In the United States, 2.8 million people suffer a traumatic brain injury (TBI) annually. Between 25%-50% of TBI injuries happen under alcohol intoxication. It is not understood how alcohol impacts patient outcomes via secondary injury pathways. Secondary injury pathways offer a window for therapeutic interventions, but there has been little success finding effective medications. Slice cultures offer a way to study secondary injury mechanisms in a controlled manner. The transection injury can model excitotoxicy seen following TBI. The current studies examined the effect of alcohol intoxication and withdrawal at the time of injury, and the effect of a calpain inhibitor (MDL-28170) on cell death following a transection injury. Intoxication had no effect on cell death compared to the TBI condition. In the ethanol withdrawal (EWD) study, EWD did not increase cell death following the TBI except at 72 hours. There was no effect of MDL on cell death. The severity of the model may have caused a ceiling effect. Additionally, imaging points may not have been sufficient for proper characterization. Future studies should use a different injury mechanism and other imaging times should be considered.

Exposure to ethanol (ETOH) during fetal development results in a range of cognitive/behavioral deficits. There are differences in sensitivity to the effects of ETOH that could be explained by other factors, such as hypoxia. Similar mechanisms of damage underlie both ETOH, more specifically ETOH withdrawal, and hypoxia. Based on this overlap, it was hypothesized that sub threshold levels of these insults may interact to produce increased damage in sensitive brain regions. This study used a rodent organotypic hippocampal slice culture model to investigate the interaction of hypoxia and ETOH withdrawal and to determine possible developmental differences in the sensitivity to these insults. The combination of ETOH and hypoxia produced greater damage in the CA1 and CA3 hippocampal regions, as measured by propidium iodide uptake. Differences in outcome were noted between on postnatal (PND) 2 and PND 8 tissue. ETOH alone caused damage as measured by the neuronal marker NeuN, suggesting the ETOH/hypoxia interaction involves different cell types and that caution should be taken when determining appropriate levels of exposure. This data could explain why some offspring appear more sensitive to ETOH and/or hypoxic challenges during early life.

La maladie d’Alzheimer (AD), pathologie neurodégénérative progressive, est caractérisée par des dépôts β-amyloïdes extracellulaires, des enchevêtrements neurofibrillaires intracellulaires de Tau et une dégénérescence neuronale. A travers les nombreux modèles transgéniques AD disponibles, les connaissances sur les peptides amyloïdes et la protéine Tau ne cessent de progresser. Mais contrairement aux cas génétiques, l’étiologie des cas sporadiques d’AD reste à ce jour idiopathique, rendant difficile d’établir une stratégie thérapeutique efficace. Au cours d’une étude sur l’implication des protéines kinases dans la pathogénèse d’AD, des collaborateurs ont fait une observation totalement inattendue, mais très intéressante: une molécule de faible poids moléculaire, serait capable d’induire une production spécifique d’Aβ1-42 sans altérer les niveaux d’Aβ1-40 dans un modèle de lignée cellulaire. Dans ce contexte, le projet de thèse portait sur l’utilisation de dérivé purique (PD1) pour développer des modèles AD induits chimiquement sur différents supports (culture primaire de neurones, culture organotypique d’hippocampe et souris) et en investiguer les mécanismes sous-jacents à l’augmentation des peptides A1-42 (issus du métabolisme de l’APP (Amyloid precursor protein)).La première partie du projet de thèse a permis de mettre en évidence dans un contexte in vitro (culture primaire de neurones et culture organotypique d’hippocampe) que PD1 à forte dose induisait une augmentation du ratio Aβ42/40 et de manière répétable. Fort de ces résultats, nous avons voulu étudier les mécanismes d’action de PD1 autour de deux hypothèses : interaction dans le métabolisme de l’APP et implication des cellules gliales. Contrairement à nos premières hypothèses, nous avons montré que PD1 aurait de potentiels effets anti-inflammatoires (i.e. IL-1β) in vitro et in vivo. La voie de signalisation de l’IL-1β étant de plus en plus incriminée dans la pathogenèse d’Alzheimer; nous nous sommes interrogés sur l’effet dual de PD1 : outil pharmacologique alzheimerigène ou candidat médicament pour le traitement d’AD? / Alzheimer’s disease (AD), a progressive neurodegenerative disorder, appears to be associated with an increase in a particular form of β-amyloid deposits, intracellular Tau tangles and neuronal degeneration. Through many available transgenic AD models, knowledge about amyloid peptides and Tau protein continues to increase. However, in contrast to the genetic cases of AD, the etiology of sporadic AD cases remains unknown, making the establishment of an effective therapeutic strategy difficult.During the course of a study on the role of protein kinase involved in AD, our collaborators made an unexpected but very interesting observation. They identified a low molecular weight compound able to induce production of Aβ1-42 while the level of the much less toxic form Aβ1-40 remained constant. This selective induction of Aβ1-42 versus Aβ1-40 was observed in a cell line model. Therefore, the overall goal of the project thesis was based on the use of purine derivative (PD1) to understand the molecular mechanisms underlying the selective production of Aβ1-42. This would allow us to establish cellular assays and a chemically-induced animal AD model relevant to studies on the treatment and prevention of AD.The first part of this project allowed us to demonstrate in vitro that PD1, at high dose, repeatedly induced an increase in Aβ42/40 ratio in primary neurons and in neuronal hippocampal slice culture (OHSCs). Based on these facts, we analyzed the amyloid profile by focusing on APP metabolism and on glial cell activity. In contrary to our hypothesis, we highlighted whether PD1 exhibits potential anti-inflammatory properties (i.e. IL-1β) both in vitro and in vivo. The IL-1β pathway is more and more linked in the AD pathogen which leads us to consider that PD1 could have a dual effect : alzheimerogenic pharmacological tool or potential drug candidate for the treatment of AD ?

In der Pathogenese der Temporallappenepilepsie wird kindlichen hippokampalen Schädigungen eine wesentliche Rolle zugeschrieben. Epileptische Krämpfe und perinatale Asphyxie sind zwei häufige Ursachen dieser Schädigungen. Anhaltende epileptiforme Aktivität im Niedrig-Mg2+-Modell als einer experimentellen Form epileptischer Krämpfe führt in organotypischen hippokampalen Schnittkulturen (OHSK) der Ratte, die als Ersatzsystem des kindlichen Hippokampus verwendet werden, zu Zellschäden. Während dieser Untersuchungen ergab sich der Verdacht auf eine zusätzlich schädigende Wirkung erhöhter Sauerstoffspannungen. In meiner ersten Versuchsreihe konnte ich nachweisen, dass erhöhte Sauerstoffspannungen (60 %, 95 %) verglichen mit 20%-Sauerstoffspannung zu reversiblen und irreversiblen Zellschäden in OHSK führen. Die Zellschäden wurden über Veränderungen reizinduzierter Feldpotentiale, d.h. Abnahme der Amplitude, Zunahme der Latenz und Zunahme des Doppelpulsindex, sowie über die Propidium Jodid (PJ)-Fluoreszenzintensität bestimmt. In der zweiten Versuchsreihe konnte gezeigt werden, dass erhöhte Sauerstoffspannungen auch nach einer Hypoxie im Sinne einer hyperoxischen Reoxygenierung verglichen mit normoxischer Reoxygenierung vermehrt Zellschäden in OHSK zur Folge haben. In der dritten Versuchsreihe konnte ich ausschließen, dass erhöhte Sauerstoffspannungen eine notwendige Bedingung für Zellschäden infolge anhaltender epileptiformer Aktivität sind. Um die zellschädigende Rolle von Spreading Depressions (SDs), die während epileptiformer Aktivität auftreten, zu bestimmen, wurde in der vierten Versuchsreihe eine Methode etabliert, SD-ähnliche Ereignisse isoliert und zuverlässig in normoxischen OHSK auszulösen. Auf diese Weise wiederholt ausgelöste SD-ähnliche Ereignisse führten zu Zellschäden, bestimmt über die Veränderung elektrophysiologischer Eigenschaften von SD-ähnlichen Ereignissen, Abnahme der Feldpotentialamplitude und PJ-Fluoreszenzintensität. / Hippocampal damage during infancy is thought to play an important role in the pathogenesis of temporal lobe epilepsy. Epileptic seizures and perinatal asphyxia are two frequent causes of these damages. Sustained epileptiform activity induced in the low Mg2+-model of epileptic seizures leads to cell damage in organotypic hippocampal slice cultures (OHSC) of the rat, which are used as a surrogate for the infantile hippocampus. During a previous study utilising this model the suspicion arose that increased oxygen tension could have an additional damaging effect. My first series of experiments proved that increased oxygen tension (60 %, 95 %) lead to reversible and irreversible cell damage in OHSC compared to 20%-oxygen tension. Cell damage was determined by alterations of evoked field potentials, i.e. decrement of amplitude, increment of latency and paired pulse index, as well as by propidium iodide fluorescence. The second series of experiments showed that increased oxygen tension applied after an hypoxic period (hyperoxic reoxygenation) result in augmented cell damage compared to normoxic reoxygenation. With the third series of experiments it could be excluded that increased oxygen tension is an essential condition for the occurrence of cell damage due to sustained epileptiform activity. In order to elucidate the damaging role of spreading depressions (SD), which emerge during epileptiform activity, a method was established in the fourth series of experiments that allowed the reliable induction of SD-like events in normoxic OHSC. Repetitive SD-like events induced by this method led to cell damage, assessed by alterations of electrophysiological characteristics of SD-like events, decrement of evoked field potential amplitude and propidium iodide fluorescence.