, et plus spécifiquement du rapport de forces, prévalant dans la réalité sociale comme facteur générateur de la création et de l’interprétation de la règle de droit. Ici encore, on détecte une certaine ambiguïté chez les auteurs analysés, lesquels ont recours en certaines occasions au droit comme à un instrument de lutte qui s’opposerait à la force et à la puissance. Finalement, la confrontation des approches normativiste et critique laisse apparaître un fil conducteur :le principe ex injuria jus non oritur n’est que le révélateur des difficultés, non seulement en pratique (comme l’a montré le volet empirique) mais aussi en théorie, de concilier les exigences idéalistes du respect du droit avec les impératifs réalistes de prendre en compte la force du fait.

En conclusion, le principe ex injuria jus non oritur se caractérise surtout par cette tension entre le droit et le fait, qui permet également d’expliquer les ambiguïtés observées dans la première partie, le principe n’étant admis en droit positif que moyennant une définition restrictive ouvrant à une certaine souplesse. Cette tension renvoie d’ailleurs à la question de l’existence même du droit international, lequel peut être présenté comme une forme sophistiquée de discours, et non comme un corps de règles régissant effectivement la réalité sociale. Dans cette perspective, il est intéressant de constater que, au-delà des stratégies discursives des Etats qui tentent de justifier certains faits accomplis sans remettre en cause le principe de légalité, il est certains précédents (comme celui du Bangladesh) où ces Etats restent tout simplement silencieux par rapport à cette question. Ainsi, l’analyse du principe ex injuria jus non oritur à l’épreuve de la pratique internationale tendrait peut-être, non pas à reconnaître la portée du principe en toute hypothèse, mais à montrer qu’au-delà d’un certain seuil de tension, le droit disparaît dans la mesure où le discours qui s’y rapporte disparaît. En définitive, la tension entre la légalité (l’existence formelle d’un ordre juridique international) et l’effectivité (laquelle ne témoigne pas toujours de l’existence de cet ordre juridique) est aussi celle qui habite le spécialiste de droit de droit international, parfois confronté aux limites de son activité et de sa discipline.

Doctorat en droit
info:eu-repo/semantics/nonPublished

Il non riconoscimento nel diritto internazionale il non riconoscimento presenta diverse questioni che devono essere risolte sia alla luce della sua natura, sia del suo contenuto sia dei suoi effetti. La prassi internazionali offre diversi esempi di non riconoscimento. Il presente lavoro cerca di dare ordine a questa varietà di casi cercando di capire come un’analisi di caso in caso sia l’approccio migliore per riaffermare l’importanza del non riconoscimento. Il fatto che esso presenti un contenuto diverso in base alla situazione non determina la sua non importanza. Esso anzi è uno strumento imprescindibile per garantire la tenuta dell’ordine internazionale.
Non-recognition in international law presents several questions that need to be resolved both in the light of its nature, its content and its effects. International practice offers several examples of non-recognition. The present work tries to give order to this variety of cases trying to understand how a case by case analysis is the best approach to reaffirm the importance of non-recognition. The fact that it presents a different content according to the situation which is the object of non-recognition does not determine its irrelevance. In fact, it turns out to be an indispensable tool to guarantee the preservation of the international order.

This PhD dissertation is about legal fictions in private law. A legal fiction, broadly, is a false assumption knowingly relied upon by the courts. The main aim of the dissertation is to formulate a test for which fictions should be accepted and which rejected. Subsidiary aims include a better understanding of the fiction as a device and of certain individual fictions, past and present. This research is undertaken, primarily, to establish a rigorous system for the treatment of fictions in English law - which is lacking. Secondarily, it is intended to settle some intractable disputes, which have plagued the scholarship. These theoretical debates have hindered progress on the practical matters which affect litigants in the real world. The dissertation is divided into four chapters. The first chapter is a historical study of common-law fictions. The conclusions drawn thereform are the foundation of the acceptance test for fictions. The second chapter deals with the theoretical problems surrounding the fiction. Chiefly, it seeks precisely to define 'legal fiction', a recurrent problem in the literature. A solution, in the form of a two-pronged definition, is proposed, adding an important element to the acceptance test. The third chapter analyses modern-day fictions and recommends retention or abolition for each fiction. In the fourth chapter, the findings hitherto are synthesised into a general acceptance test for fictions. This test, which is the thesis of this work, is presented as a flowchart. It is the author's hope that this project will raise awareness as to the merits and demerits of legal fictions, de-mystify the debate and bring about reform.

Background. Apoptosis is postulated as a mechanism involved in lung ischemia-reperfusion (IR) injury, however, the relative contributions of ischemia and reperfusion are unclear.
Methods. Heart-lung blocks were harvested from New Zealand white rabbits (3.0--4.0 kg) and exposed to 0, 6, or 18 hours of cold ischemia (4°C), followed by 3 hours of reperfusion in an ex vivo model. Terminal dUTP nick-end labeling (TUNEL), the technique used most often for detection of apoptosis, was performed on the tissue sections.
Results. TUNEL demonstrated minimal apoptosis in lungs exposed to 0, 6, or 18 hours of ischemia with insignificant differences (p = 0.6 for 0 h vs. 18 h). After one hour of reperfusion, the level of TUNEL in the 18 hour ischemic tissue was significantly increased (p < 0.05 for 0 h vs. 18 h). During the period of reperfusion, the extent of apoptosis increased in direct proportion to the duration of ischemia; the level of TUNEL staining after 2 hours of reperfusion was significantly greater in the 18 hour ischemic tissue compared to the 6 hour ischemic tissue (p < 0.05), as was the 6 hour compared to the 0 hour (p < 0.01). The hallmark of apoptosis, nucleosomal ladders of 180--200 base pair DNA fragments, corresponded in intensity on gel electrophoresis to the quantitation of TUNEL. The characteristics of apoptotic cells including cell membrane blebbing, chromatin condensation and fragmentation were confirmed by electron microscopy.
Conclusions. These results provide evidence that apoptosis may be a specific feature of IR injury in pulmonary tissue.

A growing body of evidence shows that presynaptic nerve terminals throughout the nervous system are vulnerable to a range of traumatic, toxic and disease-related neurodegenerative stimuli. The aim of this study was to further characterise this vulnerability by examining the response of mouse α-motor nerve terminals at the neuromuscular junction (NMJ) to hypoxia-reperfusion injury. To address this aim, a novel model system was generated in which ex vivo skeletal muscle preparations could be maintained in an hypoxic environment, at an O2 concentration below in vivo normoxic values (<0.25% O2), for 2hr followed by 2hr reperfusion (2H-2R). Using this model system combined with quantitative assessment of immunohistological preparations as well as some ultrastructural observations, I present evidence to show that α-motor nerve terminals are rapidly and selectively vulnerable to hypoxia-reperfusion injury with no apparent perturbations to postsynaptic endplates or muscle fibres. I show that the severity of α-motor nerve terminal pathology is age and muscle type/location dependent: in 8-12wk old mice, nerve terminals in fast-twitch lumbrical muscles are more vulnerable than predominantly slow-twitch transversus abdominis and triangularis sterni. In 5-6 week old mice however, there is an age dependent increase in vulnerability of α-motor nerve terminals from the predominantly slow-twitch muscles while the fast-twitch lumbricals remained unaffected by age. The functional, morphological and ultrastructural pathology observed in α-motor nerve terminals following 2H-2R is indicative of selective and ongoing nerve terminal disassembly but, occurs via a mechanism distinct from Wallerian degeneration, as the neuroprotective slow Wallerian degeneration (Wlds) gene did not protect nerve terminals from these pathological changes. I also provide provisional evidence to show that 1A/II muscle spindle afferents and γ-motor nerve terminals are more resistant to hypoxia-reperfusion injury compared with α-motor nerve terminals. In addition to this, I also report preliminary finding that indicate that the oxygen storing protein, neuroglobin, maybe expressed at the mouse NMJ and report the difficulties of using mice that express yellow fluorescent protein (YFP) in their neurons for repeat/live imaging studies. Overall, these data show that the model of hypoxia-reperfusion injury developed in this study is robust and repeatable, that it induces rapid, quantitative changes in α-motor nerve terminals and that it can be used to further examine the mechanisms regulating nerve terminal vulnerability in response to hypoxia-reperfusion injuries. These findings have clinical implications for the use of surgical tourniquets and in the aetiology of many neurodegenerative diseases and neuropathic sequelae where mechanisms relating to hypoxia and hypoxia-reperfusion injury have been implicated.

Autologous hMSCs are a promising therapeutic candidate for ischemic tissue injury such as that resulting from myocardial infarction. In recognition of the poor functional characterisation of hMSCs used in clinic and lack of priming methods to enhance cell function, the work presented in this thesis describes several ex vivo strategies designed to assess and enhance hMSC responses. Firstly, growth kinetics of hMSCs isolated from 10 healthy donors were mapped over 5 passages and inter-donor variability was determined. A process operating window was identified between passage 2 and passage 3 where different donors displayed no PDL and cell density differences. Expansion potential at the end of passage 2 was 17.29x106 ± 5.19x106 and at the end of passage 3 was 50.40x106 ± 8.59x106. Next, functional effects of sJag1 and sDll4 pre-conditioning on hMSCs attachment and migration responses were studied. The effect of low oxygen was also assessed, to more closely reflect the physiologic environment. In low oxygen conditions, sJag1 pre-conditioning significantly increased hMSC attachment to fibronectin after just 20 minutes compared with unconditioned cells. However, in ambient oxygen, the stimulatory effect was not seen until 40 minutes attachment time. sJag1 pre-conditioning also significantly enhanced hMSC chemotaxis through fibronectin in response to SDF-1α in low oxygen. Comparable effects were not detected when using a type I collagen substrate. Finally, sJag1 also enhanced the production of angiogenic factors VEGF-A165 and HGF by hMSCs on fibronectin. We assessed the effects of sJag1 and sDll4 on vascular support capacity of hMSCs using in vitro co-culture assays with endothelial cells. Pre-conditioning hMSCs with sJag1 resulted in significantly increased branching within co-culture networks and enhanced vessel-like network formation and structural maintenance over 96 hours in the low oxygen environment. Finally, the requirement for functional Nrp1 in MSCs was studied using Nrp1 cytoKO mice. Although cell attachment was impaired in MSCs lacking fully functional Nrp1, sJag1 was still able to enhance cell attachment. Since hMSCs are widely used in clinical trials with limited positive outcomes, pre-conditioning with sJag1 may be a tool for priming immature cells to improve retention and therapeutic efficacy in ischemic tissues.

Objectif De nombreux travaux ont souligné le rôle de la Cyclosporine A (CsA) dans la prévention des lésions d'ischémie-reperfusion (I/R) mais aucun n'a été effectué sur poumons isolés de grands mammifères. Notre objectif était de mesurer pour la première fois les effets de la CsA sur les lésions d'I/R dans un modèle de poumons porcins reperfusés ex vivo, en évaluant plusieurs doses de CsA pour différents temps d'ischémie. Méthodes L'expérimentation A a été conduite sur 4 groupes de 8 paires de poumons chacune : un groupe contrôle et 3 groupes recevant différentes concentrations de CsA (1, 10 ou 30 μM) au moment de l'ischémie et au début de la reperfusion, après 2 heures d'ischémie. L'expérimentation B a été conduite sur 3 groupes de 5 paires de poumons chacune. Les poumons de chaque paire étaient séparés juste après le début de l'ischémie. Les premiers poumons étaient évalués après une ischémie de 2 heures (jour 0), sans CsA. Les seconds poumons étaient évalués après une ischémie de 24 heures (jour 1), soit sans soit avec CsA (1 ou 5 μM), administrée le cas échéant au début de la reperfusion. Résultats La CsA augmentait le rapport PO2/FiO2 avec un effet dose mais augmentait également la pression artérielle pulmonaire, la pression capillaire et les résistances vasculaires pulmonaires, à 10 et 30 μM mais pas à 1 ni 5 μM. Les poumons qui recevaient 30 μM de CsA affichaient des concentrations élevées en cytokines pro-inflammatoires. La concentration en RAGE (receptor for advanced glycation endproducts) dans le lavage broncho-alvéolaire diminuait avec la CsA à J1 en comparaison à J0. Conclusions Lors de l'I/R pulmonaire, les bénéfices cellulaires des doses élevées de CsA sont contrebalancés par ses effets hémodynamiques sur la microvascularisation. A faibles doses, la CsA semble améliorer la fonction pulmonaire
Objective Several works highlighted the role of Cyclosporine A (CsA) in the prevention of ischemia reperfusion (I/R) injuries but none on isolated lungs of big mammals. Our objective was to measure for the first time the effects of CsA in I/R injuries in an ex vivo reperfused pig lungs model, by evaluating several doses of CsA for different times of ischemia. Methods Experimentation A was performed on 4 groups of 8 pairs of lungs each: a control group and 3 groups receiving different concentrations of CsA (1, 10 or 30 μM) at the time of ischemia and at the beginning of the reperfusion, after a 2 hours ischemia. Experimentation B was performed on 3 groups of 5 pairs of lungs each. Lungs from each pair were separated just after the beginning of ischemia. The first lungs were evaluated after a 2 hours ischemia (day 0), without CsA. The second lungs were evaluated after a 24 hours ischemia (day 1), either without or with CsA (1 or 5 μM), administered when appropriate at the beginning of the reperfusion. Results CsA improved the PO2/FiO2 ratio with a dose dependent effect but increased pulmonary arterial pressure, capillary pressure, and pulmonary vascular resistances, at 10 and 30 μM but neither at 1 or 5 μM. Lungs receiving 30 μM of CsA displayed elevated concentrations in pro-inflammatory cytokines. Concentrations in RAGE (receptor for advanced glycation endproducts) in broncho-alveolar lavage decreased with CsA at day 1 compared to day 0. Conclusions During pulmonary I/R, the cellular benefits of high doses of CsA are counterbalanced by its hemodynamic effects on microvascularisation. At low doses, CsA seems to improve lung function

INTRODUÇÃO: As técnicas de preservação pulmonar visam a melhorar a qualidade do enxerto e aumentar sua tolerância ao período de isquemia fria. A técnica mais usada atualmente consiste na perfusão da artéria pulmonar com Perfadex. O alto custo associado à importação dessa solução e as dificuldades logísticas dos portos e aeroportos brasileiros com relação a materiais médicohospitalares têm causado problemas para os centros de transplante pulmonar brasileiros. Daí a necessidade de uma solução de preservação pulmonar produzida no Brasil. O objetivo desse estudo é comparar a solução Perfadex com a solução de fabricação nacional LPD-G, quanto ao grau de lesão de isquemia-reperfusão, em um modelo de perfusão pulmonar ex vivo (PPEV). MÉTODOS: Foram usados doadores em morte cerebral, cujos pulmões foram recusados. Cada caso era incluído aleatoriamente em um dos grupos: Grupo 1, a preservação pulmonar era realizada com Perfadex, e Grupo 2, era usado o LPD-G, solução fabricada no Brasil com composição idêntica a do Perfadex. Após a captação, os pulmões eram armazenados a 4 °C por 10 horas. A reperfusão ocorria em um sistema de PPEV, no qual o bloco pulmonar era ventilado e perfundido por uma solução acelular a 37 °C por 60 minutos. A lesão de isquemia-reperfusão era medida através de parâmetros funcionais (gasometria, resistência vascular pulmonar, complacência pulmonar, relação peso úmido/peso seco) e histológicos. Foram feitas biópsias pulmonares em 3 tempos: antes da captação, após o período de isquemia fria e depois da reperfusão. Vários critérios foram usados (edema alveolar, edema intersticial, hemorragia etc.) para criar um Escore de Lesão Pulmonar (ELP). A contagem de células apoptóticas foi feita usando a metodologia TUNEL (TdT-mediated dUTP nick end labeling). RESULTADOS: Após a reperfusão, a capacidade de oxigenação média foi de 405,3 mmHg no Grupo 1 e 406,0 mmHg no Grupo 2 (p = 0,98). A mediana da resistência vascular pulmonar nos pulmões do Grupo 1 foi de 697,6 dina.s.cm-5, enquanto no Grupo 2, esse valor foi de 378,3 dina.s.cm-5 (p = 0,035). A complacência pulmonar média ao final da reperfusão foi de 46,8 cmH2O no Grupo 1 e de 49,3 ml/cmH2O no Grupo 2 (p = 0,816). A razão entre o peso úmido e o peso seco foi em média 2,06 e 2,02 nos Grupos 1 e 2, respectivamente (p = 0,87). Na biópsia realizada após reperfusão, o ELP médio foi de 4,37 e 4,37 nos Grupos 1 e 2, respectivamente (p = 1,0); a contagem de células apoptóticas foi de 118,75/mm2 e 137,50/mm2 nos Grupos 1 e 2, respectivamente (p = 0,71). CONCLUSÕES: A qualidade da preservação pulmonar obtida com a solução LPD-G nacional é semelhante a obtida com o Perfadex. A aplicação clínica da nova solução pode reduzir custos, facilitando a manutenção e a abertura de centros de transplante pulmonar
INTRODUCTION: Pulmonary preservation techniques aim at improving graft quality and increasing tolerance during reperfusion and cold ischemia times. Currently, the most used technique consists of pulmonary artery anterograde perfusion with Perfadex. The high cost associated with the importation of this solution and the logistical difficulties of our ports and airports regarding medical supplies have caused problems for lung transplant centers in Brazil. Therefore there is need for a preservation solution manufactured in Brazil. The aim of this study is to compare the pulmonary preservation solutions Perfadex and LPD-G manufactured in Brazil in an ex vivo lung perfusion (EVLP) model. METHODS: Donors with brain death, whose lungs had been declined by transplantation teams were used. Cases were randomized into two groups: in Group 1, Perfadex was used for pulmonary preservation. In Group 2, LPDnac, a solution manufactured in Brazil and whose compositon is identical to Perfadex, was used. After harvesting, lungs were stored at 4 °C for 10 hours. An EVLP system was used and the pulmonary block was ventilated and perfused by an acellular solution at 37 °C for 60 minutes. Ischemic-reperfusion injury was measured by functional (blood gas, pulmonary vascular resistance, lung compliance, wet/dry weight ratio) and histological parameters. Pulmonary biopsies were performed at three time points: before harvesting, 10 hours after cold ischemia and 60 minutes after reperfusion. Samples were prepared for light microscopy analysis. Several criteria were used (alveolar edema, interstitial edema, hemorrhage etc.) to create a lung injury score (LIS). Apoptotic cell count was carried out using the TUNEL methodology (TdT-mediated dUTP nick end labeling). RESULTS: After reperfusion, mean oxygenation capacity was 406.0 mmHg in Group 2 and 405.3 mmHg in Group 1 (p = 0.98). Mean pulmonary vascular resistance in Group 2 lungs was 378.3 dina.s.cm-5, whereas in Group 1 it was 697.6 dina.s.cm-5 (p = 0.035). Mean pulmonary compliance by the end of reperfusion was 49.3 ml/cmH2O in Group 2 and 46.8 cmH2O in Group 1 (p = 0.816). Mean wet/dry weight ratio was 2.02 and 2.06 in Groups 2 and 1, respectively (p = 0.87). Mean LIS for the biopsy performed after reperfusion was 4.37 and 4.37 in Groups 2 and 1, respectively (p= 1.0); apoptotic cell count was 137.50/mm2 and 118.75/mm2 in Groups 2 and 1, respectively (p = 0.71). CONCLUSION: The preservation solution manufactured in Brazil proved to be as good as Perfadex. The clinical application for the new solution may reduce costs, favoring the maintenance and opening of pulmonary transplantation centers

The peripheral nerve disorder Guillain-Barré syndrome (GBS) accounts for the most common cause of acute acquired paralysis in the Western World. Circulating anti-ganglioside antibodies (Abs) are considered important mediators of this disease. Research conducted in a mouse model of GBS has the revealed the neuromuscular junction (NMJ) as a potential site of anti-ganglioside Ab-binding, due to this structure lying outside the blood-nerve barrier. The ganglioside composition of the neural and glial components of the NMJ determines which of these structures are bound and in the following subjected to complement-mediated injury. Some patients suffering from the acute motor axonal neuropathy (AMAN) forms of GBS recovery very rapidly from paralysis; it has been proposed that in these patients the injury was restricted to the distal motor axons and nerve terminals (NTs), which are able to regenerate over a short time-frame. To test this hypothesis, the mouse model of GBS was combined with in and ex vivo imaging of the NMJ in the ventral neck muscles of mice expressing cytosolic fluorescent proteins in their axons (cyan fluorescent protein: CFP) and Schwann cells (green fluorescent protein: GFP). Following confirmation of the stability of NMJs in these mice over time, optimisation of in vivo imaging procedures and determination of the most advantageous Abs for these kinds of investigations, 45 mice were subjected to a single in vivo topical application of anti-ganglioside Ab followed by a source of complement. Group A (n=15) received Ab that selectively bound to the NTs, group B (n=15) received Abs that bound both to the NTs and the perisynaptic Schwann cells (pSCs) and group C (control animals; n=15) only received complement. Evolution of the injury was documented by in vivo imaging, and following euthanasia the muscles were reimaged ex vivo both quantitatively and qualitatively, either immediately, or after 1, 2, 3 or 5 days of regeneration (each n=3 per group). Within 15 minutes of complement application, a rapid loss of CFP overlying the NMJ could be seen; in group A, the GFP signal remained unchanged, whereas in group B the GFP signal was also lost. In group C no changes to either CFP or GFP were observed. At 24h, 6% of the superficial NMJs in group A and 12% of the NMJs in group B exhibited CFP; the CFP-loss extended proximally until the axons formed little bundles. In both groups, CFP returned within the next five days (group A: 93.5%, group B: 94%; p=0.739), with the recovery of CFP being preceded by a return of GFP-positive cells overlying the NMJ in group B. Both in groups A and B, the pSCs exhibited processes which extended beyond the normal NMJ boundaries and very occasionally accompanied by axonal sprouts. This behavior was similar to that of pSCs challenged by traumatic denervation of their NMJ, albeit the changes observed in the Ab-mediated injury were lower in frequency and less dramatic when compared to those observed following traumatic denervation. The rate of motor NT-regeneration corresponds well to rates observed following the application of spider or snake toxins, which mediate selective injury to the NTs. Auxiliary investigations revealed that the loss of CFP at the NMJ correlated with a loss of axonal architectural proteins (neurofilament (NF) heavy and light) and a return of CFP at the NMJ was accompanied by a return of NF heavy. Ultrastructurally, the injured NTs resembled NTs undergoing degeneration following a traumatic denervation of the endplate and following five days of regeneration, the NMJs exhibited a physiological morphology. The results described above indicate that following a single anti-ganglioside Ab-mediated and complement-mediated attack, independent of whether there are healthy and mature pSCs overlying the NMJ, the murine NT is capable of recovering its architectural, axolemmal and ultrastructural integrity very rapidly. This data supports the notion that an equivalent mechanism may account for the rapid recovery seen in some clinical cases of AMAN.

Flexor tendon repair continues to provide surgeons with a challenge. Injuries occurring in zone II are a particular challenge due to the complex arrangement of the anatomy in this area. Poor results postoperatively are costly and lead to even more complex second operations, resulting in long time periods where patients are unable to use their hands fully. The repair techniques are numerous, as are the materials available for such repairs, and the training for surgeons in this field is largely based on practice on simulation models and the apprenticeship method. The literature review in this dissertation highlights the numerous repair variables, the type of repair, suture size and material, number of strands of suture crossing the repair site, as well as testing models. The use of a non-absorbable suture like braided polyester (Ticron) and a monofilament of polypropylene (Prolene) are commonly used in clinical practice, and these are both investigated for effect on tensile properties of the repair, as is the learning curve of one of the investigators. One aim of this research was to develop a realistic, low cost bench-top model for tensile testing of tendon repairs, to enable the primary of the study; an investigation of a new core suture and a new peripheral repair method. A porcine model is investigated for anatomical similarity with human flexor tendons, and a mechanical testing protocol established, that allows investigation of tendon repairs for tensile tests as well as internal work of friction. A comparison of the well-known and tested Pennington Modification of the Modified Kessler (MK) technique is performed with a new repair type that incorporates a ventral locking loop to the standard Modified Kessler (LMK). In addition the work introduces a PolyCaprolactone (PCL) sheet to the repair, as a new peripheral repair method. Early findings showed higher values for the Ticron repairs and a clear learning curve with regards to mechanical properties of the repairs. The importance of conditioning of the tendons was also investigated in the preliminary studies and a clear effect on the work of flexion was found for each cycle tested. No difference was seen in the tensile properties between the repairs, but for the two strand core suture repairs the LMK had lower work of flexion values. This was investigated further by measuring the cross sectional diameters of the two repair types to see if the change in repair technique had influenced the size of the repair. No difference was found. The four strand core suture repairs showed a higher force to gap formation of the LMK repair compared to the MK. The addition of the PCL sheet resulted in lower force to gap formation values. With regards to the work of flexion the PCL resulted in an almost doubling of the values seen with MK or LMK repairs, however no statistical difference was seen with regards to work of flexion between the MK and LMK. This work has shown that the porcine model is similar to human and provides a good model for tendon repair. A mechanical testing procedure for tensile testing as well as work of flexion has been established that allows for testing of tendon repairs. The model would also allow for trainees to practice and get qualitative feedback on their repairs. No clear biomechanical advantage was seen with the addition of the ventral locking loop when compared to the MK, however the MK configuration used had an increased locking loop diameter than those presented in the literature which means that the LMK may provide a biomechanical advantage. Further studies are warranted to investigate how the LMK compares to MK with smaller locking loop areas, and other commonly used repair techniques. The addition of the PCL sheet to the repair, caused a large increase in the work of flexion of the repairs, and the PCL sheets showed large deformation after low loads during tensile testing which means that they would not have any clinical application for zone 2 tendon repairs. The possibility of using the PCL sheet as a tissue engineering scaffold/matrix for tendon repairs out with the hand could be investigated in further studies, however it is likely that modifications need to be done to the PCL before it proves useful in clinical practice.

Der renale Ischämie-/Reperfusionsschaden (IRI) stellt in der Transplantationsmedizin ein grosses Problem dar. Fucoidin, ein potenter Antagonist der Selektin-vermittelten Leukozytenaggregation, verbesserte an der Rattenleber (in einer Konzentration von 360mg/l) das Ausmass der leukozytären Gewebeinfiltration in der frühen Phase nach Ischämie und Reperfusion. In einem Modell der isoliert hämoperfundierten Schweineniere sollte die Wirkung von Fucoidin auf die postischämische Organfunktion untersucht werden. Hierzu wurden 24 Versuche durchgeführt. Dem Blut der Versuchsgruppen wurde vor Beginn der Reperfusion Fucoidin in einer Konzentration von 100 mg/l zugesetzt. Es zeigte sich unter Fucoidin ein signifikanter Abfall des renalen Blutflusses (55 ( 28 vs. 143 ( 97 ml*min-1*100g-1, p
Renal postischemic reperfusion injury constitutes a significant problem after kidney transplantation. The polysaccharide fucoidin (360 mg/l) improves postischemic function in Ratliver, presumably by blocking selectin-mediated leukocyte adhesion. Twelve pairs of ischemic pig kidneys were reperfused in an ex vivo model with autologous blood with or without fucoidin (100 mg/L). Fucoidin resulted in a significant decrease of renal blood flow (55 ( 28 vs. 143 ( 97 mL*min-1*100g-1, p < 0.001) and increased vascular resistance (2.9 ( 2.8 vs. 1.1 ( 1.5 mmHg*mL-1*min-1*100g-1, p < 0.001). Compared to untreated control kidneys significantly more interstitial and intravascular leucocytes were found in fucoidin treated kidneys. Intraglomerular fibrinogen and thrombocytic aggregates were also increased significantly. Granulocytic emboli were present in afferent glomerular arteries of 10/12 fucoidin-treated kidneys and in 2/12 controls (p < 0.001). L-selectin-dependent granulocytic aggregation under shear stress in vitro was prevented by fucoidin in a dose-dependent fashion. However similar concentrations used in reperfused kidneys caused large granulocytic aggregates. The observed formation of embolizing granulocytic aggregates indicates limited effectiveness of fucoidin as an inhibitor of selectin-mediated leukocyte adhesion.

INTRODUÇÃO: O transplante pulmonar é terapia reconhecida de tratamento de doenças terminais pulmonares. Os pulmões, entretanto, são muito susceptíveis às transformações hormonais e hidroeletrolíticas ocorridas no doador após a morte encefálica. As baixas taxas de aproveitamento dos pulmões alavancam pesquisas e meios de utilizar pulmões considerados nãoideais. Um desses modelos é o recondicionamento pulmonar ex-vivo concebido por Steen, no qual se utiliza uma solução hiperosmolar (Steen Solution®) para avaliação e melhora dos pulmões doados. Consideramos que o desenvolvimento de uma solução de recondicionamento pulmonar produzida no Brasil seria conveniente aos serviços de transplante e aos pacientes. Foram comparadas a solução de recondicionamento Steen Solution® e uma solução de fabricação nacional em modelo de ex vivo de pulmões humanos não aceitos para transplante, através da avaliação da mecânica ventilatória, hemodinâmica, trocas gasosas e histologia. MÉTODOS: Foram utilizados 16 pulmões de doadores em morte encefálica, considerados inadequados para o transplante pulmonar. Os pulmões foram submetidos à captação habitual e acondicionados sob isquemia fria por 10 horas. Após este período, os pulmões foram designados, por sorteio, para reperfusão com a solução padrão (Steen solution®) ou a solução nacional por 1h em modelo ex vivo. A lesão pulmonar foi estudada através de parâmetros gasométricos, resistência pulmonar e complacência pulmonar. Foram medidos os pesos em três tempos e relação peso úmido/peso seco após a reperfusão para avaliação de edema. A partir de biópsias seriadas era calculado um score de lesão tecidual e grau de apoptose. RESULTADOS: A capacidade de oxigenação foi de 498,00 ± 37,53 mmHg no grupo STEEN e 521,00 ± 55,43 mmHg no grupo SRNac (p = 0,348). A capacidade relativa de oxigenação calculada ao final do recondicionamento foi 501,37 ± 207,77 no grupo STEEN e 470,30 ± 232,41 no grupo SRNac (p=0,782). Os pesos dos pulmões nos três momentos de avaliação foram: início da isquemia: STEEN = 1.026 ± 451 g, SRNac = 745 ± 282 g (p = 0,180); fim da isquemia: STEEN = 998 ± 391 g, SRNac = 738 ± 316 g (p = 0,184); e fim da reperfusão: STEEN = 1.097 ± 526 g, SRNac = 743 ± 248 g (p = 0,163). A relação peso úmido/peso seco foi 3,63 ± 1,26 no grupo SRNac e 2,06 ± 0,28 no grupo STEEN (p = 0,009). A resistência vascular pulmonar foi 787,99 ± 367,23 dina.s.cm-5 no grupo STEEN e 1.026,81 ± 1.112,53 dina.s.cm-5 no grupo SRNac (p = 0,575). A complacência pulmonar média foi 46,75 ± 20,99 mL/cmH2O no grupo STEEN e 49,74 ± 26,11 cmH2O no grupo SRNac (p = 0,809). O Escore de Lesão Pulmonar foi: STEEN = 4,38 ± 1,51 e SRNac = 4,50 ± 1,77 (p = 0,881). O número de células apoptóticas foi: STEEN = 2,4 ± 2,0 cel/mm2 e SRNac = 4,8 ± 6,9 cel/mm2 (p = 0,361). CONCLUSÕES: Os pulmões reperfundidos com a solução de recondicionamento de fabricação nacional apresentaram características morfológicas e funcionais similares aos que foram reperfundidos com a solução STEEN®, apesar do maior edema encontrado no grupo da solução nacional
INTRODUCTION: Lung transplantation is routine treatment of end-stage lung diseases. The lungs, however, are very susceptible to hormonal and electrolyte changes occurred in the donor after brain death. The low recovery rates of the lungs leverage researches and ways to use lungs considered non-ideal. One such model is the lung ex vivo reconditioning designed by Steen, in which using a hyperosmolar solution (Steen Solution®) for evaluation and improvement of donor lungs. We believe that the development of a pulmonary reconditioning solution produced in Brazil would be convenient to transplantation service and patients. Were compared the standard Steen Solution® and a national manufacturing solution in ex vivo model with human lungs not accepted for transplant, through the evaluation of respiratory mechanics, hemodynamics, gas exchange and histology. METHODS: 16 brain-dead donors lungs, considered unsuitable for lung transplantation, were used. The lungs were harvest as usual, packed and stored in cold ischemia for 10 hours. After this period, the lungs were appointed by randomization to reperfusion with the standard solution (Steen Solution®) or national solution for 1 h in ex vivo model. Lung injury was accessed by blood gas parameters, lung resistance and lung compliance. The weights were measured in three times and the after reperfusion wet weight / dry weight ratio for evaluation of edema. The degree of apoptosis and tissue injury score was calculated from serial biopsies. RESULTS: The oxygenation capacity was 498.00 ± 37.53mmHg in STEEN group and 521.00 ± 55.43mmHg in SRNac group (p = 0.348). The relative oxygenation capacity calculated at the end of the reconditioning was 501.37 ± 207.77 in the STEEN group and 470.30 ± 232.41 in the SRNac group (p = 0.782). The weights of lungs in the three stages of evaluation were: onset of ischemia: STEEN = 1,026 ± 451g, SRNac = 745 ± 282g (p = 0.180); end of ischemia: STEEN = 998 ± 391g, SRNac = 738 ± 316g (p = 0.184); and the end of reperfusion: STEEN = 1,097 ± 526g, SRNac = 743 ± 248g (p = 0.163). The wet weight / dry weight ratio was 3.63 ± 1.26 in SRNac group and 2.06 ± 0.28 in STEEN group (p = 0.009). Pulmonary vascular resistance was 787.99 ± 367.23dina.s.cm-5 in STEEN group and 1026.81 ± 1112.53dina.s.cm-5 in SRNac group (p = 0.575). The Lung Injury Score was: STEEN = 4.38 ± 1.51 and SRNac = 4.50 ± 1.77 (p = 0.881). The number of apoptotic cells was: STEEN = 2.4 ± 2.0 cells / mm 2 and SRNac = 4.8 ± 6.9 cells / mm2 (p = 0.361). CONCLUSIONS: The lungs reperfused with national manufacturing reconditioning solution presented morphological and functional characteristics similar to those reperfused with STEEN® solution despite the greater edema found in the group of national solution

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

La démyélinisation et la mort oligodendrocytaire sont bien connues dans la sclérose en plaques (SEP). Au cours de ces dernières années, plusieurs études ont également décrit ce type de lésion après un traumatisme crânien (TC), participant à l’aggravation des lésions de la substance blanche, responsables des dysfonctionnements cognitifs et moteurs. Malgré de nombreux efforts, aucune thérapie efficace n’est disponible à ce jour pour traiter les lésions de la substance blanche. Dans ce contexte, une stratégie thérapeutique prometteuse serait de freiner la neuro-inflammation et la démyélinisation, en plus de promouvoir la maturation des oligodendrocytes afin de favoriser la remyélinisation des axones et de limiter ainsi leur dégénérescence. Notre choix de stratégie porte sur la stimulation des processus de réparation endogène via la protéine neuroprotectrice et neurotrophique sAPPα, forme soluble de la protéine βAPP libérée par l’action des α-sécrétases (ADAM10). Dans ce contexte, l’objectif de mes travaux de thèse était d’étudier l’intérêt thérapeutique de l’étazolate, un activateur desα-sécrétases, sur les conséquences biochimiques, histologiques et fonctionnelles, dans différents modèles de TC et de SEP in vivo chez la souris, et ex vivo sur des tranches organotypiques de cervelet. Les résultats obtenus sur le modèle de TC par percussion mécanique chez la souris ont montré pour la première fois le potentiel anti-inflammatoire de l’étazolate, associé à la restauration du taux de la sAPPα. De plus, l’étazolate s’est également opposé aux troubles fonctionnels post-TC tels que l’hyperactivité locomotrice et le déficit cognitif à court et à long terme. Par la suite, j’ai développé un nouveau modèle ex vivo de TC par percussion mécanique sur des tranches organotypiques de cervelet. Nous avons montré pour la première fois que l’étazolate était neuroprotecteur dans le tissu cérébelleux, et qu’il s’opposait à la démyélinisation post-traumatique. Par ailleurs, les effets bénéfiques de l’étazolate sur les gaines de myéline ont été reproduits dans un modèle ex vivo de démyélinisation induite par la lysolécithine, modèle ex vivo de SEP. De façon intéressante nous avons montré que l’étazolate exerçait un effet remyélinisant en stimulant la différenciation des oligodendrocytes. Cet effet a été reproduit in vitro dans des cultures primaires mixtes de cellules gliales issues de souris PLP-eGFP, où la maturation morphologique des oligodendrocytes a été favorisée en présence d’étazolate. L’ensemble des effets bénéfiques exercés par l’étazolate a été inhibé en présence d’un inhibiteur pharmacologique spécifique d’ADAM10, le GI254023X, suggérant que l’effet remyélinisant de l’étazolate dépend, au moins en partie, d’ADAM10. Par la suite, l’effet remyélinisant de l’étazolate a été étudié dans un modèle in vivo de démyélinisation chronique induite par la cuprizone. Dans ce modèle, l’étazolate a été capable de promouvoir la remyélinisation en stimulant la différenciation des oligodendrocytes, confirmant nos résultats in vitro et ex vivo. L’ensemble de mon travail permet de considérer le potentiel thérapeutique de l’étazolate, en visant l’ADAM10 comme nouvelle cible thérapeutique neuroprotectrice et remyélinisante. Cela aura pour intérêt de limiter la neuro-inflammation, la démyélinisation, ainsi que de promouvoir la différenciation des oligodendrocytes et la remyélinisation, afin de favoriser la récupération fonctionnelle suite aux lésions de la substance blanche survenant après un TC ou la SEP chez l’homme
Demyelination and oligodendrocyte cell death are well established in multiple sclerosis (MS) and are increasingly described after traumatic brain injury (TBI), participating in the aggravation of white matter injury responsible of motor and cognitive deficits. Despite many efforts in clinical research, no efficient therapy against white matter injury progression is available nowadays. Thus, promoting remyelination and counteracting neuroinflammation and demyelination are major therapeutic strategies in order to restore white matter integrity. Here, we studied the stimulation of endogenous repair mechanisms through the neuroprotective and neurotrophic protein sAPPα, the soluble form of βAPP protein released by the α-secretases (ADAM10). In this context, the aim of this work was to evaluate the therapeutic potential of etazolate, an α-secretase activator on short- and long-term biochemical, histological and functional outcome in different mouse models of TBI and MS in vivo, and ex vivo on organotypic cerebellar slices. The results obtained from the TBI mouse model by mechanical percussion showed for the first time the anti-inflammatory effect of etazolate associated to a restoration of sAPPα levels. The same treatment was able to attenuate functional deficits (hyperactivity, cognitive deficit). We also developed a new ex vivo model of TBI by mechanical percussion on organotypic cerebellar slices. We confirmed the neuroprotective effect of etazolate on cerebellar tissue reducing the lesion size. Interestingly, etazolate treatment attenuated post-traumatic ex vivo demyelination. Moreover, the beneficial effect of etazolate on myelin sheaths have been well reproduced after lysolecithin-induced demyelination, an ex vivo model of MS. Interestingly, etazolate was able to enhance remyelination promoting oligodendrocyte differentiation. This effect has been reproduced in the primary mixed glial culture from PLP-eGFP mice, enhancing oligodendrocyte morphological maturation. However, etazolate failed to promote its beneficial effects in the presence of GI254023X, a specific ADAM10 (α-secretase) inhibitor, suggesting that the mechanism of action of etazolate is partly through the activation of ADAM10. Furthermore, etazolate reproduced in vivo the oligodendrocyte differentiation, accompanied by an increase of the myelinated axons, observed by electron microscopy in a mouse model of cuprizone-induced chronic demyelination. Taken together, the findings of this work provide a first evidence for the therapeutic potential of etazolate, with ADAM10 as new therapeutic target in white matter repair. The interest of this approach is to attenuate neuroinflammation and demyelination and to enhance oligodendrocyte differentiation and thus remyelination, in order to promote functional recovery following white matter lesions arising after TBI or MS

Traumatic brain injury (TBI) may result in significant impairment in an individual’s physical, cognitive and psychosocial functioning, and is acknowledged to be the leading cause of long-term disability in young adults (DSM III; 1999). An increasing body of high-quality evidence now exists for the effectiveness of rehabilitation interventions for TBI of all severities (Powell, Heslin, & Greenwood, 2002; Turner-Stokes, 2008; Wade, Crawford, Wenden, King, & Moss, 1997). However, there is a need for a more comprehensive description of the types of care allocated at the end of the acute phase of TBI, and the factors that influence variation in referral and access to services. Much of the literature focuses on people with severe TBI receiving specialist rehabilitation, and considers only hospitalised cases. The aims of the present research were to investigate patterns of referral to outpatient rehabilitation services in a population-based sample, to describe factors related to progress in rehabilitation for those referred to public community rehabilitation, and to increase understanding of TBI and its management, by developing a model of rehabilitation pathways. Study 1 looked at some demographic, injury-related, and post-injury characteristics of the participants of the Tasmanian Neurotrauma Register (TNTR) research project (N = 1226), and examined differences in the groups referred to public and private rehabilitation. Studies 2, 3 and 4 looked at the sub-sample of individuals (n = 175) referred for public multidisciplinary rehabilitation at the Community Rehabilitation Unit (CRU). These three studies considered how a range of variables were related to referral to CRU’s clinical disciplines, to the likelihood of being offered appointments, and to attendance or non-attendance at initial appointments, when offered them. Study 5 looked further at how rehabilitation services contribute to TBI patients’ recovery by considering the amount and nature of therapy participants received at CRU. This research provides clinicians and researchers with a clearer picture of some of the factors that affect the post-acute rehabilitation process, in a sample of TBI patients that is more representative of adult TBI than those found in the overwhelming majority of studies, which typically consider only moderate to severe TBI and/or hospitalised cases. The rehabilitation pathways and processes outlined will be valuable for rehabilitation clinicians who wish to identify people at risk of poor outcomes. The findings of this research provide a foundation upon which a number of avenues for further research can be based. These include looking at different measures of outcome in TBI samples referred for community rehabilitation, identifying effective interventions that are compatible with existing rehabilitation services, and comparing outcomes in matched samples referred and not-referred for rehabilitation.

Intricate neural circuits underlie all brain functions. However, these neural circuits are highly dynamic. The ability to change, or the plasticity, of the brain has long been demonstrated at the level of isolated single synapses under artificial conditions. Circuit organization and brain function has been extensively studied by correlating neuronal activity with information input. The primary visual cortex has become an important model brain region for the study of sensory processing, in large part due to the ease of manipulating visual stimuli. Much has been learned from studies of visual cortex focused on understanding the signal-processing of visual inputs within neural circuits. Many of these findings are generalizable to other sensory systems and other regions of cortex. However, few studies have directly demonstrated the orchestrated neural-circuit plasticity occurring during behavioral experience.

It is vital to measure the precise circuit connectivity and to quantitatively characterize experience-dependent circuit plasticity to understand the processes of learning and memory formation. Moreover, it is important to study how circuit connectivity and plasticity in neurological and psychiatric disease states deviates from that in healthy brains. By understanding the impact of disease on circuit plasticity, it may be possible to develop therapeutic interventions to alleviate significant neurological and psychiatric morbidity. In the case of neural trauma or ischemic injury, where neurons and their connections are lost, functional recovery relies on neural-circuit repair. Evaluating whether neurons are reconnected into the local circuitry to re-establish the lost connectivity is crucial for guiding therapeutic development.

There are several major technical hurdles for studies aiming to quantify circuit connectivity. First, the lack of high-specificity circuit stimulation methods and second, the low throughput of the gold-standard patch-clamp technique for measuring synaptic events have limited progress in this area. To address these problems, we first engineered the patch-clamp experimental system to automate the patching process, increasing the throughput and consistency of patch-clamp electrophysiology while retaining compatibility of the system for experiments in ex vivo brain slices. We also took advantage of optogenetics, the technology that enables control of neural activity with light through ectopic expression of genetically encoded photo-sensitive channels in targeted neuronal populations. Combining optogenetic stimulation of pre-synaptic axonal terminals and whole-cell patch-clamp recording of post-synaptic currents, we mapped the distribution and strength of synaptic connections from a specific group of neurons onto a single cell. With the improved patch-clamp efficiency using our automated system, we efficiently mapped a significant number of neurons in different experimental conditions/treatments. This approach yielded large datasets, with sufficient power to make meaningful comparisons between groups.

Using this method, we first studied visual experience-dependent circuit plasticity in the primary visual cortex. We measured the connectivity of local feedback and recurrent neural projections in a Fragile X syndrome mouse model and their healthy counterparts, with or without a specific visual experience. We found that repeated visual experience led to increased excitatory drive onto inhibitory interneurons and intrinsically bursting neurons in healthy animals. Potentiation at these synapses was absent or abnormal in Fragile X animals. Furthermore, recurrent excitatory input onto regular spiking neurons within the same layer remained stable in healthy animals but was depressed in Fragile X animals following repeated visual experience. These results support the hypothesis that visual experience leads to selective circuit plasticity which may underlie the mechanism of visual learning. This circuit plasticity process is impaired in a mouse model of Fragile X syndrome.

In a separate study, in collaboration with the laboratory of Dr. Gong Chen, we applied the circuit-mapping method to measure the effect of a novel brain-repair therapy on functional circuit recovery following ischemic injury, which locally kills neurons and creates a glial scar. By directly reprogramming astrocytes into neurons within the region of the glial scar, this gene-therapy technology aims to restore the local circuit and thereby dramatically improve behavioral function after devastating neurological injury. We found that direct reprogramming converted astrocytes into neurons, and importantly, we found that these newly reprogrammed neurons integrated appropriately into the local circuit. The reprogramming also improved connections between surviving endogenous neurons at the injury site toward normal healthy levels of connectivity. Connections formed onto the newly reprogrammed neurons spontaneously remodeled, the process of which resembled neural development. By directly demonstrating functional connectivity of newly reprogrammed neurons, our results suggest that this direct reprogramming gene-therapy technology holds significant promise for future clinical application to restore circuit connectivity and neurological function following brain injury.