Aside from preventing traumatic brain injuries (TBIs) altogether, treatment options for TBI typically focus on the secondary biochemical processes that occur in response to the primary mechanical insult. These secondary injuries can lead to apoptosis and necrosis in the days and weeks that follow a TBI. Therefore, finding a treatment that can prevent, reduce, and repair secondary damage is instrumental in the recovery of TBI patients. The flavonoid 7,8-dihydroxyflavone (7,8-DHF) has been identified as a TrkB agonist that mimics the effects of brain derived neurotrophin factor (BDNF). Upon binding to the TrkB receptor, signaling cascades are initiated that can promote neuronal survival and neural differentiation. The use of 7,8-DHF in the treatment of TBI is favorable due to its long half-life and ability to pass the blood-brain barrier (BBB). In this study, we evaluated the dosage time frame of 7,8-DHF that would allow for the greatest impact in recovery after a focal TBI. Adult Sprague-Dawley rats were subjected to a moderate cortical impact injury and administered a 5mg/kg dose of 7,8-DHF i.p. for five days starting on day 0, 2, 3, or 5 post injury. Sensorimotor function was evaluated with beam walk and rotarod test. Morris Water Maze (MWM) and fear conditioning test were used to analyze cognitive function. Biotinylated dextran amine (BDA) was injected into the contralateral cerebral cortex 14 days after injury and animals were sacrificed 28 dpi. Brain sections were processed for Giemsa histological staining to assess cortical lesion volume and the total number of surviving neurons. Parallel sections were processed for BDA staining to assess changes of axon sprouting in the injured cortex. VGlut-1 staining of the hippocampus was used to identify presynaptic plasticity. We found that the administration of 7,8- DHF starting at one hour after TBI could provide protection against motor and cognitive dysfunction. Histological examination showed a significant reduction of cortical lesion volume and higher number of survival neurons in the injured hippocampus when 7,8-DHF administration began one hour and two days after injury. BDA staining of intracortical axon sprouting and VGlut-1 staining of the hippocampus highlighted a trend that 7,8-DHF administration starting day five post brain injury may enhance neuronal plasticity. Collectively, the results indicate that 7,8-DHF can provide the better neuronal protection when administration begins one hour after TBI.

Following traumatic brain injury (TBI), the hippocampus is particularly vulnerable to damage, and BDNF, an endogenous neurotrophin that activates the TrkB receptor, has been shown to play a key role in the brain’s neuroprotective response. Activation of the TrkB signaling pathway by BDNF in the CNS promotes cell survival and aids in cell growth. However, due to its inability to cross the blood brain barrier (BBB), the therapeutic advantages of BDNF treatment following TBI are limited. 7,8-Dihydroxyflavone (7,8-DHF) is a flavonoid that mimics the effects of BDNF, is a potent TrkB receptor agonist, and can successfully cross the BBB. Our lab has previously demonstrated that administration of 7,8-DHF post-TBI results in improved cognitive functional recovery, increased neuronal survival, and reduced lesion volume. The current study examined the effects of 7,8-DHF on neurogenesis and neuronal migration in the dentate gyrus following TBI. In this study, adult male Sprague-Dawley rats were subjected to moderate controlled cortical impact injury (CCI) or sham surgery. Injured animals received 5 daily single doses of 7,8-DHF treatment (i.p) or vehicle starting either 60 mins after injury or 2 days after injury. BrdU was administered in 3 doses at 2 days post-injury for animals sacrificed at day 15, and single daily doses at days 1-7 post-injury for animals sacrificed at day 28 to label cell proliferation. Animals were sacrificed at 15 days or 28 days post-injury to examine cell proliferation, generation of new neurons, and differentiation of newly generated cells using proliferation marker Ki67, immature neuronal marker DCX, and BrdU double-labeling with markers for mature neurons (NeuN), astrocytes (GFAP) and microglia (Iba1). We found that administration of 5 doses (5mg/kg) of 7,8-DHF beginning two days post-injury had the strongest effect on neurogenesis and migration, but did not have a significant prolonged effect on cell proliferation at 15 days post-injury. We also found that 7,8-DHF treatment given early or 2 days post-TBI did not affect the neuronal differentiation in the granule cell layer. However, a higher percentage of BrdU/GFAP+ and BrdU/IBa1+ cells were found in the hilus regions in 7,8-DHF treated animals, suggesting newly generated cells in this region are mostly glial cell types. Our results suggest that 7,8-DHF has neurotrophic-like therapeutic effects following injury, and due to increased neurogenesis (compared to injured animals treated with vehicle), may effectively contribute to greater cell survival long-term. Additionally, potential long-term survival coupled with increased outward migration from the subgranular zone may result in increased integration of newly formed neurons into existing hippocampal circuitry, further contributing to cognitive recovery.

Ce mémoire présente mes travaux ayant menés au développement d’une première génération de radioligands marqués au fluor-18 (t1/2 = 110 min) et au carbone-11 (t1/2 = 20.4 min) destinés à l’imagerie cérébrale in vivo du récepteur tyrosine kinase neurotrophique de type 2 (TrkB) en tomographie par émission de positons (TEP). Ces travaux reposent sur l’identification récente de ligands de TrkB non peptidiques à hautes affinités dérivés du 7,8-dihydroxyflavone. La synthèse d’une série de dérivés du 7,8-dihydroxyflavone non-radioactifs de même que des précuseurs à l’incorporation du fluro-18 et du carbone-11 a d’abord été effectuée. Partant des précurseurs adéquats synthétisés, la radiosynthèse de deux radioligands, l’un marqué au fluor-18 et l’autre au carbone-11, a été développée. Ces radiosynthèses reposent respectivement sur une 18F-radiofluorination nucléophile aromatique nouvelle et hautement efficace et sur une 11C-méthylation N-sélective. Les radiotraceurs de TrkB ainsi obtenus ont ensuite été évalués in vitro en autoradiographie et in vivo en tant que traceurs TEP dans des rats. L’évaluation des propriétés physico-chimique de même que de la stabilité in vitro des radiotraceurs sont présentées. Partant d’une série d’analogues cristallisés de ces flavones synthétiques, une étude de relation structure-activité a été menée. La combinaison de cette étude, de pair avec l’évaluation in vivo de la première génération de radiotraceurs de TrkB a aussi permis d’investiguer les pharmacophores nécessaires à l’affinité de ces ligands de même que d’identifier des fragments structurels associés au métabolisme des radiotraceurs. La radiosynthèse d’un troisième radioligand de TrkB et son évaluation TEP in vivo de même que la mise en lumière des modifications structurelles utiles au développement d’une seconde génération de radioligands de TrkB avec des propriétés optimisées pour fin d’imagerie TEP sont aussi détaillés. / This thesis describes my contribution leading to the development of the first-generation positron emission tomography (PET) radioligands labeled with fluorine-18 (t1/2 = 110 min) or carbon-11 (t1/2 = 20.4 min) for the in vivo brain imaging of tropomyosin-related kinase B (TrkB). This research follows from the recent discovery of non-peptidic, high-affinity TrkB ligands derived from 7,8-dihydroxyflavone. The synthesis of non-radioactive 7,8-dihydroxyflavone derivatives and radiolabeling precursors amenable to fluorine-18 and carbon-11 incorporation was performed. Two synthesized compounds have been brought forward as precursors for radiolabeling with either fluorine-18 or carbon-11. Radiosynthesis involved either a novel nucleophilic aromatic subsitution with [18F]fluoride, or N-methylation with [11C]methyl iodide or [11C] methyl triflate. The resulting radiotracers were assessed in vitro by autoradiography and in vivo by PET scans of rats. The physicochemical properties and serum stability of these tracers were also evaluated. X-ray crystal structures of a series of synthetic flavone analogues were used as basis for structure-activity relationship (SAR) analysis. In combination with the above in vivo PET evaluation of these compounds, certain pharmacophores were shown essential for ligand binding affinity. In addition, some structural fragments were associated with in vivo ligand metabolism. The development and radiosynthesis of a third TrkB radiotracer, along with its in vivo PET evaluation and structural analysis, is also described here. In all, better understanding of these tracers have led to the design of potential second-generation TrkB ligands with more optimal properties as PET radiotracers.

Ce mémoire présente mes travaux ayant menés au développement d’une première génération de radioligands marqués au fluor-18 (t1/2 = 110 min) et au carbone-11 (t1/2 = 20.4 min) destinés à l’imagerie cérébrale in vivo du récepteur tyrosine kinase neurotrophique de type 2 (TrkB) en tomographie par émission de positons (TEP). Ces travaux reposent sur l’identification récente de ligands de TrkB non peptidiques à hautes affinités dérivés du 7,8-dihydroxyflavone. La synthèse d’une série de dérivés du 7,8-dihydroxyflavone non-radioactifs de même que des précuseurs à l’incorporation du fluro-18 et du carbone-11 a d’abord été effectuée. Partant des précurseurs adéquats synthétisés, la radiosynthèse de deux radioligands, l’un marqué au fluor-18 et l’autre au carbone-11, a été développée. Ces radiosynthèses reposent respectivement sur une 18F-radiofluorination nucléophile aromatique nouvelle et hautement efficace et sur une 11C-méthylation N-sélective. Les radiotraceurs de TrkB ainsi obtenus ont ensuite été évalués in vitro en autoradiographie et in vivo en tant que traceurs TEP dans des rats. L’évaluation des propriétés physico-chimique de même que de la stabilité in vitro des radiotraceurs sont présentées. Partant d’une série d’analogues cristallisés de ces flavones synthétiques, une étude de relation structure-activité a été menée. La combinaison de cette étude, de pair avec l’évaluation in vivo de la première génération de radiotraceurs de TrkB a aussi permis d’investiguer les pharmacophores nécessaires à l’affinité de ces ligands de même que d’identifier des fragments structurels associés au métabolisme des radiotraceurs. La radiosynthèse d’un troisième radioligand de TrkB et son évaluation TEP in vivo de même que la mise en lumière des modifications structurelles utiles au développement d’une seconde génération de radioligands de TrkB avec des propriétés optimisées pour fin d’imagerie TEP sont aussi détaillés. / This thesis describes my contribution leading to the development of the first-generation positron emission tomography (PET) radioligands labeled with fluorine-18 (t1/2 = 110 min) or carbon-11 (t1/2 = 20.4 min) for the in vivo brain imaging of tropomyosin-related kinase B (TrkB). This research follows from the recent discovery of non-peptidic, high-affinity TrkB ligands derived from 7,8-dihydroxyflavone. The synthesis of non-radioactive 7,8-dihydroxyflavone derivatives and radiolabeling precursors amenable to fluorine-18 and carbon-11 incorporation was performed. Two synthesized compounds have been brought forward as precursors for radiolabeling with either fluorine-18 or carbon-11. Radiosynthesis involved either a novel nucleophilic aromatic subsitution with [18F]fluoride, or N-methylation with [11C]methyl iodide or [11C] methyl triflate. The resulting radiotracers were assessed in vitro by autoradiography and in vivo by PET scans of rats. The physicochemical properties and serum stability of these tracers were also evaluated. X-ray crystal structures of a series of synthetic flavone analogues were used as basis for structure-activity relationship (SAR) analysis. In combination with the above in vivo PET evaluation of these compounds, certain pharmacophores were shown essential for ligand binding affinity. In addition, some structural fragments were associated with in vivo ligand metabolism. The development and radiosynthesis of a third TrkB radiotracer, along with its in vivo PET evaluation and structural analysis, is also described here. In all, better understanding of these tracers have led to the design of potential second-generation TrkB ligands with more optimal properties as PET radiotracers.