Arterial spin labeling is a completely non-invasive method for blood-flow measurement techniques. Alzheimer's disease pathology includes microvascular abnormalities in addition to practically all risk factors having a vascular component that reduces cerebral perfusion. Hemodynamic parameters of cerebral blood flow and arterial transit time were estimated via single-compartment modeling of pseudo continuous arterial spin labeling data and neurocognitive test scores (Alzheimer's disease assessment scale and mini-mental state examination) were compared between a group of healthy (N=20) and early Alzheimer's disease (N=25) subjects before and six months after the Alzheimer's subjects began treatment with cholinesterase inhibitors. The early Alzheimer's group showed improved CBF after 6 months' treatment in every Alzheimer's-prone region except the medial and lateral temporal lobes. No difference in arterial transit time was found between groups, indicating that the pathophysiological process causing hypoperfusion in Alzheimer's disease may differ from vascular dementia.

O objetivo geral deste estudo foi verificar os efeitos terapêuticos do enriquecimento ambiental e do exercício físico forçado sobre as conseqüências funcionais e cognitivas da Hipoperfusão Encefálica Crônica (HEC) em ratos Wistar. Além disso, padronizamos protocolo alternativo de HEC, a fim de aumentar a taxa de sobrevivência característica deste modelo, juntamente com a análise comportamental e imunohistoquímica do sétimo ao sexto mês após a cirugia, analisando o comportamento de neurônios e astrócitos no hipocampo. Os resultados mostraram que o protocolo modificado, ou seja, com a oclusão das carótidas realizadas com uma semana de intervalo, apresentam resultados comportamentais e morfológicos similares aos do protocolo padrão; todavia, a taxa de sobrevida dos animais no protocolo modificado é significativamente maior quando comparados ao padrão. Além disso, em ambos os protocolos, os animais hipoperfundidos apresentam um déficit cognitivo no Teste Water Maze três meses após a cirurgia, mas os grupos isquêmicos não apresentaram diferenças morfológicas quando comparados ao seus controles em relação a volume total Hipocampal e área Estriatal. Ao analisar o tempo pós- lesão do déficit cognitivo, verificamos que a HEC causa danos comportamentais a longo prazo, chegando a seis meses após o evento isquêmico. Enquanto a cognição é afetada na fase crônica, as células neuronais e astrogliais são acometidas nas fases mais agudas deste modelo, começando a aparecer morte neuronal (através da quantificação de NeuN) e astrogliose reativa (através da quantificação do imunoconteúdo de GFAP) já nos primeiros dias, permanecendo até aproximadamente 3 meses após a lesão. Após o dano estabelecido, testamos estratégias terapêuticas com o objetivo de amenizar principalmente o dano cognitivo apresentado pelos animais a partir dos três meses após cirurgia. Inicialmente foi analisado o efeito pré e pós - isquemia do exercício físico forçado através de uma esteira para ratos na memória espacial e de trabalho, através do Water Maze e bioquimicamente no estado oxidativo cerebral. Posteriormente, os mesmos parâmetros foram testados, só que com a intervenção do enriquecimnto ambiental tanto pré quanto pós-isquemia também. O modelo de hipoperfusão cerebral crônica causa como citado anteriormente, um déficit bastante importante na memória e no apredizado. Somado a isso, em relação ao estresse oxidativo, a quantidade de radicais livres e o estado de lipoperoxidação celular encontra-se bastante aumentados no hipocampo de animais submetidos a isquemia, assim como as enzimas antioxidantes. O protocolo de atividade física regular forçada (3 vezes por semana, durante 20 minutos num período de 3 meses), sobretudo nos grupos pré e pré+pós-isquemia, preveniu os efeitos cognitivos e bioquímicos da HEC. Tal reversão também ocorreu com o uso do enriquecimento ambiental realizado 3 vezes por semana, 1 hora durante 3 meses em todos os grupos estimulados. / The aim of this study was to verify the experimental therapeutic effects of environmental enrichment and forced physical exercise on the consequences of cognitive and functional chronic cerebral hypoperfusion (CCH) in Wistar rats. To do that, a CCH alternative protocol was standardized, in order to increase the survival rate of this model, together with the behavioral and immunohistochemistry analysis done up to the sixth month after the surgery, analyzing hippocampus neurons and astrocytes. Results showed that the modified protocol, with occlusion of both carotid arteries performed with a one-week interval, presents morphological and behavioral results similar to the standard protocol; however, the rate of survival after the modified protocol was significantly greater when compared to the standard one. In addition, both protocols produced ischemic animals with cognitive deficits in the Water Maze Task, three months after the surgery, however with no gross morphological lesion, as assessed by hippocampal volume and estriatal area. The cognitive deficit in CCH rats is long lasting, reaching six months after the ischemic event. While the cognition is affected in the chronic phase of hypoperfusion, the neuronal and astrogliais cells are affected in acute phases of this model; neuronal death (through quantification of NeuN) and reactive astrogliosis (through quantification of imunocontent of GFAP) are already present in the first days and remain until approximately 3 months after the lesion. We tested therapeutic strategies with the aim of alleviating mainly the cognitive damage presented by animals, as from three months after surgery. Initially, the effects of preand post-ischemia forced physical exercise in spatial and working memory, through the Water Maze Tak and oxidative stress parameters were analyzed. Subsequently, we tested the effects of environmental enrichment, both pre- and post-ischemia. The model of chronic cerebral hypoperfusion as previously mentioned, causes an important deficit in spatial learning and memory; the free radicals content and cellullar lipid peroxidation is also substantially increased in the hippocampus of animals submitted to hypoperfusion, as well as the antioxidant enzymes. Forced regular physical activity protocol (20-min, 3 times per week during 12 weeks – moderate), especially in groups pre and pre+post-ischemia, warned cognitive and biochemical preventive effects in CCH rats; the same occurred with the use of enrichment environmental performed 1- hr, 3 times per week during 12 weeks in all groups stimulated.

O objetivo geral deste estudo foi verificar os efeitos terapêuticos do enriquecimento ambiental e do exercício físico forçado sobre as conseqüências funcionais e cognitivas da Hipoperfusão Encefálica Crônica (HEC) em ratos Wistar. Além disso, padronizamos protocolo alternativo de HEC, a fim de aumentar a taxa de sobrevivência característica deste modelo, juntamente com a análise comportamental e imunohistoquímica do sétimo ao sexto mês após a cirugia, analisando o comportamento de neurônios e astrócitos no hipocampo. Os resultados mostraram que o protocolo modificado, ou seja, com a oclusão das carótidas realizadas com uma semana de intervalo, apresentam resultados comportamentais e morfológicos similares aos do protocolo padrão; todavia, a taxa de sobrevida dos animais no protocolo modificado é significativamente maior quando comparados ao padrão. Além disso, em ambos os protocolos, os animais hipoperfundidos apresentam um déficit cognitivo no Teste Water Maze três meses após a cirurgia, mas os grupos isquêmicos não apresentaram diferenças morfológicas quando comparados ao seus controles em relação a volume total Hipocampal e área Estriatal. Ao analisar o tempo pós- lesão do déficit cognitivo, verificamos que a HEC causa danos comportamentais a longo prazo, chegando a seis meses após o evento isquêmico. Enquanto a cognição é afetada na fase crônica, as células neuronais e astrogliais são acometidas nas fases mais agudas deste modelo, começando a aparecer morte neuronal (através da quantificação de NeuN) e astrogliose reativa (através da quantificação do imunoconteúdo de GFAP) já nos primeiros dias, permanecendo até aproximadamente 3 meses após a lesão. Após o dano estabelecido, testamos estratégias terapêuticas com o objetivo de amenizar principalmente o dano cognitivo apresentado pelos animais a partir dos três meses após cirurgia. Inicialmente foi analisado o efeito pré e pós - isquemia do exercício físico forçado através de uma esteira para ratos na memória espacial e de trabalho, através do Water Maze e bioquimicamente no estado oxidativo cerebral. Posteriormente, os mesmos parâmetros foram testados, só que com a intervenção do enriquecimnto ambiental tanto pré quanto pós-isquemia também. O modelo de hipoperfusão cerebral crônica causa como citado anteriormente, um déficit bastante importante na memória e no apredizado. Somado a isso, em relação ao estresse oxidativo, a quantidade de radicais livres e o estado de lipoperoxidação celular encontra-se bastante aumentados no hipocampo de animais submetidos a isquemia, assim como as enzimas antioxidantes. O protocolo de atividade física regular forçada (3 vezes por semana, durante 20 minutos num período de 3 meses), sobretudo nos grupos pré e pré+pós-isquemia, preveniu os efeitos cognitivos e bioquímicos da HEC. Tal reversão também ocorreu com o uso do enriquecimento ambiental realizado 3 vezes por semana, 1 hora durante 3 meses em todos os grupos estimulados. / The aim of this study was to verify the experimental therapeutic effects of environmental enrichment and forced physical exercise on the consequences of cognitive and functional chronic cerebral hypoperfusion (CCH) in Wistar rats. To do that, a CCH alternative protocol was standardized, in order to increase the survival rate of this model, together with the behavioral and immunohistochemistry analysis done up to the sixth month after the surgery, analyzing hippocampus neurons and astrocytes. Results showed that the modified protocol, with occlusion of both carotid arteries performed with a one-week interval, presents morphological and behavioral results similar to the standard protocol; however, the rate of survival after the modified protocol was significantly greater when compared to the standard one. In addition, both protocols produced ischemic animals with cognitive deficits in the Water Maze Task, three months after the surgery, however with no gross morphological lesion, as assessed by hippocampal volume and estriatal area. The cognitive deficit in CCH rats is long lasting, reaching six months after the ischemic event. While the cognition is affected in the chronic phase of hypoperfusion, the neuronal and astrogliais cells are affected in acute phases of this model; neuronal death (through quantification of NeuN) and reactive astrogliosis (through quantification of imunocontent of GFAP) are already present in the first days and remain until approximately 3 months after the lesion. We tested therapeutic strategies with the aim of alleviating mainly the cognitive damage presented by animals, as from three months after surgery. Initially, the effects of preand post-ischemia forced physical exercise in spatial and working memory, through the Water Maze Tak and oxidative stress parameters were analyzed. Subsequently, we tested the effects of environmental enrichment, both pre- and post-ischemia. The model of chronic cerebral hypoperfusion as previously mentioned, causes an important deficit in spatial learning and memory; the free radicals content and cellullar lipid peroxidation is also substantially increased in the hippocampus of animals submitted to hypoperfusion, as well as the antioxidant enzymes. Forced regular physical activity protocol (20-min, 3 times per week during 12 weeks – moderate), especially in groups pre and pre+post-ischemia, warned cognitive and biochemical preventive effects in CCH rats; the same occurred with the use of enrichment environmental performed 1- hr, 3 times per week during 12 weeks in all groups stimulated.

O objetivo geral deste estudo foi verificar os efeitos terapêuticos do enriquecimento ambiental e do exercício físico forçado sobre as conseqüências funcionais e cognitivas da Hipoperfusão Encefálica Crônica (HEC) em ratos Wistar. Além disso, padronizamos protocolo alternativo de HEC, a fim de aumentar a taxa de sobrevivência característica deste modelo, juntamente com a análise comportamental e imunohistoquímica do sétimo ao sexto mês após a cirugia, analisando o comportamento de neurônios e astrócitos no hipocampo. Os resultados mostraram que o protocolo modificado, ou seja, com a oclusão das carótidas realizadas com uma semana de intervalo, apresentam resultados comportamentais e morfológicos similares aos do protocolo padrão; todavia, a taxa de sobrevida dos animais no protocolo modificado é significativamente maior quando comparados ao padrão. Além disso, em ambos os protocolos, os animais hipoperfundidos apresentam um déficit cognitivo no Teste Water Maze três meses após a cirurgia, mas os grupos isquêmicos não apresentaram diferenças morfológicas quando comparados ao seus controles em relação a volume total Hipocampal e área Estriatal. Ao analisar o tempo pós- lesão do déficit cognitivo, verificamos que a HEC causa danos comportamentais a longo prazo, chegando a seis meses após o evento isquêmico. Enquanto a cognição é afetada na fase crônica, as células neuronais e astrogliais são acometidas nas fases mais agudas deste modelo, começando a aparecer morte neuronal (através da quantificação de NeuN) e astrogliose reativa (através da quantificação do imunoconteúdo de GFAP) já nos primeiros dias, permanecendo até aproximadamente 3 meses após a lesão. Após o dano estabelecido, testamos estratégias terapêuticas com o objetivo de amenizar principalmente o dano cognitivo apresentado pelos animais a partir dos três meses após cirurgia. Inicialmente foi analisado o efeito pré e pós - isquemia do exercício físico forçado através de uma esteira para ratos na memória espacial e de trabalho, através do Water Maze e bioquimicamente no estado oxidativo cerebral. Posteriormente, os mesmos parâmetros foram testados, só que com a intervenção do enriquecimnto ambiental tanto pré quanto pós-isquemia também. O modelo de hipoperfusão cerebral crônica causa como citado anteriormente, um déficit bastante importante na memória e no apredizado. Somado a isso, em relação ao estresse oxidativo, a quantidade de radicais livres e o estado de lipoperoxidação celular encontra-se bastante aumentados no hipocampo de animais submetidos a isquemia, assim como as enzimas antioxidantes. O protocolo de atividade física regular forçada (3 vezes por semana, durante 20 minutos num período de 3 meses), sobretudo nos grupos pré e pré+pós-isquemia, preveniu os efeitos cognitivos e bioquímicos da HEC. Tal reversão também ocorreu com o uso do enriquecimento ambiental realizado 3 vezes por semana, 1 hora durante 3 meses em todos os grupos estimulados. / The aim of this study was to verify the experimental therapeutic effects of environmental enrichment and forced physical exercise on the consequences of cognitive and functional chronic cerebral hypoperfusion (CCH) in Wistar rats. To do that, a CCH alternative protocol was standardized, in order to increase the survival rate of this model, together with the behavioral and immunohistochemistry analysis done up to the sixth month after the surgery, analyzing hippocampus neurons and astrocytes. Results showed that the modified protocol, with occlusion of both carotid arteries performed with a one-week interval, presents morphological and behavioral results similar to the standard protocol; however, the rate of survival after the modified protocol was significantly greater when compared to the standard one. In addition, both protocols produced ischemic animals with cognitive deficits in the Water Maze Task, three months after the surgery, however with no gross morphological lesion, as assessed by hippocampal volume and estriatal area. The cognitive deficit in CCH rats is long lasting, reaching six months after the ischemic event. While the cognition is affected in the chronic phase of hypoperfusion, the neuronal and astrogliais cells are affected in acute phases of this model; neuronal death (through quantification of NeuN) and reactive astrogliosis (through quantification of imunocontent of GFAP) are already present in the first days and remain until approximately 3 months after the lesion. We tested therapeutic strategies with the aim of alleviating mainly the cognitive damage presented by animals, as from three months after surgery. Initially, the effects of preand post-ischemia forced physical exercise in spatial and working memory, through the Water Maze Tak and oxidative stress parameters were analyzed. Subsequently, we tested the effects of environmental enrichment, both pre- and post-ischemia. The model of chronic cerebral hypoperfusion as previously mentioned, causes an important deficit in spatial learning and memory; the free radicals content and cellullar lipid peroxidation is also substantially increased in the hippocampus of animals submitted to hypoperfusion, as well as the antioxidant enzymes. Forced regular physical activity protocol (20-min, 3 times per week during 12 weeks – moderate), especially in groups pre and pre+post-ischemia, warned cognitive and biochemical preventive effects in CCH rats; the same occurred with the use of enrichment environmental performed 1- hr, 3 times per week during 12 weeks in all groups stimulated.

Au cours du vieillissement, la rigidification des artères élastiques entraine une augmentation de l'amplitude de la pression pulsée centrale, qui se propage dans la microcirculation cérébrale. De façon chronique, l’élévation anormale de la pression pulsée endommage les fonctions vasculaires et cérébrales, pouvant être impliquée dans le développement d’une déficience cognitive d’origine vasculaire. Ceci est supporté par l’observation d’anomalies cérébrovasculaires chez les individus atteints de démence vasculaire et de la maladie d’Alzheimer. De plus, les individus exposés aux facteurs de risque vasculaires (hypertension, obésité, diabète, athérosclérose), démontrent une vascularisation fragilisée, une augmentation de la pression pulsée centrale et présentent un déclin cognitif. Il est donc probable qu’en association avec l’âge, les facteurs de risque vasculaires favorisent de façon mécanistique la propagation de la pression pulsée dans la circulation cérébrale et révèlent de façon prématurée le déclin cognitif. Le lien mécanistique entre l’augmentation de la pression pulsée cérébrale, les facteurs de risque vasculaires, les dommages cérébrovasculaires et l’incidence de la démence reste à être plus clairement investigué. La présente thèse vise ainsi à étudier l’hypothèse biomécanique du rôle délétère de la pression pulsée dans la détérioration des fonctions cérébrovasculaires et cognitives, avec l’âge et en association avec les facteurs de risque vasculaires, en élucidant la cascade des événements pathologiques depuis l’élévation de la pression pulsée centrale jusqu’à l’incidence de la démence. Afin de vérifier notre hypothèse, nous avons entrepris dans une première étude d’étudier chez la souris WT, l’impact de l’augmentation in vivo d’un stress mécanique pulsatile central (par chirurgie TAC) sur la vasculature cérébrale, le tissu cérébral et les fonctions cognitives. Ce stress a été induit en parallèle dans le modèle de souris transgénique APP/PS1 de la maladie d’Alzheimer. Nous avons pu démontrer que les vaisseaux cérébraux des souris WT et APP/PS1 sont vulnérables au stress mécanique de la pression pulsée, caractérisé par une diminution de la réponse vasodilatatrice des artères piales, une raréfaction des capillaires due à une apoptose, l’incidence de micro-hémorragies, une rupture de la barrière hémato-encéphalique et une hypoperfusion cérébrale. Ces dommages cumulatifs à la microcirculation cérébrale sont associés à une inflammation cérébrale et à une diminution des performances d’apprentissage et de mémoire de travail et spatiale des souris. De plus, le phénotype Alzheimer des souris APP/PS1 est exacerbé en présence du stress vasculaire, exprimé par l’augmentation des dépôts béta-amyloïdes, ainsi que la dysfonction endothéliale cérébrale et l’inflammation cérébrale déjà présentes dans ce modèle. Dans une deuxième étude, nous avons caractérisé les fonctions cérébrovasculaires et cognitives des souris transgéniques LDLR-/-;hApoB100+/+ avec l’ajout ou non d’un stress mécanique pulsatile central in vivo (par chirurgie TAC). Ces souris présentent des facteurs de risques des maladies cardiovasculaires (hypertension et dyslipidémie) menant au développement d’athérosclérose et miment un vieillissement artériel central accéléré (rigidité aortique et des carotides, dysfonction endothéliale, augmentation de la pression pulsée). Nous avons démontré que les souris LDLR-/-;hApoB100+/+ exhibent des anomalies cérébrovasculaires structurelles et fonctionnelles, dont une atrophie cérébrale, une dysfonction endothéliale cérébrale, une hypoperfusion cérébrale, une augmentation de la perméabilité de la barrière hémato-encéphalique, des micro-hémorragies corticales, la présence d’inflammation, de sénescence et de stress oxydant au niveau vasculaire et parenchymateux. L’ensemble de ces altérations majoritairement vasculaires, sont associées à une diminution des performances cognitives et sont exacerbées en présence d’un stress vasculaire additif. Nos deux études chez la souris démontrent qu’en présence d’une pression pulsée élevée, les dommages à la microvasculature cérébrale conduisent à une perte fonctionnelle de l’homéostasie cérébrale et à un déclin cognitif, dont l’incidence est accélérée soit dans un modèle de démence ou soit de vieillissement artériel central et en présence de facteurs de risque des maladies cardiovasculaires. Nos études fournissent la démonstration mécanistique d’un continuum entre une augmentation de pression pulsée et un déclin cognitif vasculaire. / With advancing age, the large elastic arteries undergo significant stiffening, resulting in increased central pulse pressure waves that penetrates deeper the cerebral microcirculation and may result in cerebrovascular and neuronal tissue damages, likely contributing to the development of cognitive impairment from vascular injury origin. This is compatible with strong evidence between impaired cerebrovascular structure and function in the brain of patients with vascular dementia or Alzheimer’s disease. In addition, elderly individuals are subjected in their lifetime to multiple vascular risk factors (hypertension, obesity, diabetes, atherosclerosis), all of which are known to be deleterious to the vascular function, are associated with an increase in central pulse pressure and with cognitive decline. Therefore, it is likely that with age, risk factors for vascular diseases may mechanistically promote the propagation of pulse pressure into the cerebrovascular system and reveal prematurely the brain susceptibility to cognitive decline. The present thesis was conducted to study the biomechanical hypothesis of the deleterious role of the pulse pressure in the deterioration of cerebrovascular and cognitive functions, with age and in association with vascular risk factors, by elucidating the cascade of pathological events linking the increase in central pulse pressure to the expression of dementia. To validate our hypothesis, we first studied in mice the impact of the in vivo increase of central pulsatile mechanical stress (achieved by trans-aortic constriction surgery) on the cerebral vasculature, brain tissue and cognitive functions. This stress was also induced in the APP/PS1 transgenic mouse model of Alzheimer's disease. We have shown that cerebral vessels of WT and APP/PS1 mice are vulnerable to the mechanical stress of the increased pulse pressure, which is characterized by a decrease in the vasodilatory response of the pial arteries, a rarefaction of the capillaries due to apoptosis, the incidence of micro-hemorrhages, a rupture of the blood-brain barrier and cerebral hypoperfusion. These cumulative damages to the cerebral microcirculation are associated with brain inflammation and poorer learning and working and spatial memory performances in mice. The Alzheimer's phenotype of APP/PS1 mice was exacerbated in the presence of elevated pulse pressure, as shown by the increase in beta-amyloid deposits, the decreased in endothelial cerebral vasodilatory responses and brain inflammation, which are already present in this model. In a second study, we sought to characterize the cerebrovascular and cognitive functions in the transgenic mouse model LDLR-/-;hApoB100+/+, subjected or not in vivo to a central pulsatile mechanical stress (by trans-aortic constriction surgery). These mice exhibit risk factors for cardiovascular diseases (hypertension and dyslipidemia), develop atherosclerosis and mimic premature central arterial aging (aortic and carotid stiffness, endothelial dysfunction, increased pulse pressure). We reported that LDLR-/-;hApoB100+/+ mice were characterized by structural and functional brain vascular abnormalities, including cerebral hypoperfusion, increased permeability of the blood-brain barrier, endothelial cerebral dysfunction, microhemorrhages, but also cerebral atrophy and the presence of inflammation, senescence and high oxidative stress at the vascular and parenchymal level. In addition, all these alterations, which are mainly vascular, were associated with a decrease in the cognitive performance of mice. Also, these vascular, parenchymal and cognitive changes were exacerbated in the presence of the vascular stress induced by transverse aortic constriction. Altogether, our two studies in mice demonstrated that, in the presence of an increase in pulse pressure, the damages to the micro-cerebrovascular system lead to loss of cerebral homeostasis and to cognitive decline, which are accelerated in a model of dementia or a model of central vascular aging and in presence or vascular risk factors. Our studies highlight the mechanistic demonstration of a continuum between an increase in pulse pressure and vascular cognitive decline.