Choroidal and retinal structural, cellular and vascular changes in a rat model of Type 2 diabetes

Increasing evidence points to inflammation as a key factor in the pathogenesis of diabetic retinopathy (DR). Choroidal changes in diabetes have been reported and several attempts were made to validate in vivo choroidal thickness (CT) as a marker of retinopathy. We aimed to study choroidal and retinal changes associated with retinopathy in an animal model of spontaneous Type 2 diabetes, Goto-Kakizaki (GK) rats. Sclerochoroidal whole mounts and cryosections were prepared from 52-week-old GK and age-matched control Wistar Han rats. CT was measured by optical coherence tomography. Microglia reactivity, pericyte and endothelial cells distribution, and immunoreactivity of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) were evaluated by immunofluorescence. Choroidal vessels were visualized by direct perfusion with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil). Choroidal vascular density was evaluated by fluorescence microscopy. GK rats had increased CT (58.40 ± 1.15 μm versus 50.90 ± 1.58 μm, p < 0.001), reduced vascular density of the choriocapillaris (CC) (p = 0.045), increased Iba1+ cells density in the outer retina (p = 0.003) and increased VEGFR2 immunoreactivity in most retinal layers (p = 0.021 to 0.037). Choroidal microglial cells and pericytes showed polarity in their distribution, sparing the innermost choroid. This cell-free gap in the inner choroid was more pronounced in GK rats. In summary, GK rats have increased CT with decreased vascular density in the innermost choroid, increased VEGFR2 immunoreactivity in the retina and increased Iba1+ cells density in the outer retina.info:eu-repo/semantics/publishedVersio

Retinal aging in 3× Tg-AD mice model of Alzheimer's disease

The retina, as part of the central nervous system (CNS), can be the perfect target for in vivo, in situ, and noninvasive neuropathology diagnosis and assessment of therapeutic efficacy. It has long been established that several age-related brain changes are more pronounced in Alzheimer’s disease (AD). Nevertheless, in the retina such link is still under-explored. This study investigates the differences in the aging of the CNS through the retina of 3×Tg-AD and wild-type mice. A dedicated optical coherence tomograph imaged mice’s retinas for 16 months. Two neural networks were developed to model independently each group’s ages and were then applied to an independent set containing images fromboth groups. Our analysis shows amean absolute error of 0.875±1.1×10−2 and 1.112 ± 1.4 × 10−2 months, depending on training group. Our deep learning approach appears to be a reliable retinal OCT aging marker. We show that retina aging is distinct in the two classes: the presence of the three mutated human genes in the mouse genome has an impact on the aging of the retina. For mice over 4 months-old, transgenic mice consistently present a negative retina age-gap when compared to wildtype mice, regardless of training set. This appears to contradict AD observations in the brain. However, the ‘black-box” nature of deep-learning implies that one cannot infer reasoning. We can only speculate that some healthy age-dependent neural adaptations may be altered in transgenic animals.This study was supported by The Portuguese Foundation for Science and Technology (FCT) through PTDC/EMD-EMD/28039/2017, UIDB/04950/2020, PestUID/NEU/04539/2019, and by FEDER-COMPETE through POCI-01-0145-FEDER-028039.info:eu-repo/semantics/publishedVersio

Normative mice retinal thickness: 16-month longitudinal characterization of wild-type mice and changes in a model of Alzheimer's disease

Animal models of disease are paramount to understand retinal development, the pathophysiology of eye diseases, and to study neurodegeneration using optical coherence tomography (OCT) data. In this study, we present a comprehensive normative database of retinal thickness in C57BL6/129S mice using spectral-domain OCT data. The database covers a longitudinal period of 16 months, from 1 to 16 months of age, and provides valuable insights into retinal development and changes over time. Our findings reveal that total retinal thickness decreases with age, while the thickness of individual retinal layers and layer aggregates changes in different ways. For example, the outer plexiform layer (OPL), photoreceptor inner segments (ILS), and retinal pigment epithelium (RPE) thickened over time, whereas other retinal layers and layer aggregates became thinner. Additionally, we compare the retinal thickness of wild-type (WT) mice with an animal model of Alzheimer's disease (3 × Tg-AD) and show that the transgenic mice exhibit a decrease in total retinal thickness compared to age-matched WT mice, with statistically significant differences observed at all evaluated ages. This normative database of retinal thickness in mice will serve as a reference for future studies on retinal changes in neurodegenerative and eye diseases and will further our understanding of the pathophysiology of these conditions

Stage-independent biomarkers for Alzheimer’s disease from the living retina: an animal study

The early diagnosis of neurodegenerative disorders is still an open issue despite the many efforts to address this problem. In particular, Alzheimer’s disease (AD) remains undiagnosed for over a decade before the first symptoms. Optical coherence tomography (OCT) is now common and widely available and has been used to image the retina of AD patients and healthy controls to search for biomarkers of neurodegeneration. However, early diagnosis tools would need to rely on images of patients in early AD stages, which are not available due to late diagnosis. To shed light on how to overcome this obstacle, we resort to 57 wild-type mice and 57 triple-transgenic mouse model of AD to train a network with mice aged 3, 4, and 8 months and classify mice at the ages of 1, 2, and 12 months. To this end, we computed fundus images from OCT data and trained a convolution neural network (CNN) to classify those into the wild-type or transgenic group. CNN performance accuracy ranged from 80 to 88% for mice out of the training group’s age, raising the possibility of diagnosing AD before the first symptoms through the non-invasive imaging of the retina.Tis study was supported by Te Portuguese Foundation for Science and Technology (FCT) through PTDC/EMD-EMD/28039/2017, UIDB/04950/2020, UIDB/04539/2020, Pest-UID/NEU/04539/2019, and by FEDERCOMPETE through POCI-01-0145-FEDER-028039.info:eu-repo/semantics/publishedVersio

Retinal imaging in animal models: Searching for biomarkers of neurodegeneration

There is a pressing need for novel diagnostic and progression biomarkers of neurodegeneration. However, the inability to determine disease duration and stage in patients with Alzheimer’s disease (AD) hinders their discovery. Because animal models of disease allow us to circumvent some of these limitations, they have proven to be of paramount importance in clinical research. Due to the clear optics of the eye, the retina combined with optical coherence tomography (OCT) offers the perfect opportunity to image neurodegeneration in the retina in vivo, non-invasively, directly, quickly, and inexpensively. Based on these premises, our group has worked towards uncovering neurodegeneration-associated changes in the retina of the triple-transgenic mouse model of familial AD (3×Tg-AD). In this work, we present an overview of our work on this topic. We report on thickness variations of the retina and retinal layers/layer aggregates caused by healthy aging and AD-like conditions and discuss the implications of focusing research efforts solely on retinal thickness. We explore what other information is embedded in the OCT data, extracted based on texture analysis and deep-learning approaches, to further identify biomarkers that could be used for early detection and diagnosis. We were able to detect changes in the retina of the animal model of AD as early as 1 month of age. We also discuss our work to develop an optical coherence elastography system to measure retinal elasticity, which can be used in conjunction with conventional OCT. Finally, we discuss the potential application of these technologies in human patients and the steps needed to make OCT a helpful screening tool for the detection of neurodegeneration

Microglial depletion has no impact on disease progression in a mouse model of machado–joseph disease

Machado–Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disorder (ND). While most research in NDs has been following a neuron-centric point of view, microglia are now recognized as crucial in the brain. Previous work revealed alterations that point to an increased activation state of microglia in the brain of CMVMJD135 mice, a MJD mouse model that replicates the motor symptoms and neuropathology of the human condition. Here, we investigated the extent to which microglia are actively contributing to MJD pathogenesis and symptom progression. For this, we used PLX3397 to reduce the number of microglia in the brain of CMVMJD135 mice. In addition, a set of statistical and machine learning models were further implemented to analyze the impact of PLX3397 on the morphology of the surviving microglia. Then, a battery of behavioral tests was used to evaluate the impact of microglial depletion on the motor phenotype of CMVMJD135 mice. Although PLX3397 treatment substantially reduced microglia density in the affected brain regions, it did not affect the motor deficits seen in CMVMJD135 mice. In addition to reducing the number of microglia, the treatment with PLX3397 induced morphological changes suggestive of activation in the surviving microglia, the microglia of wild-type animals becoming similar to those of CMVMJD135 animals. These results suggest that microglial cells are not key contributors for MJD progression. Furthermore, the impact of PLX3397 on microglial activation should be taken into account in the interpretation of findings of ND modification seen upon treatment with this CSF1R inhibitor.Fundação para a Ciência e a Tecnologia (FCT) (PTDC/NEUNMC/3648/2014) and COMPETE-FEDER (POCI-01-0145-FEDER-016818). It was also supported by Portuguese funds through FCT in the framework of the Project POCI-01-0145-FEDER-031987 (PTDC/MED-OUT/31987/2017). A.B.C. was supported by a doctoral fellowship from FCT (PD/BD/127828/2016). B.C. was also supported by FCT (2020.03898.CEECIND). This work was funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI (Portuguese Platform of Bioimaging) (PPBIPOCI-01-0145-FEDER-022122), and by National funds, through FCT—project UIDB/50026/2020 and UIDP/50026/2020

Profiling microglia in a mouse model of Machado-Joseph disease

Microglia have been increasingly implicated in neurodegenerative diseases (NDs), and specific disease associated microglia (DAM) profiles have been defined for several of these NDs. Yet, the microglial profile in Machado–Joseph disease (MJD) remains unexplored. Here, we characterized the profile of microglia in the CMVMJD135 mouse model of MJD. This characterization was performed using primary microglial cultures and microglial cells obtained from disease-relevant brain regions of neonatal and adult CMVMJD135 mice, respectively. Machine learning models were implemented to identify potential clusters of microglia based on their morphological features, and an RNA-sequencing analysis was performed to identify molecular perturbations and potential therapeutic targets. Our findings reveal morphological alterations that point to an increased activation state of microglia in CMVMJD135 mice and a disease-specific transcriptional profile of MJD microglia, encompassing a total of 101 differentially expressed genes, with enrichment in molecular pathways related to oxidative stress, immune response, cell proliferation, cell death, and lipid metabolism. Overall, these results allowed us to define the cellular and molecular profile of MJD-associated microglia and to identify genes and pathways that might represent potential therapeutic targets for this disorder.This work was supported by Fundação para a Ciência e a Tecnologia (FCT) (PTDC/NEUNMC/3648/2014) and COMPETE-FEDER (POCI-01-0145-FEDER-016818). It was also supported by Portuguese funds through FCT in the framework of the Project POCI-01-0145-FEDER-031987 (PTDC/MED-OUT/31987/2017). A.B.C. was supported by a doctoral fellowship from FCT (PD/BD/ 127828/2016). S.P.N. was also supported by FCT (PD/BD/114120/2015). Work in the JBR laboratory was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT in the framework of the Project POCI-01-0145- FEDER030647 (PTDC/MED-NEU/31318/2017). This work was funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI (Portuguese Platform of Bioimaging) (PPBIPOCI-01-0145-FEDER-022122), and by National funds, through FCT—project UIDB/50026/2020 and UIDP/50026/2020

PROT-OVT: Plano Regional de Ordenamento do Territorio do Oeste e Vale do Tejo

Os princípios, objectivos e orientações consagrados no Programa Nacional da Política de Ordenamento do Território (PNPOT), aprovado pela Lei n.º 58/2007, de 4 de Setembro, são desenvolvidos nos vários planos regionais de ordenamento do território (PROT) que, por sua vez, constituem um quadro de referência estratégico para os planos directores municipais (PDM). No processo de elaboração e revisão articulada destes três pilares fundamentais do sistema de gestão territorial, em que assenta a política de ordenamento do território e do urbanismo, cabe aos PROT uma posição de charneira fundamental. O significado e as potencialidades dos PROT são ainda significativamente ampliados pela articulação com a revisão dos PDM. Face a uma nova geração de PDM, que se pretendem mais estratégicos, os PROT fornecem um quadro de referência estratégica de longo prazo que permite aos municípios estabelecerem as suas opções de desenvolvimento e definirem regras de gestão territorial compatíveis com o modelo consagrado para a região. Os PROT, além de um pilar da política de desenvolvimento territorial, são documentos fundamentais para a definição dos programas de acção das intervenções co-financiadas pelos Fundos Estruturais e de Coesão da União Europeia. Os PROT são instrumentos de desenvolvimento territorial e de natureza estratégica. Em matéria de conteúdo, estabelecem a estrutura regional do sistema urbano, das redes de infra‑estruturas e dos equipamentos de interesse regional e definem os objectivos e princípios quanto à localização das actividades e os grandes investimentos públicos; as suas normas fixam o quadro estratégico, as orientações de carácter genérico e as directrizes para o ordenamento do território regional. O PROT do Oeste e Vale do Tejo (PROT OVT) visa, neste contexto, a espacialização de estratégias de desenvolvimento territorial nos territórios das NUTS III do Oeste, Médio Tejo e Lezíria do Tejo. Por isso, ocupa, entre o nível nacional e o nível municipal, uma posição chave para a definição das estratégias e das opções de desenvolvimento e de ordenamento regional. O PROT OVT é pois um instrumento privilegiado para promover a reflexão estratégica do desenvolvimento do Oeste e do Vale do Tejo e acolher a tomada de decisão quanto às opções de desenvolvimento territorial [...]info:eu-repo/semantics/publishedVersio

Mechanisms of action of antiepileptic drugs and neurotoxicity in hippocampus

Tese de doutoramento em Biologia (Biologia Celular) apresentada à Fac. de Ciências e Tecnologia de CoimbraNesta dissertação apresentam-se resultados relativos à investigação dos mecanismos de acção de dois fármacos novos com propriedades antiepilépticas, desenvolvidos pela BIAL, (S)-(-)-10-acetoxi-10,11-dihidro-5H-dibenzo[b,f ]azepina-5-carboxamida (BIA 2-093) and 10,11-dihidro-10-hidroxiimino-5H-dibenzo[b,f ] azepina-5-carboxamida (BIA 2-024), que foram comparados com os mecanismos de acção de dois fármacos antiepilépticos que são utilizados no tratamento da epilepsia, carbamazepina (CBZ) e oxcarbazepina (OXC). Também investigámos o papel dos receptores ionotrópicos do glutamato na excitotoxicidade, assim como o perfil neurotóxico/neuroprotector dos fármacos antiepilépticos. Os estudos foram efectuados em neurónios do hipocampo de rato em cultura e em terminais nervosos isolados do hipocampo de rato. Os resultados mostram que os fármacos antiepilépticos inibem canais de sódio, e que a CBZ também pode inibir canais de Ca2+ do tipo L, mas os fármacos antiepilépticos não afectam a activação dos receptores ionotrópicos do glutamato, não inibem os canais de cálcio que estão acoplados à libertação exocitótica de glutamato nem o transportador do glutamato. Os fármacos antiepilépticos novos são menos tóxicos do que a CBZ ou a OXC. Os antiepilépticos causam condensação da cromatina nuclear em alguns neurónios, característica de apoptose, e aumentam a actividade das enzimas tipo caspase-3, essencialmente nos neurónios tratados com CBZ ou com OXC. Além disso, os fármacos antiepilépticos, em concentrações que não causam toxicidade, não protegem os neurónios da toxicidade causada por cainato, veratridina ou por condições que mimetizam a isquémia. Também verificámos que existem várias populações de receptores de AMPA nos neurónios do hipocampo, com diferente sensibilidade para os agonistas, antagonistas e moduladores, e que a dessensibilização dos receptores de AMPA actua como um mecanismo neuroprotector