Pre-clinical investigation of brain mechanisms associated with Parkinson’s disease: The impact of diet

Obesity among the population is increasing worldwide, as are the related disorders. An association has already been found between obesity and neurodegenerative diseases. Knowledge about the relationship between obesity and brain disorders and a better understanding of the early stages of disease can also facilitate the development of new treatments and diagnostic tools. Lifestyle changes may also help prevent or delay neurodegenerative diseases. Reali Nazario's goal was to understand how certain brain processes are involved in Parkinson's disease. For this she initially used zebrafish. In addition, it was also examined whether it is possible to use zebrafish for molecular imaging using PET scans. Then she investigated how diet can influence the course of the disease in a rat model for Parkinson's disease.She found that the high-fat diet can influence the course of Parkinson's disease in the animal, particularly by increasing inflammatory processes in the brain and decreasing receptors associated with the disease. The gut microbiota was also affected by the high-fat diet, and this change in gut microbiota was related to inflammatory processes in the brain.The topics studied in this thesis are important for the understanding the progression of Parkinson's disease and in helping to find an effective treatment or cure for the disease

Potential PET tracers for imaging of tumor-associated macrophages

The increasing incidence of cancer over the years is one of the most challenging problems in healthcare. As cancer progresses, the recruitment of several immune cells is triggered. Infiltration of tumor-associated macrophages (TAMs) is correlated with poor patient prognosis. Since TAMs constitute a big portion of the tumor mass, targeting these cells seems to be an attractive approach for cancer immunotherapy. Additionally, TAM assessment using non-invasive imaging techniques, such as positron emission tomography (PET), might provide a better understanding of the role of TAMs in cancer, and a means for tumor profile characterization, patient selection for individualized immunotherapy and treatment monitoring. Imaging of TAMs using PET tracers is still in its infancy. TAMs have several characteristics that could be exploited as potential targets for imaging. Various PET tracers for these TAM biomarkers have been developed, although often in the context of (neuro)inflammatory diseases rather than cancer. Since macrophages in inflammatory diseases express similar biomarkers as TAMs, these PET tracers could potentially also be applied for the assessment of TAMs in the tumor microenvironment. Therefore, the present review provides an overview of the TAM biomarkers, for which potential PET tracers are available and discusses the status of these tracers

A single dose of ketamine cannot prevent protracted stress-induced anhedonia and neuroinflammation in rats

Worldwide, millions of people suffer from treatment-resistant depression. Ketamine, a glutamatergic receptor antagonist, can have a rapid antidepressant effect even in treatment-resistant patients. A proposed mechanism for the antidepressant effect of ketamine is the reduction of neuroinflammation. To further explore this hypothesis, we investigated whether a single dose of ketamine can modulate protracted neuroinflammation in a repeated social defeat (RSD) stress rat model, which resembles features of depression. To this end, male animals exposed to RSD were injected with ketamine (20 mg/kg) or vehicle. A combination of behavioral analyses and PET scans of the inflammatory marker TSPO in the brain were performed. Rats submitted to RSD showed anhedonia-like behavior in the sucrose preference test, decreased weight gain, and increased TSPO levels in the insular and entorhinal cortices, as observed by [11C]-PK11195 PET. Whole brain TSPO levels correlated with corticosterone levels in several brain regions of RSD exposed animals, but not in controls. Ketamine injection 1 day after RSD disrupted the correlation between TSPO levels and serum corticosterone levels, but had no effect on depressive-like symptoms, weight gain or the protracted RSD-induced increase in TSPO expression in male rats. These results suggest that ketamine does not exert its effect on the hypothalamic-pituitary-adrenal axis by modulation of neuroinflammation

Ultrafast Photoclick Reaction for Selective 18F-Positron Emission Tomography Tracer Synthesis in Flow

The development of very fast, clean, and selective methods for indirect labeling in PET tracer synthesis is an ongoing challenge. Here we present the development of an ultrafast photoclick method for the synthesis of short-lived 18F-PET tracers based on the photocycloaddition reaction of 9,10-phenanthrenequinones with electron-rich alkenes. The respective precursors are synthetically easily accessible and can be functionalized with various target groups. Using a flow photo-microreactor, the photoclick reaction can be performed in 60 s, and clinically relevant tracers for prostate cancer and bacterial infection imaging were prepared to demonstrate practicality of the method

Estudo do papel da ecto-5'-nucleotidase no contexto da inflama??o avaliando par?metros citol?gicos, bioqu?micos, moleculares e imagens de ?PET/CT em Zebrafish

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS ([email protected]) on 2016-08-25T16:23:46Z No. of bitstreams: 1 DIS_LUIZA_REALI_NAZARIO_PARCIAL.pdf: 804114 bytes, checksum: d4d4d3525f9a5a4a62c1c8080197776f (MD5)Made available in DSpace on 2016-08-25T16:23:46Z (GMT). No. of bitstreams: 1 DIS_LUIZA_REALI_NAZARIO_PARCIAL.pdf: 804114 bytes, checksum: d4d4d3525f9a5a4a62c1c8080197776f (MD5) Previous issue date: 2016-03-21Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPESFunda??o de Amparo ? Pesquisa do Estado do Rio Grande do Sul - FAPERGSThe LPS mechanism of action is still not completely elucidated on vertebrates like fish, and indeed differs from higher vertebrates. In zebrafish, LPS is capable of increasing the recruitment of immune cells and the expression of genes related to the immune response. The purinergic system has a great relation to the regulation of the immune system and inflammatory responses. The nucleotide ATP is able to induce cytokine secretion, recruitment and differentiation of immune cells. ATP can be dephosphorylated sequentially generating adenosine. In the context of inflammation adenosine serves as an innate immunomodulatory molecule. The control of adenosine extracellular levels is performed by nucleoside transporters and ecto-5'-nucleotidase. The ecto-5'-nucelotidase is an ectonucleotidase with pacemaker role in the production of adenosine and is one of the focuses of this study. Considering the analysis of the input images approach to the study of inflammation context, it is known that in rodents the uptake of 18F-FDG, an analogue of glucose, is increased under inflammation, which generates a differential image. The micro Positron Emission Tomography/ Computed Tomography (?PET /CT) is used for research in small animals and take images using a radiopharmaceutical. The use of ?PET/CT contributes with information at the molecular, structural and functional level and allows too monitor the effect of drugs in physiological / pathological situations in the range of a small animal as the zebrafish. The technology ?PET/CT is relatively new and so far there are no published scientific studies applying radiopharmaceuticals in zebrafish. In this context, the aim of the project was to study the involvement of the enzyme ecto-5'-nucleotidase in the development of inflammation induced by LPS using the cytological, biochemical, molecular and image (?PET) in different tissues of adult zebrafish (Danio rerio). To induce inflammation in zebrafish, the animals were injected with a solution of LPS (10 ug/g body weight, i.p) after being subjected to anesthesia (tricaine 0.1 g/L). The animals were kept for 2 hours or 24 hours in this treatment. For confirmation of inflammation were analyzed the gene expression of specific markers (tnf-? and cox-2) in encephalon, heart, kidney and intestine and differential counts of cells of the immune system. The activity and expression of ecto-5'-nucleotidase enzyme was analyzed in the encephalon, heart, kidney and intestine of control and treated animals. To keep the animals in ?PET/CT was performed anesthetic concentration curve (tricaine - 0.1, 0.12, 0.15 g/L) and standardized an apparatus to keep the fish in the presence of water, but yet still. A curve of time after injection of 18F-FDG was performed to obtain images in ?PET/CT (0, 10, 20 and 30 min) for standardizing the radiation quantitation in a gamma counter (15, 30, 60 90 and 120 min). Exposure to the LPS was able to increase the tnf-? expression in nearly all tissues studied (heart, kidney and intestine) and cox-2 in the kidney. The number of active peripheral blood white cells was also increased, confirming the induction of the inflammatory response. Hydrolysis of AMP in animals injected with LPS was increased in the heart in 24 hpi [72% compared to control] with no change in gene expression of ecto-5'-nucleotidase. The gene expression of ecto-5'-nucleotidase was adjusted temporarily in the kidney and intestine without altering the enzyme activity. After patterning images with ?PET/ CT and quantitation radiation by gamma counter for each organ examined, 30 minutes was defined as the best time for the biodistribution of 18F-FDG. After acquiring inflamed animal ?PET images it was not identified changes in the uptake of 18F-FDG compared to the control. Tissue quantification radiation registered a decrease in bone samples in animals treated with LPS, while other tissues have not changed. These data indicate that zebrafish responds to LPS by altering gene expression of specific markers, especially in kidney, and activation of white blood cells. The inflammation appears to be accompanied by a fine adjustment tissue-specific expression and activity of ecto-5'-nucleotidase in response to the inflammatory process. Although inflammation has been confirmed, the registration images by ?PET and radiation determination in different tissues have not been able to register differences in metabolic activity in animals treated with LPS. However, standardization of these techniques provides an advance in the use of radiopharmaceuticals in small animals, such as zebrafish.O mecanismo de a??o do LPS ainda n?o ? completamente elucidado em vertebrados como os peixes, e se diferencia dos vertebrados superiores. Em zebrafish, o LPS ? capaz de aumentar o recrutamento de c?lulas imunes e a express?o de genes relacionados com a resposta imune. O sistema purin?rgico tem uma grande rela??o com a regula??o do sistema imune e as respostas inflamat?rias. O ATP ? um nucleot?deo importante na secre??o de citocinas e no recrutamento e diferencia??o de c?lulas imunes, podendo ser sequencialmente desfosforilado gerando adenosina. No contexto da inflama??o, a adenosina atua como uma mol?cula imunomodulat?ria inata. Participam do controle dos n?veis extracelulares da adenosina, os transportadores de nucleos?deos e a ecto-5?-nucleotidase. A ecto-5?-nucleotidase ? uma enzima com papel de marcapasso na produ??o de adenosina e constitui um dos focos do presente estudo. Considerando a contribui??o da abordagem de an?lise de imagens no contexto do estudo da inflama??o, ? sabido que a capta??o do radiof?rmaco 18F-FDG, um an?logo da glicoseest? aumentada em roedores submetidos ? inflama??o, o que gera uma imagem diferenciada. O micro Tom?grafo por Emiss?o de P?sitron/Tomografia Computadorizada (?PET/CT) ? utilizado para pesquisas em animais de pequeno porte e obt?m imagens utilizando um radiof?rmaco. O uso da ?PET/CT contribui com informa??es a n?vel molecular, funcional e estrutural em tempo real e permite acompanhar o efeito de f?rmacos em situa??es fisiol?gicas/patol?gicas na escala de um animal diminuto como o zebrafish. A tecnologia do ?PET/CT ? relativamente nova e at? o momento n?o existem estudos cient?ficos publicados aplicando radiof?rmacos em zebrafish. Neste contexto, o objetivo do projeto foi estudar o envolvimento da enzima ecto-5?-nucleotidase no desenvolvimento de inflama??o induzida por LPS utilizando-se de par?metros citol?gicos, bioqu?micos, moleculares e de imagem por ?PET em diferentes tecidos de zebrafish adulto (Danio rerio). Para induzir inflama??o no zebrafish, os animais foram injetados com uma solu??o de LPS (10 ?g/g de peso corporal; i.p) ap?s terem sido submetidos ? anestesia (trica?na 0.1 g/L). Os animais permaneceram por 2 h ou 24 h neste tratamento. Para confirma??o da inflama??o foram analisadas a express?o g?nica de marcadores espec?ficos (tnf-? e cox-2) em enc?falo, cora??o, rim e intestino e contagem diferencial de c?lulas do sistema imune. A atividade e express?o da enzima ecto-5?-nucleotidase foi analisada no enc?falo, cora??o, rim e intestino dos animais controle e tratados. Para manter os animais no ?PET/CT foi realizada uma curva de concentra??o de anest?sico MS-222 (0.1, 0. 12, 0.15 g/L) e determinado um aparato para manter o peixe na presen?a de ?gua mas, ainda im?vel. Uma curva de tempo ap?s a inje??o de 18F-FDG foi realizada para a obten??o de imagens em ?PET/CT (0, 10, 20 e 30 min) e para a padroniza??o da quantifica??o de radia??o em um Gamma counter (15, 30, 60, 90 e 120 min). A exposi??o ao LPS foi capaz de aumentar a express?o de tnf-? em quase todos os tecidos estudados (cora??o, rim e intestino) e de cox-2 no rim. O n?mero de c?lulas brancas ativas do sangue perif?rico tamb?m foi aumentado, confirmando a indu??o da resposta inflamat?ria. A hidr?lise de AMP em animais injetados com LPS foi aumentada no cora??o em 24 hpi [72% em rela??o ao controle] com nenhuma altera??o na express?o g?nica da ecto-5?-nucleotidase. A express?o g?nica do ecto-5?-nucleotidase foi ajustada temporalmente no rim e intestino sem altera??o da atividade enzim?tica. Ap?s a padroniza??o de imagens com ?PET/CT e da quantifica??o de radia??o por Gamma counter para cada ?rg?o analisado, definiu-se 30 min como o melhor tempo para a biodistribui??o do 18F-FDG. Ap?s a aquisi??o de imagens em ?PET de animais inflamados n?o se identificou altera??es na capta??o do 18F-FDG comparado com o controle. A quantifica??o tecidual de radia??o registrou uma queda nas amostras de ossos nos animais tratados com LPS, embora os demais tecidos n?o tenham sido alterados. Estes dados indicam que o zebrafish responde ao LPS alterando express?o g?nica de marcadores espec?ficos, especialmente no rim e ativando c?lulas brancas do sangue. A inflama??o induzida parece estar acompanhada por um t?nue ajuste tecido-espec?fico da atividade e express?o da ecto-5?-nucleotidase em resposta ao processo inflamat?rio. Ainda que a inflama??o tenha sido confirmada, o registro de imagens por ?PET e a determina??o de radia??o nos diferentes tecidos n?o foram capazes de registrar diferen?as na atividade metab?lica em animais tratados com LPS. Entretanto, a padroniza??o destas t?cnicas oferece um avan?o no uso de radiof?rmacos em animais de pequeno porte, como o zebrafish

Chronic harmine treatment has a delayed effect on mobility in control and socially defeated rats

INTRODUCTION: Depression is characterized by behavioral, cognitive and physiological changes, imposing a major burden on the overall wellbeing of the patient. Some evidence indicates that social stress, changes in growth factors (e.g., brain-derived neurotrophic factor (BDNF)), and neuroinflammation are involved in the development and progression of the disease. The monoamine oxidase A inhibitor drug harmine was suggested to have both antidepressant and anti-inflammatory properties and may, therefore, be a potential candidate for treatment of depression. AIM: The goal of this study was to assess the effects of harmine on behavior, brain BDNF levels, and microglia activation in control rats and a rat model of social stress. MATERIAL AND METHODS: Rats were submitted to 5 consecutive days of repeated social defeat (RSD) or control conditions. Animals were treated daily with harmine (15 mg/kg) or vehicle from day 3 until the end of the experiment. To assess the effects of harmine treatment on behavior, the sucrose preference test (SPT) was performed on days 1, 6, and 15, the open field test (OFT) on days 6 and 14, and the novel object recognition test (NOR) on day 16. Brain microgliosis was assessed using [11C]PBR-28 PET on day 17. Animals were terminated on day 17, and BDNF protein concentrations in the hippocampus and frontal cortex were analyzed using ELISA. RESULTS: RSD significantly decreased bodyweight and increased anxiety and anhedonia-related parameters in the OFT and SPT on day 6, but these behavioral effects were not observed anymore on day 14/15. Harmine treatment caused a significant reduction in bodyweight gain in both groups, induced anhedonia in the SPT on day 6, and significantly reduced the mobility and exploratory behavior of the animals in the OFT mainly on day 14. PET imaging and the NOR test did not show any significant effects on microglia activation and memory, respectively. BDNF protein concentrations in the hippocampus and frontal cortex were not significantly affected by either RSD or harmine treatment. DISCUSSION: Harmine was not able to reverse the acute effects of RSD on anxiety and anhedonia and even aggravated the effect of RSD on bodyweight loss. Moreover, harmine treatment caused unexpected side effects on general locomotion, both in RSD and control animals, but did not influence glial activation status and BDNF concentrations in the brain. In this model, RSD-induced stress was not strong enough to induce long-term effects on the behavior, neuroinflammation, or BDNF protein concentration. Thus, the efficacy of harmine treatment on these delayed parameters needs to be further evaluated in more severe models of chronic stress

Automated Radiosynthesis, Preliminary In Vitro/In Vivo Characterization of OncoFAP-Based Radiopharmaceuticals for Cancer Imaging and Therapy

FAP-targeted radiopharmaceuticals represent a breakthrough in cancer imaging and a viable option for therapeutic applications. OncoFAP is an ultra-high-affinity ligand of FAP with a dissociation constant of 680 pM. OncoFAP has been recently discovered and clinically validated for PET imaging procedures in patients with solid malignancies. While more and more clinical validation is becoming available, the need for scalable and robust procedures for the preparation of this new class of radiopharmaceuticals continues to increase. In this article, we present the development of automated radiolabeling procedures for the preparation of OncoFAP-based radiopharmaceuticals for cancer imaging and therapy. A new series of [68Ga]Ga-OncoFAP, [177Lu]Lu-OncoFAP and [18F]AlF-OncoFAP was produced with high radiochemical yields. Chemical and biochemical characterization after radiolabeling confirmed its excellent stability, retention of high affinity for FAP and absence of radiolysis by-products. The in vivo biodistribution of [18F]AlF-NOTA-OncoFAP, a candidate for PET imaging procedures in patients, was assessed in mice bearing FAP-positive solid tumors. The product showed rapid accumulation in solid tumors, with an average of 6.6% ID/g one hour after systemic administration and excellent tumor-to-healthy organs ratio. We have developed simple, quick, safe and robust synthetic procedures for the preparation of theranostic OncoFAP-compounds based on Gallium-68, Lutetium-177 and Fluorine-18 using the commercially available FASTlab synthesis module.ISSN:1424-824

Proton therapy induces a local microglial neuroimmune response

BACKGROUND AND PURPOSE: Although proton therapy is increasingly being used in the treatment of paediatric and adult brain tumours, there are still uncertainties surrounding the biological effect of protons on the normal brain. Microglia, the brain-resident macrophages, have been shown to play a role in the development of radiation-induced neurotoxicity. However, their molecular and hence functional response to proton irradiation remains unknown. This study investigates the effect of protons on microglia by comparing the effect of photons and protons as well as the influence of age and different irradiated volumes.MATERIALS AND METHODS: Rats were irradiated with 14 Gy to the whole brain with photons (X-rays), plateau protons, spread-out Bragg peak (SOBP) protons or to 50 % anterior, or 50 % posterior brain sub-volumes with plateau protons. RNA sequencing, validation of microglial priming gene expression using qPCR and high-content imaging analysis of microglial morphology were performed in the cortex at 12 weeks post irradiation.RESULTS: Photons and plateau protons induced a shared transcriptomic response associated with neuroinflammation. This response was associated with a similar microglial priming gene expression signature and distribution of microglial morphologies. Expression of the priming gene signature was less pronounced in juvenile rats compared to adults and slightly increased in rats irradiated with SOBP protons. High-precision partial brain irradiation with protons induced a local microglial priming response and morphological changes.CONCLUSION: Overall, our data indicate that the brain responds in a similar manner to photons and plateau protons with a shared local upregulation of microglial priming-associated genes, potentially enhancing the immune response to subsequent inflammatory challenges.</p