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Intracerebral hemorrhage (ICH) is a devastating form of stroke that results from rupture of a blood vessel in the brain. Tissue inside the hematoma is irreversibly damaged soon after ICH onset and when this thesis research began, there was a dearth of information regarding pathological changes outside the hematoma. Inflammation is often proposed as a mechanism of injury, but very little information was available to show that inflammatory cells were in the right place at the right time to cause secondary brain injury. Using the collagenase-induced model of ICH, this work sought to better define spatial and temporal relationships between secondary brain injury and the inflammatory response after ICH. To test the hypothesis that reducing inflammation can protect the brain from secondary injury, minocycline, an antibiotic with established anti-inflammatory effects, was administered 6 hours after ICH onset. A small number of neurons die in the parenchyma bordering the hematoma between 6 hours and 3 days after ICH onset. This area was not associated with neutrophil infiltration, and most activated microglia/macrophages did not accumulate until after most neuron death had occurred. Despite a pronounced microglial response and prolonged increase in expression of many inflammatory genes, including complement receptor-3, interleukin-1 beta, interleukin-6, and interleukin-1 converting enzyme, no dying neurons were observed further outside the hematoma at any time. Interestingly, less early neuron death was observed in aged than in young animals, without a concomitant difference in the amount of tissue lost at 28 days. However, aged animals had less early microglial activation and a larger residual lesion, which might have resulted from impaired phagocytosis by activated microglia/macrophages. Minocycline was less effective in reducing microglial activation in aged animals, and did not reduce neuron death in either young or aged animals. Edema and BBB disruption was associated with degradation of the basal lamina protein, collagen type IV, and that damaged vessels are associated with tumor necrosis factor-alpha (TNFα)-positive neutrophils and active matrix metalloprotease-12 (MMP-12), all of which were reduced by delayed minocycline treatment. In contrast to ischemic stroke, there is a limited ‘penumbra’ outside the hematoma. Nevertheless, BBB damage in this region appears to be a potential target for protection. Furthermore, the prominent inflammatory response that continues for days after ICH does not appear to be associated with damage to other areas of the brain. Minocycline appears to protect the BBB by reducing neutrophil infiltration and the MMP-12 mediated basal lamina degradation. Future studies should investigate other targets for protection (i.e., white matter injury), and seek drugs that modulate the inflammatory response in aged animals and promote lesion resolution.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Glutamate is the primary excitatory neurotransmitter in the central nervous system. Extracellular glutamate concentrations are tightly regulated to avoid over-stimulation of glutamate receptors, which leads to a cascade of deleterious processes collectively known as excitotoxicity. Excitotoxicity is common to several neurodegenerative disorders and CNS injuries, including stroke and Alzheimer’s disease (AD). The projects described in this thesis were designed to uncover novel protective pathways in excitotoxic neurodegeneration. Excessive activation of the DNA repair enzyme, poly(ADP-ribose) polymerase-1 (PARP-1), is a convergence point for neuron death signaling in excitotoxic pathways. In AD, the peptide amyloid-β1-42 (Aβ1-42) is aberrantly produced, leading to excitotoxic neuron death in vitro. To investigate links between Aβ1-42 and PARP, we treated cultured cortical neurons with Aβ1-42 and determined whether PARP-1 contributes to neuron death. Increased neuron death was observed after Aβ1-42 exposure. A non-selective PARP-1/2 inhibitor significantly reduced Aβ1-42-induced death while elimination of PARP-1 alone was not neuroprotective. This suggests that PARP-2 or combined effects of PARP-1 and PARP-2 are required for Aβ1-42-induced neuron death. A hallmark of PARP over-activation is depletion of intracellular NAD+ and ATP levels, yet nearly all studies examining adenine nucleotide levels use separate biochemical samples to measure nucleotides individually. We developed two HPLC methods for simultaneous separation of NAD+, ATP, ADP and AMP. We determined that PARP-1 activation in astrocytes leads to near complete NAD+ depletion, followed by partial loss of ATP pools and total adenine nucleotide pools. Finally, we hypothesized that conjugated linoleic acid (CLA), a naturally occurring polyunsaturated fatty acid, is capable of enhancing neuron survival after an excitotoxic insult. Cultured cortical neurons were exposed to glutamate in the presence and absence of CLA. CLA levels likely achievable in human plasma and brain tissue during dietary supplementation regimens, protected neurons against glutamate excitotoxicity when given during or up to five hours after glutamate exposure. Several markers of mitochondrial damage and intrinsic apoptosis were examined. CLA stabilized mitochondrial membrane potential and permeability, shedding light on the mechanism of CLA neuroprotection. Overall, our research suggests a role for PARP in Aβ1-42 toxicity and identifies a novel role for CLA in neuroprotection following excitotoxicity.

Die Forschungsergebnisse der letzten Jahre beweisen, dass die Informationsverarbeitung im ZNS auf eine ausgewogene Interaktion zwischen den Neuronen und den Astrozyten im Sinne eines funktionellen Netzwerkes angewiesen ist. Allerdings liegen nur unzureichende Erkenntnisse über den strukturellen Charakter dieser symbiotischen Beziehung vor. Zu den grundlegenden Aufgaben der Astrozyten gehört die Modulation der synaptischen Aktivität von Neuronen, Aufrechterhaltung der extrazellulären Homoöstase, Ausbildung der Blut-Hirn-Schranke, Pufferung der lokalen Kaliumkonzentration und die Synthese von Zytokinen, Wachstumsfaktoren sowie Neurotransmittern. Ein Hauptbestandteil des Zytoskelettes der Astrozyten stellen die Intermediärfilamente (z.B. GFAP, Vimentin) dar. Funktionell dienen sie dem Aufbau von Zell-Zell- Kontakten, der Stabilisierung des Zytoskelettes und der Verankerung der Zellorganellen im Zytoplasma. Charakteristisch für den strukturellen Aufbau der Astrozyten ist das GFAP, welches hauptsächlich in den Zellfortsätzen lokalisiert ist. Aktuelle Forschungen legen nahe, dass es sowohl unter physiologischen Alterungsprozessen als auch im Rahmen von pathologischen Vorgängen im ZNS (beispielsweise chronischer Alkoholabusus, Alexanderkrankheit, Ischämien und Epilepsie) zu einem prozentualen Anstieg der GFAP-Expression kommt, wobei der Einfluss dieser erhöhten GFAP-Synthese auf die Funktionsfähigkeit der Astrozyten noch nicht umfassend geklärt werden konnte. Im Zentrum dieser Dissertation steht deshalb die Fragestellung, haben Alter und Intermediärfilamente (GFAP, Vimentin) Einfluss auf Morphologie und Zellzahl der Astrozyten im Hippokampus einer Maus? Initial erfolgte die Volumenbestimmung der Astrozytendomänen mittels LSM- Aufnahmen im Reflexionsmodus sowie Vermessung nach Bearbeitung der Präparate mit der Silber Imprägnationstechnik. Die Domäne eines Astrozyten stellt das von einem Astrozyten mit Soma und allen Fortsätzen okkupierte Volumen dar. Nach der Auswahl von S100ß als immunhistochemischen Astrozytenmarker wurde die Zellzählung in entsprechenden Hippokampusarealen durchgeführt. Aus den ermittelten Daten wurde rechnerisch der Überlappungsgrad der hippokampalen Astrozytendomänen bestimmt. Von entscheidendem Interesse war dabei die Möglichkeit einer Überschneidung benachbarter Astrozytendomänen. Aus den Messungen resultiert ein ca 1,6 fach grösseres Volumen der Wildtyp-Tiere im Vergleich zu den Doppel-knock-out-Mäusen. Im Gegensatz dazu zeigten die Ergebnisse der Zellzählung sowohl in der Kontrollgruppe als auch in der Gruppe der Versuchstiere eine vergleichbare Astrozytenanzahl pro mm3. Entsprechend lag der Überlappungsfaktor bei den Doppel-Knock-out-Tieren (0,49) unter dem der Wildtyp-Tiere (0,7). Die abschließende Auswertung erbrachte in allen Untersuchungsgruppen einen Überlappungsfaktor < 1. Ausgehend von der essentiellen Bedeutung der Astrozyten für die Funktionsfähigkeit der Nervenzellen käme es z.B. im Rahmen pathologischer Prozesse, welche mit Gliazellschäden einhergehen, bei einem Überlappungsfaktor < 1 zu erheblichen Engpässen in der neuronalen Versorgung, sowie in der Kompensation äußerer Einflüsse. Die Wahrscheinlichkeit irreversibler Schädigung der Nervenzellen steigt.

Glutamate is the primary excitatory neurotransmitter in the central nervous system. Extracellular glutamate concentrations are tightly regulated to avoid over-stimulation of glutamate receptors, which leads to a cascade of deleterious processes collectively known as excitotoxicity. Excitotoxicity is common to several neurodegenerative disorders and CNS injuries, including stroke and Alzheimer’s disease (AD). The projects described in this thesis were designed to uncover novel protective pathways in excitotoxic neurodegeneration. Excessive activation of the DNA repair enzyme, poly(ADP-ribose) polymerase-1 (PARP-1), is a convergence point for neuron death signaling in excitotoxic pathways. In AD, the peptide amyloid-β1-42 (Aβ1-42) is aberrantly produced, leading to excitotoxic neuron death in vitro. To investigate links between Aβ1-42 and PARP, we treated cultured cortical neurons with Aβ1-42 and determined whether PARP-1 contributes to neuron death. Increased neuron death was observed after Aβ1-42 exposure. A non-selective PARP-1/2 inhibitor significantly reduced Aβ1-42-induced death while elimination of PARP-1 alone was not neuroprotective. This suggests that PARP-2 or combined effects of PARP-1 and PARP-2 are required for Aβ1-42-induced neuron death. A hallmark of PARP over-activation is depletion of intracellular NAD+ and ATP levels, yet nearly all studies examining adenine nucleotide levels use separate biochemical samples to measure nucleotides individually. We developed two HPLC methods for simultaneous separation of NAD+, ATP, ADP and AMP. We determined that PARP-1 activation in astrocytes leads to near complete NAD+ depletion, followed by partial loss of ATP pools and total adenine nucleotide pools. Finally, we hypothesized that conjugated linoleic acid (CLA), a naturally occurring polyunsaturated fatty acid, is capable of enhancing neuron survival after an excitotoxic insult. Cultured cortical neurons were exposed to glutamate in the presence and absence of CLA. CLA levels likely achievable in human plasma and brain tissue during dietary supplementation regimens, protected neurons against glutamate excitotoxicity when given during or up to five hours after glutamate exposure. Several markers of mitochondrial damage and intrinsic apoptosis were examined. CLA stabilized mitochondrial membrane potential and permeability, shedding light on the mechanism of CLA neuroprotection. Overall, our research suggests a role for PARP in Aβ1-42 toxicity and identifies a novel role for CLA in neuroprotection following excitotoxicity.

Intracerebral hemorrhage (ICH) is a devastating form of stroke that results from rupture of a blood vessel in the brain. Tissue inside the hematoma is irreversibly damaged soon after ICH onset and when this thesis research began, there was a dearth of information regarding pathological changes outside the hematoma. Inflammation is often proposed as a mechanism of injury, but very little information was available to show that inflammatory cells were in the right place at the right time to cause secondary brain injury. Using the collagenase-induced model of ICH, this work sought to better define spatial and temporal relationships between secondary brain injury and the inflammatory response after ICH. To test the hypothesis that reducing inflammation can protect the brain from secondary injury, minocycline, an antibiotic with established anti-inflammatory effects, was administered 6 hours after ICH onset. A small number of neurons die in the parenchyma bordering the hematoma between 6 hours and 3 days after ICH onset. This area was not associated with neutrophil infiltration, and most activated microglia/macrophages did not accumulate until after most neuron death had occurred. Despite a pronounced microglial response and prolonged increase in expression of many inflammatory genes, including complement receptor-3, interleukin-1 beta, interleukin-6, and interleukin-1 converting enzyme, no dying neurons were observed further outside the hematoma at any time. Interestingly, less early neuron death was observed in aged than in young animals, without a concomitant difference in the amount of tissue lost at 28 days. However, aged animals had less early microglial activation and a larger residual lesion, which might have resulted from impaired phagocytosis by activated microglia/macrophages. Minocycline was less effective in reducing microglial activation in aged animals, and did not reduce neuron death in either young or aged animals. Edema and BBB disruption was associated with degradation of the basal lamina protein, collagen type IV, and that damaged vessels are associated with tumor necrosis factor-alpha (TNFα)-positive neutrophils and active matrix metalloprotease-12 (MMP-12), all of which were reduced by delayed minocycline treatment. In contrast to ischemic stroke, there is a limited ‘penumbra’ outside the hematoma. Nevertheless, BBB damage in this region appears to be a potential target for protection. Furthermore, the prominent inflammatory response that continues for days after ICH does not appear to be associated with damage to other areas of the brain. Minocycline appears to protect the BBB by reducing neutrophil infiltration and the MMP-12 mediated basal lamina degradation. Future studies should investigate other targets for protection (i.e., white matter injury), and seek drugs that modulate the inflammatory response in aged animals and promote lesion resolution.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thèse (M.Sc.)--Université Laval, 2008. / Bibliogr.: f. 60-63. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 116-138).