Traumatic brain injury (TBI) is a devastating disease causing disability and death, and currently there are no effective treatments available. Therefore, there is an utmost need to improve our understanding of the pathophysiology of TBI and to identify potential therapies that can provide neuroprotection after injury. The aims of this thesis were to develop an in vivo and in vitro model of TBI, in which to assess the potential neuroprotective effects of an omega-3 polyunsaturated fatty acid (PUFAs), docosahexaenoic acid (DHA). Method The controlled cortical impact (CCI) in vivo model of TBI was optimized and performed in mice. Both a behavioural (Morris water maze (MWM) for cognitive deficits) and histological endpoints (astrogliosis, lesion size and activated microglia) were used to assess severity and neuroprotective effects of DHA. An in vitro model of mechanical TBI was also set up and optimized. This model employed 3D astrocyte cultures obtained from GFP positive rat pups. The CCI impactor from the in vivo studies was used to damage the cultures, and at 24 hours, 5 days and 10 days the astrogliosis and cell number was measured. Results The optimization of the in vivo studies demonstrated that at impaction depth of 2.2 mm produced an injury that was significantly different to the sham injury, in MWM performance and increased astrogliosis. Interestingly, there was an increase in the amount of astrogliosis on the contralateral side of the brain. A second study performed using the 2.2 mm injury parameters was performed, where an injection of DHA was administered via the tail vein 30 min after injury. The DHA-treated group did not demonstrate any neuroprotection compared to the injury-only group. However, there was an increase in the amount of astrogliosis in the contralateral hippocampus of the DHA-treat group. In the fat-1 studies it was shown that older male mice performed worse in the MWM, that the fat-1 gene did not confer neuroprotection but did lead to increased astrogliosis. The in vitro study revealed that astrocytes in the lesioned gels demonstrated an increase in astrogliosis, there was also an increase in the number of cell in the cultures following the lesion. Conclusion In conclusion, the in vivo model of CCI replicated components of the human TBI including a behavioural deficit and pathophysiological changes. Omega-3 PUFAs failed to demonstrate functional neuroprotection in this model, but histologically, promoted an increase in reactive astrogliosis. The development of a novel in vitro model of focal injury in a 3D culture system, that elicits reactive astrogliosis, could be used to support further studies of the astrocytic responses to mechanical injury.

No description available.

Chondroitin sulphate proteoglycans (CSPGs) from the glial scar inhibit axonal regeneration following spinal cord injury. CSPG expression can be induced by transforming growth factor β (TGFβ), which suggests that inhibition of TGFβ may reduce CSPG levels. Astrocytes were treated with cyclic AMP (cAMP), which reduced TGFβ signaling protein Smad2 in astrocytes. However, cAMP-treated astrocytes showed strong neurocan expression following TGFβ treatment, which suggests that TGFβ may mediate CSPG expression through non-Smad pathways. Smad2 or Smad4 were knocked down in astrocytes using siRNA and TGFβ-induced neurocan, brevican and aggrecan expression were still observed, indicating that Smad signaling is not required for CSPG expression. Administration of a PI3K/Akt inhibitor produced significant reductions in neurocan, brevican and aggrecan expression in astrocytes, which suggests that PI3K/Akt pathway mediates CSPG expression. Erk1/2 inhibitor treatment did not reduce CSPG expression significantly. Targeting non-Smad signaling pathways may therefore be effective strategies to reduce CSPG expression following injury.

abstract: Specificity and affinity towards a given ligand/epitope limit target-specific delivery. Companies can spend between $500 million to $2 billion attempting to discover a new drug or therapy; a significant portion of this expense funds high-throughput screening to find the most successful target-specific compound available. A more recent addition to discovering highly specific targets is the application of phage display utilizing single chain variable fragment antibodies (scFv). The aim of this research was to employ phage display to identify pathologies related to traumatic brain injury (TBI), particularly astrogliosis. A unique biopanning method against viable astrocyte cultures activated with TGF-β achieved this aim. Four scFv clones of interest showed varying relative affinities toward astrocytes. One of those four showed the ability to identify reactive astroctyes over basal astrocytes through max signal readings, while another showed a statistical significance in max signal reading toward basal astrocytes. Future studies will include further affinity characterization assays. This work contributes to the development of targeting therapeutics and diagnostics for TBI. / Dissertation/Thesis / M.S. Bioengineering 2013

Mild traumatic brain injury (mTBI)/ concussion accounts for 70-90% of all reported TBI cases in the United States and can cause long-term neurological outcomes that negatively impact quality of life. Previous studies revealed that increased blood-brain barrier (BBB) leakage is correlated with poor neurological outcomes after mTBI, yet the biological mechanisms linking BBB damage to the onset of neurological deficits after mTBI are not well understood. Previously, we found that astrocytes lose expression of homeostatic proteins after mTBI, characterizing the changes in astrocytic protein expression as an "atypical astrocyte response." Yet, the upstream mechanisms that induce this atypical astrocyte response after mTBI have yet to be elucidated. In models of more severe TBI, exposure to blood-borne factors triggers astrogliosis via upregulation in markers, such as glial fibrillary acidic protein (GFAP), but how exposure to blood-borne factors affects astrocyte protein expression in the context of mTBI is not well understood. Therefore, we hypothesized that mTBI-induced BBB damage causes atypical astrocytes via exposure to blood-borne factors. To test this hypothesis, we use a mTBI mouse model, two-photon microscopy, an endothelial cell-specific genetic ablation model, and serum-free primary astrocyte cultures. Here, we found that mTBI causes BBB damage through the loss of proteins involved in maintaining the BBB's physical and metabolic barriers, and BBB damage is sustained long-term after injury. Also, we demonstrated that leakage of blood-borne factors is sufficient to trigger atypical astrocytes, and plasma exposure triggers a similar response in vitro. Overall, these findings suggest that mTBI induces long-term BBB damage, and exposure to blood-borne factors triggers the loss of key homeostatic astrocytic proteins involved in maintaining healthy neuronal function. / Doctor of Philosophy / Mild traumatic brain injury (mTBI)/ concussion makes up 70-90% of all TBI cases reported in the United States and is commonly observed after car crashes, sports-related tackles, and blast exposure during military combat. People who experience mTBI develop debilitating long-term neurological consequences, such as sleep disturbances, depression, and dementia. Clinical data suggests mTBI causes damage to the barrier between the brain and blood, known as the blood-brain barrier (BBB). This damage has been correlated to the onset of poor neurological deficits, yet how damage to this barrier is causally linked to long-term neurological consequences remains to be fully understood. In our lab, we found that mTBI causes loss of proteins important for maintaining a healthy environment in the brain in specialized cells called astrocytes. However, the biological events that trigger the loss of protein expression in astrocytes after mTBI have yet to be fully investigated. Thus, we hypothesized that mTBI causes loss of these proteins via leakage of blood-borne factors. To test this hypothesis, we used a mTBI mouse model, two-photon microscopy, genetic manipulation, and cell cultures. In our studies, we found that mTBI triggers BBB damage via loss of proteins that make up its protective properties. Also, we demonstrated that leakage of blood-borne factors is sufficient to cause loss of astrocyte-specific proteins both in brain and cell cultures. Altogether, we show that a single mTBI is sufficient to cause loss of astrocyte-specific protein expression via exposure to blood-borne factors. These findings may point to targeting either the blood-borne factor(s) or their corresponding receptor pathways in astrocytes to halt the progression of long-term neurological deficits after mTBI.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A diminuição do aporte de oxigênio e nutrientes na vida perinatal resulta em danos, como astrogliose, morte de neurônios e de células proliferativas. Déficits cognitivos podem estar relacionados a danos no hipocampo. Neste trabalho avaliamos a citoarquitetura do giro dentado (DG) durante o desenvolvimento e a memória de ratos submetidos à HI. Para tal, utilizamos a técnica de imunohistoquímica para marcador de proliferação celular (KI67), neurônio jovem (DCX), de astrócitos (GFAP) e de óxido nítrico sintase neuronal (NOSn). Para avaliar a memória de curta e de longa duração foi utilizado o teste de reconhecimento de objetos (RO). Ratas Wistar grávidas em E18 foram anestesiadas (tribromoetanol) e as quatro artérias uterinas foram ocluídas com grampos de aneurisma (Grupo HI). Após 45 minutos, os grampos foram removidos e foi feita a sutura por planos anatômicos. Os animais do grupo controle (SHAM) sofreram o mesmo procedimento, excetuando a oclusão das artérias. Os animais nasceram a termo. Animais com idades de 7 a 90 dias pós-natal (P7 a P90), foram anestesiados e perfundido-fixados com paraformaldeído a 4%, e os encéfalos submetidos ao processamento histológico. Cortes coronais do hipocampo (20m) foram submetidos à imunohistoquímica para KI67, DCX, GFAP e NOSn. Animais P90 foram submetidos ao RO. Os procedimentos foram aprovados pelo comitê de ética (CEA/019/2010). Observamos menor imunomarcação para KI67 no giro dentado de animais HI em P7. Para a marcação de DCX nesta idade não foi observada diferença entre os grupos. Animais HI em P15, P20 e P45 tiveram menor imunomarcação para DCX e Ki67 na camada granular. Animais P90 de ambos os grupos não apresentaram marcação para KI67 e DCX. Vimos aumento da imunomarcação para GFAP nos animais HI em todas as idades. A imunomarcação para NOSn nos animais HI foi menor em todas as idades. O maior número de células NOSn positivas foi visto em animais P7 em ambos os grupos na camada polimórfica. Em P15, animais HI apresentam células NOSn+ em todo o DG. Em P30 animais HI apresentam células NOSn+ nas camadas polimórfica e sub-granular. Animais adultos (P90) de ambos os grupos apresentam células NOSn positivas apenas nas camadas granular e sub-granular. Embora animais HI P90 não apresentaram déficits de memória, estes apresentaram menor tempo de exploração do objeto. Comportamento correspondente a déficits de atenção em humanos. Nossos resultados sugerem que HI perinatal diminui a população de células proliferativas, de neurônios jovens, de neurônios NOSn+, além de causar astrogliose e possivelmente déficits de atenção. O modelo demonstrou ser útil para a compreensão dos mecanismos celulares das lesões hipóxico-isquêmicas e pode ser usado para testar estratégias terapêuticas. / The supply of oxygen and nutrients decreasing in perinatal life may results in CNS damage such as deficits in memory and attention and increased susceptibility to epileptic disorders in adulthood. Perinatal hypoxia-ischemia ( HI ) results in astrogliosis in white matter and loss of cortical neurons (Robinson et al, 2005). Cognitive deficits may be related to hippocampal damage. In this study we evaluate the cytoarchitecture of the dentate gyrus (DG) during development and memory in rats submeted to HI. We used the immunohistochemistry marker of cell proliferation (Ki67), young neuron (DCX), astrocytes (GFAP) and neuronal nitric oxide synthase (nNOS). To evaluate the short-memory and long-lasting the recognition of objects (RO) test was used. Pregnant Wistar rats on E18 were anesthetized (tribromoethanol) and the four uterine arteries were occluded with aneurysm clips (Group HI). After 45 minutes, the clips were removed and the incision was sutured to the anatomical planes. The control group (SHAM) underwent the same procedure, except the occlusion of arteries. The animals were born at term. Animals aged 7 to 90 days postnatal (P7 to P90) were anesthetized and perfused-fixed with 4% paraformaldehyde, and their brains were subjected to histological processing. Coronal sections of the hippocampus (20μm) were subjected to immunohistochemistry for Ki67, DCX, GFAP and nNOS. Animals were subjected to RO P90. The procedures were approved by the ethics committee ( CEA/019/2010 ). We observed lower Ki67 immunostaining in the dentate gyrus of animals HI at P7. For marking DCX at this age is no difference between the groups was observed. HI animals at P15, P20 and P45 had less immunostaining for DCX and Ki67 in the granular layer. Animals P90 in both groups showed no labeling for Ki67 and DCX. We have seen an increase in GFAP immunostaining HI in animals at any age. The immunostaining for nNOS in HI animals was lower at all ages. The greater number of positive cells was seen in nNOS P7 animals in both groups in the polymorphic layer. In P15 animals HI nNOS + cells present in the whole DG. In P30 animals HI feature nNOS + cells in the polymorphic layer and sub-granular. Adult animals (P90) of both groups have positive nNOS granular cell layers, and only in the sub-granular. Although HI P90 animals showed no memory deficits, these patients had shorter holding the object. Corresponding to attention deficits in human behavior. Our results suggest that perinatal HI decreases the population of proliferative cells, young neurons, nNOS+ neurons, and astrocytic and possibly cause attention deficits. The model proved to be useful for understanding the cellular mechanisms of hypoxic- ischemic injury and can be used to test therapeutic strategies.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A diminuição do aporte de oxigênio e nutrientes na vida perinatal resulta em danos, como astrogliose, morte de neurônios e de células proliferativas. Déficits cognitivos podem estar relacionados a danos no hipocampo. Neste trabalho avaliamos a citoarquitetura do giro dentado (DG) durante o desenvolvimento e a memória de ratos submetidos à HI. Para tal, utilizamos a técnica de imunohistoquímica para marcador de proliferação celular (KI67), neurônio jovem (DCX), de astrócitos (GFAP) e de óxido nítrico sintase neuronal (NOSn). Para avaliar a memória de curta e de longa duração foi utilizado o teste de reconhecimento de objetos (RO). Ratas Wistar grávidas em E18 foram anestesiadas (tribromoetanol) e as quatro artérias uterinas foram ocluídas com grampos de aneurisma (Grupo HI). Após 45 minutos, os grampos foram removidos e foi feita a sutura por planos anatômicos. Os animais do grupo controle (SHAM) sofreram o mesmo procedimento, excetuando a oclusão das artérias. Os animais nasceram a termo. Animais com idades de 7 a 90 dias pós-natal (P7 a P90), foram anestesiados e perfundido-fixados com paraformaldeído a 4%, e os encéfalos submetidos ao processamento histológico. Cortes coronais do hipocampo (20m) foram submetidos à imunohistoquímica para KI67, DCX, GFAP e NOSn. Animais P90 foram submetidos ao RO. Os procedimentos foram aprovados pelo comitê de ética (CEA/019/2010). Observamos menor imunomarcação para KI67 no giro dentado de animais HI em P7. Para a marcação de DCX nesta idade não foi observada diferença entre os grupos. Animais HI em P15, P20 e P45 tiveram menor imunomarcação para DCX e Ki67 na camada granular. Animais P90 de ambos os grupos não apresentaram marcação para KI67 e DCX. Vimos aumento da imunomarcação para GFAP nos animais HI em todas as idades. A imunomarcação para NOSn nos animais HI foi menor em todas as idades. O maior número de células NOSn positivas foi visto em animais P7 em ambos os grupos na camada polimórfica. Em P15, animais HI apresentam células NOSn+ em todo o DG. Em P30 animais HI apresentam células NOSn+ nas camadas polimórfica e sub-granular. Animais adultos (P90) de ambos os grupos apresentam células NOSn positivas apenas nas camadas granular e sub-granular. Embora animais HI P90 não apresentaram déficits de memória, estes apresentaram menor tempo de exploração do objeto. Comportamento correspondente a déficits de atenção em humanos. Nossos resultados sugerem que HI perinatal diminui a população de células proliferativas, de neurônios jovens, de neurônios NOSn+, além de causar astrogliose e possivelmente déficits de atenção. O modelo demonstrou ser útil para a compreensão dos mecanismos celulares das lesões hipóxico-isquêmicas e pode ser usado para testar estratégias terapêuticas. / The supply of oxygen and nutrients decreasing in perinatal life may results in CNS damage such as deficits in memory and attention and increased susceptibility to epileptic disorders in adulthood. Perinatal hypoxia-ischemia ( HI ) results in astrogliosis in white matter and loss of cortical neurons (Robinson et al, 2005). Cognitive deficits may be related to hippocampal damage. In this study we evaluate the cytoarchitecture of the dentate gyrus (DG) during development and memory in rats submeted to HI. We used the immunohistochemistry marker of cell proliferation (Ki67), young neuron (DCX), astrocytes (GFAP) and neuronal nitric oxide synthase (nNOS). To evaluate the short-memory and long-lasting the recognition of objects (RO) test was used. Pregnant Wistar rats on E18 were anesthetized (tribromoethanol) and the four uterine arteries were occluded with aneurysm clips (Group HI). After 45 minutes, the clips were removed and the incision was sutured to the anatomical planes. The control group (SHAM) underwent the same procedure, except the occlusion of arteries. The animals were born at term. Animals aged 7 to 90 days postnatal (P7 to P90) were anesthetized and perfused-fixed with 4% paraformaldehyde, and their brains were subjected to histological processing. Coronal sections of the hippocampus (20μm) were subjected to immunohistochemistry for Ki67, DCX, GFAP and nNOS. Animals were subjected to RO P90. The procedures were approved by the ethics committee ( CEA/019/2010 ). We observed lower Ki67 immunostaining in the dentate gyrus of animals HI at P7. For marking DCX at this age is no difference between the groups was observed. HI animals at P15, P20 and P45 had less immunostaining for DCX and Ki67 in the granular layer. Animals P90 in both groups showed no labeling for Ki67 and DCX. We have seen an increase in GFAP immunostaining HI in animals at any age. The immunostaining for nNOS in HI animals was lower at all ages. The greater number of positive cells was seen in nNOS P7 animals in both groups in the polymorphic layer. In P15 animals HI nNOS + cells present in the whole DG. In P30 animals HI feature nNOS + cells in the polymorphic layer and sub-granular. Adult animals (P90) of both groups have positive nNOS granular cell layers, and only in the sub-granular. Although HI P90 animals showed no memory deficits, these patients had shorter holding the object. Corresponding to attention deficits in human behavior. Our results suggest that perinatal HI decreases the population of proliferative cells, young neurons, nNOS+ neurons, and astrocytic and possibly cause attention deficits. The model proved to be useful for understanding the cellular mechanisms of hypoxic- ischemic injury and can be used to test therapeutic strategies.

Orientadores: Fabio Rogério, Carlos Amilcar Parada / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T17:39:33Z (GMT). No. of bitstreams: 1 Ignarro_RaffaelaSilvestre_M.pdf: 3644274 bytes, checksum: 6e13f812b2d525e18878656d3ec27815 (MD5) Previous issue date: 2011 / Resumo: A injeção de ácido quinolínico (AQ), um agonista glutamatérgico do receptor N-metil-D-aspartato, no estriado de roedores induz morte seletiva de neurônios espinhosos médios, gliose reativa e neurogênese na zona subventricular, acompanhada da migração dos neurônios recém-gerados para o estriado lesado. Tais achados são também descritos na doença de Huntington (DH). Há indícios de que a via de sinalização JAK/STAT esteja envolvida no mecanismo de ação do AQ, bem como na patogênese da DH. A interação das citocinas da família da IL-6 com seus receptores desencadeia a ativação de enzimas da família das Janus-Quinases (JAKs), que por sua vez permitem o recrutamento e a ativação de fatores de transcrição da família das proteínas transdutoras de sinais e ativadoras da transcrição (STATs). Embora as principais características da DH sejam a presença da coréia e déficits na execução de movimentos voluntários, poucos testes são realizados abordando o comportamento locomotor dos animais no modelo de lesão por AQ. Neste trabalho, estudamos o efeito do AG490, um inibidor da JAK2, na gliose, perda neuronal e neurogênese no estriado de camundongos adultos C57BL/6J após a administração estereotáxica unilateral de AQ (30nmol). Imediatamente após a lesão, os animais receberam uma injeção subcutânea de AG490 (10mg/kg) ou veículo (PBS+DMSO), e injeções diárias por 6 dias adicionais. Além disso, investigamos o possível efeito da lesão por AQ na atividade física voluntária diária (AFVD) em rodas de atividade. A distância percorrida pelos camundongos foi monitorada por 28 dias após a injeção unilateral de QA (30nmol) ou PBS no estriado. Cortes coronais do cérebro (40?m) obtidos em criostato foram utilizados para quantificação de neurônios por estereologia e para a análise de expressão protéica, através de imunoistoquímica e Western Blotting para GFAP e doublecortina, marcadores de gliose e neuroblastos, respectivamente. A área total de células doublecortina-positivas (ACDP) e o número de neurônios (NN) no lado lesado (L) e contralateral à lesão (CL) foram avaliados. O Índice de Neurogênese (IN=ACDP(L)/ACDP(CL)) e o Índice de Sobrevivência Neuronal (ISN=NN(L)/NN(CL)) foram calculados. Após a administração de AQ, o estriado ipsilateral apresentou intensa gliose e células doublecortina positivas com características de células migratórias. O Western Blotting para GFAP mostrou uma redução ipsilateral de 19% nos animais tratados com AG490, em comparação aos animais do grupo tratado apenas com veículo (0.82±0.05; 1.010±0.06, n=9, p<0.05). O ISN foi 25% maior nos camundongos que receberam AG490 em comparação aos animais controles (0.75 ± 0.07; 0.60 ± 0.03; n=8, p<0.05). O IN mostrou uma diminuição de 21% no grupo AG490 em relação ao grupo de animais tratados apenas veículo de diluição (1.08±0.06; 1.37±0.09, n=5, p<0.05). A AFVD média, medida em quilômetros por dia, não se alterou nos animais que receberam injeção intra-estriatal de QA (30nmol) em comparação aos animais do grupo controle (3.97±0.34; 3.90±0.21, n=8, p>0.05). Portanto, nossos resultados suportam um papel para a JAK2 na morte neuronal, gliose, e neurogênese estriatais após lesão com AQ. O tratamento com o inibidor AG490 causou neuroproteção e diminuição da gliose, sugerindo que a reação astrocitária pode prejudicar a sobrevivência neuronal neste modelo experimental / Abstract: Injection of quinolinic acid (QA), a N-methyl-D-aspartate receptor agonist, in murine striatum induces death of medium spiny neurons, gliosis and neurogenesis in the subventricular zone with migration of newly synthesized neurons to damaged striatum. Such findings are also described in Huntington's disease (HD). The Janus-kinase (JAK) pathway would take part in QA mechanism of action and HD pathogenesis as well. The interaction of interleukin-6 family of cytokines with its receptor triggers the activation of enzymes of the family of JAKs, which in turn allow the recruitment and activation of transcription factors, known as signal transducers and activators of transcription (STATs). Although the main features of HD are the presence of chorea and deficits in performing voluntary movements, few tests are realized regarding locomotor behavioral on QA model. We studied the effect of AG490, an inhibitor of JAK isoform 2 (JAK2), on gliosis, neuronal loss and neurogenesis in the striatum of adult C57BL/6J mice after unilateral estereotaxic administration of QA (30 nmol). Immediately after injury, animals received a subcutaneous injection of AG490 (10 mg/kg) or vehicle (PBS + DMSO), and then once daily injections for 6 days. Furthermore, in a parallel experiment, we investigated the possible effect of the lesion by AQ on the voluntary daily physical activity (VDPA) in running wheels. The distance traveled by mice was monitored daily for 28 days after unilateral injection of QA (30 nmol) or PBS into the striatum. Frozen brain sections (40?m) were used for neuronal stereological quantification and immunohistochemical and Western Blotting analyses for GFAP and doublecortin, markers of gliosis and neuroblasts, respectively. The total area of doublecortin-positive cells (ADPC) and the number of neurons (NN) in the lesioned (L) and contralateral (CL) sides were evaluated. Neurogenesis index (NI = ADPC in L/ ADPC in CL) and neuronal survival ratio (NSR = NN in L/ NN in CL) were calculated. After QA administration, ipsilateral striatum showed intense gliosis and doublecortin-positive cells with few processes and ovoid bodies, morphological features corroborating a migratory activity. Western Blotting for GFAP showed an ipsilateral decrease of 19% in AG490- vs vehicle-treated animals (0.82 ± 0.05 vs 1.010 ± 0.06; n=9, p<0.05). NSR was 25% higher in mice given AG490 vs controls given vehicle (0.75 ± 0.07 vs 0.60 ± 0.03; n=8, p<0.05). NI showed a decrease of 21% in AG490- vs vehicle-treated mice (1.08 ± 0.06, 1.37 ± 0.09; n=5, p<0.05). The average VDPA, measured in kilometers per day for 28 days, has not changed in animals that received intrastriatal injection of QA (30nmol) compared to animals that received PBS (3.97 ± 0.34, 3.90 ± 0.21, n = 8, p> 0.05). In conclusion, our results support a role for JAK2 in striatal neuronal death, gliosis and neurogenesis determined by QA. AG490 caused neuroprotection and reduced gliosis suggesting that astrocytic reaction may impair neuronal survival in the present experimental model / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Parmi les innovations technologiques récentes, la téléphonie mobile a connu une progression fulgurante. Les expositions aux champs électromagnétiques radiofréquences (CEM RF) apparaissent de plus en plus tôt, lors de l’adolescence, voire dès l’enfance. Parallèlement la littérature scientifique rapporte des effets des CEM RF (GSM 900 MHz) à forts niveaux d’exposition sur l’expression de la glial fibrillary acidic protein (GFAP), le principal filament intermédiaire des astrocytes. Ces cellules jouent un rôle dans la transmission synaptique et dans la réparation des lésions cérébrales. Ce contexte nous a amené à poser l’hypothèse d’une perturbation des fonctions astrocytaires et cérébrales par des expositions aux CEM RF à niveaux élevés réalisées durant les stades de maturation cérébrale du foetus ou de l’adolescent. Nous avons posé une seconde hypothèse selon laquelle la fragilisation des organismes en développement par un épisode inflammatoire les rendrait plus vulnérables aux expositions environnementales, favorisant ainsi l’expression des effets neuro-biologiques des CEM RF.Afin de tester ces hypothèses, nous avons mimé les effets foeto-toxiques consécutifs à un état pathologique de la mère dans un modèle d’inflammation gestationnelle de rat utilisant des injections intra-péritonéales de lipopolysaccharides (LPS). Ce modèle a été exposé aux CEM RF, soit durant toute la gestation (co-exposition avec le LPS), soit durant le stade adolescent. Dans un autre groupe expérimental, nous avons mimé une astrogliose réactive consécutive à une infection ou un état neuro-pathologique au stade adolescent par micro-perfusion de LPS dans les ventricules cérébraux. Dans ce modèle, les rats adolescents ont été co-exposés au LPS aux CEM RF. Les variables d’intérêt ont été mesurées chez le jeune adulte et, dans le cas des co-expositions gestationnelles, au cours des stades juvénile et adolescent. Des paradigmes comportementaux ont été utilisés pour examiner les états émotionnels, la perception et l’adaptation à la nouveauté. Les niveaux de GFAP ont été quantifiés dans le cortex préfrontal, l’hippocampe, le striatum et l’amygdale. Nos résultats indiquent des perturbations comportementales (notamment en réponse à la nouveauté) chez le jeune adulte antérieurement exposé durant la gestation (et non pas durant l’adolescence) aux CEM RF. Une seule interaction du LPS et des CEM RF a été montrée dans le cas d’une co-exposition chez l’adolescent, par une plus faible augmentation des niveaux de GFAP à 1,5W/kg. D’un point de vue de santé publique, ces résultats sont obtenus avec des niveaux d’exposition aux CEM RF bien supérieurs (10 à 50 fois) à ceux environnementaux induits par le port du téléphone portable à proximité du foetus par la femme enceinte ou proche de l’oreille par un appel téléphonique. Dans un premier temps, il sera important de reproduire ces effets avant d’envisager des hypothèses mécanistiques d’interaction des CEM RF sur le développement foetal et sur le processus neuro-inflammatoire au stade adolescent. Il conviendra par ailleurs d’identifier si ces effets sont induits à des niveaux de CEM RF environnementaux afin de contribuer à l’évaluation du risque neuro-toxicologique des CEM RF. / The widespread use of mobile phones raises the question of the possible health effects of radiofrequency electromagnetic fields (RF EMF, GSM 900 MHz) on the brain. Acquisition of the first cell phone occurs predominantly before adolescence. Scientific literature reports effects of high levels of RF EMF exposure on the expression of the glial fibrillary acidic protein (GFAP). The GFAP is the principal intermediate filament of the astrocytes. These cells play a role in the synaptic transmission and brain damages repair. In this context, we hypothesized a disturbance of the astrocytes and brain functions by the exposure of high RF EMF levels carried out during foetal or adolescent cerebral maturation. A second assumption is made that the organisms under development sensitised by an inflammatory episode would be more vulnerable to the environmental exposures and lead the expression of the neuro-biological effects of RF EMF. To test these hypotheses, we mimicked the foetotoxic effects of a pathological state of the mother. We used a gestational inflammation model of rat obtained with intra-peritoneal injections of lipopolysaccharides (LPS). This model was exposed to RF EMF, either during all gestation (co-exposure with LPS), or during the adolescent stage. In another experimental group, we mimicked a reactive astrogliosis consecutive to an infection or a neuro-pathological state at the adolescent stage by micro-perfusion of LPS in the cerebral ventricle. In this model, adolescent rats were co-exposed to LPS and RF EMF. The different endpoints were measured in the young adult. In gestational co-exposure, endpoints were measured during juvenile and adolescent stages. Behavioural paradigms were used to examine the emotional states, the perception and the adaptation to novelty. The GFAP levels were quantified in the prefrontal cortex, the hippocampus, striatum and amygdala. Our results indicate effects on behavioural endpoints (particularly in novelty perception) in the young adult previously exposed to RF EMF during gestation (and not during adolescence). Only one interaction between the LPS and RF EMF was shown in co-exposure during adolescence. A weaker increase of the GFAP levels was shown after a 1,5W/kg exposure. These results were obtained with levels of RF EMF exposure which were much higher (10 to 50 times) than those induced by the mobile phone held near the foetus by the pregnant woman or near the ear during a phone call. It will be important to reproduce these effects before considering mechanistic interactions of RF EMF on the foetal development and the neuro-inflammatory process at the adolescent stage. In addition, it will be necessary to identify if these effects are induced at environmental RF EMF levels in order to contribute to the neuro-toxicological risk evaluation of RF EMF.

Astrocytes comprise the most abundant cell population in human brain (1). First described by Virchow as being 'glue' of the brain (2), modern research has truly extended our knowledge and understanding regarding the vast array of roles these cells execute under normal physiological conditions. Examples include neurotransmitter reuptake at the synapse (3), the regulation of blood flow at capillaries to meet neuronal energy demand (4), and maintenance/repair of the blood-brain barrier (BBB) (5), which is comprised, in part, of tight junction proteins such zonula-occludens-1 (ZO1) (6) and Claudin-5 (7). Underlying the execution of these processes is the morphological and spatial arrangement of astrocytes between neurons and endothelial cells comprising blood vessels, where comprehensively speaking, these cells form what is known as the gliovascular unit (8). Astrocytes extend large processes called endfeet that intimately associate with and enwrap up to 99% of the cerebrovascular surface (9). Disruptions to this association can occur in the form of retracted endfeet, and this has been characterized in several disease states such as major depressive disorder (10-12), ischemia (13-15), and normal biological aging (16-18). Disruption can also take the form of cellular/protein aggregate intercalation, which our lab previously characterized in a human-derived glioma model (19) and vascular amyloidosis human Amyloid Precursor Protein J20 (hAPPJ20) animal model (20). In both models, focal astrocyte-vascular disruptions coincided with perturbations to astrocyte control of blood flow, with deficits in BBB integrity present in the glioma model as well. These findings lead to the preliminary work in this dissertation where we aimed to extend BBB findings in the glioma model to the hAPPJ20 vascular amyloidosis model. Immunohistochemical analysis in two-year old hAPPJ20 animal arterioles revealed that indeed in locations of vascular amyloid buildup and endfoot separation, there was a significant reduction in a tight junction protein critical for BBB maintenance, ZO1. This reduction in ZO1 expression was accompanied by extravasation of 70kDa FITC and the ~1kDa Cadaverine, suggesting that BBB integrity was compromised. These findings led to the objective of this dissertation, which was to determine if focal ablation of an astrocyte is sufficient to disrupt BBB integrity. By utilizing the in vivo 2Phatal single-cell apoptosis induction method (21), we found that 1) focal loss of astrocyte-vascular coverage does not result in barrier deficits, but rather induces a plasticity response whereby surrounding astrocytes extend processes to reinnervate vascular vacancies no longer occupied by previously ablated astrocytes. 2) Replacement astrocytes are capable of inducing vasocontractile responses in blood vessels, and that 3) aging significantly attenuates the kinetics of this process. We then tested the hypothesis that focal loss of astrocyte-vascular coverage leads to a gliovascular structural plasticity response, in part, through the phosphorylation of signal transducer and activator of transcription 3 (STAT3) by Janus Kinase 2 (JAK2). This dissertation found that 4), this was indeed the case, and finally, 5) we determined that gliovascular structural plasticity occurs after reperfusion post-focal photothrombotic stroke. Together, the work presented in this dissertation sheds light on a novel plasticity response whereby astrocytes maintain continual cerebrovascular coverage and therefore physiological control. Future studies should aim to determine if 1) astrocytes also replace the synaptic contacts with neighboring neurons once held by a previous astrocyte, and 2) what therapeutic opportunity gliovascular structural plasticity may present regarding BBB repair following stroke. / Doctor of Philosophy / Astrocytes are the most abundant cell type in the brain. Their anatomical relationship to neurons and endothelial cells allows them to execute many vital brain functions, and comprehensively speaking, these cells form what is known as the gliovascular unit. Important for maintaining the expression of proteins preventing vascular leakage in the brain are molecules released from astrocytes processes called endfeet. These endfeet intimately enwrap blood vessels, and disruptions to endfeet-vascular coverage often coincide with vascular leakage in the brain. This dissertation therefore aimed to determine if astrocyte-vascular coverage is necessary in preventing vascular leakage. State-of-the art imaging in live animals determined this not to be the case, and rather found that focal loss of astrocyte-vascular coverage induces a plasticity response wherein neighboring astrocytes extend new endfeet to reinnervate vascular vacancies. Furthermore, we found that the kinetics of endfoot replacement are significantly reduced in aging, and that the phosphorylation of signal transducer and activator of transcription 3 (STAT3) is a critical arbiter underlying this response. Finally, given that we found endfoot replacement to occur in locations of lost astrocyte-vascular contact following reperfusion post-focal photothrombotic stroke, these findings may have implications regarding repair of the blood-brain barrier following CNS insults such as stroke.