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.

Chronic intermittent ethanol exposure (CIE) is associated with degeneration of hippocampal neurons. The present study used hippocampal cultures to examine the loss of NeuN immunoreactivity, a relaible marker or neuronal density, after 1, 2, or 3 cycles of 5 days EtOH exposure (50 mM), followed by a 24-hour period of EWD or continuous EtOH exposure. NeuN immunoreactivity was decreased by 13%, 19%, and 16% in the CA1, CA3, and dentate gyrus after 3 cycles of CIE respectively; thionine staining confirmed significant cellular losses within each hippocampal subregion. Two cycles of CIE in aged tissue cultures resulted in significant decreases in NeuN immunoreactivity in all hippocampal subregions; however continuous ethanol exposure or exposure to one cycle of CIE did not. Further, exposure to the N-Methyl-D-aspartate receptor (NMDAR) antagonist 2-amino-7-phosphonvaleric acid (APV) (30 uM) during periods of EWD attenuated the loss of NeuN in all hippocampal subregions, while exposure to APV (40 uM) prevented the loss of NeuN in the CA1 and dentate gyrus. These results suggest that the loss of mature neurons after CIE is associated with the overactivation on the NMDAR.

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.

Κύριος στόχος της εργασίας ήταν η συγκριτική μελέτη της παλίνδρομης αναστολής μεταξύ του ραχιαίου και του κοιλιακού πόλου του ιππόκαμπου αρουραίου. Χρησιμοποιήθηκε η μεθοδολογία της in vitro διατήρησης τομών ιππόκαμπου και εξωκυττάριων καταγραφών προκλητών δυναμικών πεδίου. Τα αποτελέσματά μας έδειξαν ότι η GABAA εξαρτώμενη παλίνδρομη αναστολή είναι ασθενέστερη, έχει μικρότερη διάρκεια και φθίνει πιο γρήγορα στον κοιλιακό σε σχέση με το ραχιαίο ιππόκαμπο. Χρησιμοποιώντας διάφορα φάρμακα που δρουν ενισχυτικά στον GABAA υποδοχέα δείξαμε ότι υπάρχει λειτουργική διαφοροποίηση του GABAA εξαρτώμενου ανασταλτικού μηχανισμού μεταξύ των δύο πόλων του ιππόκαμπου, ενισχύοντας την υπόθεση της λειτουργικής διαφοροποίησης στο επίπεδο του υποδοχέα μεταξύ των δύο πόλων. Στην in vivo μελέτη, χρησιμοποιώντας το μοντέλο επαγωγής επιληπτικών κρίσεων με χορήγηση πεντυλενοτετραζόλης, δείξαμε ότι η ενίσχυση της GABAA εξαρτώμενης αναστολής απο τα κατασταλτικά φάρμακα συσχετίζεται με το μέγεθος της αντιεπιληπτικής τους δράσης. Επιπλέον, η βιταμίνη D δεν παρουσίασε αντιεπιληπτική δράση στους C57BL/6J μύες, ούτε ενίσχυσε την αναστολή, κάτι που δείχνει ότι δεν έχει επίδραση στον GABAA υποδοχέα ή, τουλάχιστον, στους υπότυπούς του στον ιππόκαμπο. / The major aim of this work was the comparative study of recurrent inhibition between the dorsal and ventral pole of the rat hippocampus. We used the methodology of in vitro maintenance of hippocampal slices and recording of evoked field potentials. We showed that the GABAA mediated recurrent inhibition is weaker, lasts less and decays faster in ventral than in dorsal hippocampus. Using various drugs that act as positive allosteric modulators of the GABAA receptor, we showed that there is a functional differentiation of the GABAA inhibitory mechanism between the two hippocampal poles, strengthening the hypothesis of the functional differentiation at the level of the receptor between the two poles. In the in vivo study, using the pentylenetetrazole model for inducing epileptic seizures, we showed that the enhancement of the GABAA mediated recurrent inhibition correlates with the strength of antiepileptic action of the sedative drugs used. In addition vitamin D did not show antiepileptic action in C57BL/6J mice. Moreover it didn’t enhance recurrent inhibition, showing that it doesn’t have any action on the GABAA receptor or, at least, on its subtypes in hippocampus.

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 ?

Indiana University-Purdue University Indianapolis (IUPUI) / Acute and chronic activation of both ionotropic and metabotropic glutamate (glut) receptors is implicated in many neurodegenerative disorders including AD, dementia, epilepsy, stroke and neurotrauma. TRH and glut receptors (ionotropic & metabotropic) receptors are differentially coexpressed in granule and pyramidal neurons of the hippocampus. The author shows TRH to be protective when added to cultured pituitary adenoma (GH-3) cells and neuron-like pheochromocytoma (PC12) cells either prior to, during, or after glut-induced toxicity (Endo. Soc. Abs. 01), and also shows that the possible neuroprotective mechanism may involve heterologous downregulation of the metabotropic glut receptors, using superfused hippocampal slices and noting a reduction of Gαq/11 (SFN Abs. 02). He has also demonstrated that TRH protected against glut toxicity in fetal cortical cultures (Endo. Soc. Abs. 04). To extend these studies he used 14-day cultured rat fetal hippocampal neurons (Day E17) to determine if TRH is protective against toxicity induced by specific ionotropic and metabotropic glut agonists. Neuronal viability and integrity were assessed by trypan blue exclusion and LDH release after 18 hrs following 30 min exposure to glut agonists. Ten µM dihydroxyphenylglycine (DHPG, a Group 1 receptor agonist) + 30 µM N-methyl-D-aspartate (NMDA)-induced toxicity (42% vs contr. P<0.05); whereas, concurrent and continued treatment with 10 uM but not 1uM 3Me-HTRH resulted in less neuronal death and damage (86% vs contr P<0.05; 53% vs contr. P>0.05) respectively. DHPG treatment alone (10 µM) for 30 min. was non-toxic by both criteria (90% vs contr. P<0.05). The data suggest that TRH may be a selective modulator of glut-induced toxicity.

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.