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After brain damage, regenerative angiogenesis and neurogenesis have been shown to occur simultaneously in mammals, suggesting a close link between these processes. However, the mechanisms by which these processes interact are not well understood. In this work, we aimed to study the correlation between angiogenesis and neurogenesis after a telencephalic stab wound injury. To this end, we used zebrafish as a relevant model of neuroplasticity and brain repair mechanisms. First, using the Tg(fli1:EGFP × mpeg1.1:mCherry) zebrafish line, which enables visualization of blood vessels and microglia respectively, we analyzed regenerative angiogenesis from 1 to 21 days post-lesion. In parallel, we monitored brain cell proliferation in neurogenic niches localized in the ventricular zone by using immunohistochemistry. We found that after brain damage, the blood vessel area and width as well as expression of the fli1 transgene and vascular endothelial growth factor (vegfaa and vegfbb) were increased. At the same time, neural stem cell proliferation was also increased, peaking between 3 and 5 days post-lesion in a manner similar to angiogenesis, along with the recruitment of microglia. Then, through pharmacological manipulation by injecting an anti-angiogenic drug (Tivozanib) or Vegf at the lesion site, we demonstrated that blocking or activating Vegf signaling modulated both angiogenic and neurogenic processes, as well as microglial recruitment. Finally, we showed that inhibition of microglia by clodronate-containing liposome injection or dexamethasone treatment impairs regenerative neurogenesis, as previously described, as well as injury-induced angiogenesis. In conclusion, we have described regenerative angiogenesis in zebrafish for the first time and have highlighted the role of inflammation in this process. In addition, we have shown that both angiogenesis and neurogenesis are involved in brain repair and that microglia and inflammation-dependent mechanisms activated by Vegf signaling are important contributors to these processes. This study paves the way for a better understanding of the effect of Vegf on microglia and for studies aimed at promoting angiogenesis to improve brain plasticity after brain injury.

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Insulin is a peptide hormone that plays a central role in the regulation of circulating blood glucose in vertebrates, including zebrafish. Increasing evidence has demonstrated the important role of insulin in many brain functions. In zebrafish, two insulin receptor genes (insra and insrb) have been identified. However, their biodistribution in the adult brain as well as their cell-specific expression pattern has not been well described. Using gene expression analysis, in situ hybridization and transgenic fish, we confirmed the expression of insra, insrb, and irs1 (insulin receptor substrate 1, the downstream effector of insulin receptor) in the brain of adult zebrafish and characterized their specific expression in neurons and neural stem cells (radial glia). After demonstrating that intracerebroventricular (ICV) injection resulted in the diffusion of the injected solution within the ventricular system, we analyzed the effect of insulin ICV injection on neurogenesis. We showed that insulin promotes ventricular cell proliferation 24 h postinjection. This neurogenic effect appeared to be independent of neuroinflammatory processes. Also, after a mechanical telencephalic stab-wound injury, we highlighted the overexpression of irs1 gene 5 days postlesion notably in the ventricular zone where radial glial cells (RGCs) are localized, suggesting key roles of insulin signaling in regenerative processes. Finally, our results reinforced the expression of insulin-related proteins in the brain of adult zebrafish, highlighting the potential role of insulin signaling on neurogenesis.

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Regenerative medicine is an emerging field of research aiming to understand the wound healing mechanisms and to develop new therapeutic strategies. Nanocarriers are used to improve drug bioavailability, solubility, and therapeutic abilities. In this study, we used for the first time curcumin loaded oligo kappa-carrageenan-graft-polycaprolactone (oligoKC-g-PCL) nanomicelles to investigate their regenerative potential using a model of tail amputation in zebrafish eleutheroembryo. First, we showed that curcumin encapsulated oligoKC-g-PCL spherical micelles had a mean size of 92 ± 32 nm and that micelles were successfully loaded with curcumin. These micelles showed a slow and controlled drug release over 72 h. The toxicity of curcumin nanomicelles was then tested on zebrafish eleutheroembryo based on the survival rate after 24 h. At nontoxic concentration, curcumin nanomicelles improved tail regeneration within 3 days postamputation, compared with empty micelles or curcumin alone. Furthermore, we demonstrated that curcumin nanomicelles increased the recruitment of neutrophils and macrophages 6 h postlesion. Finally, our study highlights the efficiency of oligoKC-g-PCL nanomicelles for encapsulation of hydrophobic molecules such as curcumin. Indeed, our study demonstrates that curcumin nanomicelles can modulate inflammatory reactions in vivo and promote regenerative processes. However, further investigations will be required to better understand the mechanisms sustaining regeneration and to develop new therapeutics.

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Microglia are macrophage-like cells exerting determinant roles in neuroinflammatory and oxidative stress processes during brain regeneration. We used zebrafish as a model of brain plasticity and repair. First, by performing L-plastin (Lcp1) immunohistochemistry and using transgenic Tg(mpeg1.1:GFP) or Tg(mpeg1.1:mCherry) fish, we analyzed the distribution of microglia/immune cells in the whole brain. Specific regional differences were evidenced in terms of microglia/immune cell density and morphology (elongated, branched, highly branched, and amoeboid). Taking advantage of Tg(fli:GFP) and Tg(GFAP::GFP) enabling the detection of endothelial cells and neural stem cells (NSCs), we highlighted the association of elongated microglia/immune cells with blood vessels and rounded/amoeboid microglia with NSCs. Second, after telencephalic injury, we showed that L-plastin cells were still abundantly present at 5 days post-lesion (dpl) and were associated with regenerative neurogenesis. Finally, RNA-sequencing analysis from injured telencephalon (5 dpl) confirmed the upregulation of microglia/immune cell markers and highlighted a significant increase of genes involved in oxidative stress (nox2, nrf2a, and gsr). The analysis of antioxidant activities at 5 dpl also revealed an upregulation of superoxide dismutase and persistent H2 O2 generation in the injured telencephalon. Also, microglia/immune cells were shown to be a source of oxidative stress at 5 dpl. Overall, our data provide a better characterization of microglia/immune cell distribution in the healthy zebrafish brain, highlighting some evolutionarily conserved features with mammals. They also emphasize that 5 days after injury, microglia/immune cells are still activated and are associated to a persistent redox imbalance. Together, these data raise the question of the role of oxidative stress in regenerative neurogenesis in zebrafish.

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Obesity has reached epidemic proportions, and its prevalence tripled worldwide between 1975 and 2016, especially in Reunion Island, a French overseas region. Psiloxylon mauritianum, an endemic medicinal plant from Reunion Island registered in the French pharmacopeia, has recently gained interest in combating metabolic disorders because of its traditional lipid-lowering and "anti-diabetic" use. However, scientific data are lacking regarding its toxicity and its real benefits on metabolic diseases. In this study, we aim to determine the toxicity of an aqueous extract of P. mauritianum on zebrafish eleutheroembryos following the OECD toxicity assay (Organization for Economic Cooperation and Development, guidelines 36). After defining a non-toxic dose, we determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) that this extract is rich in gallic acid but contains also caffeoylquinic acid, kaempferol and quercetin, as well as their respective derivatives. We also showed that the non-toxic dose exhibits lipid-lowering effects in a high-fat-diet zebrafish larvae model. In a next step, we demonstrated its preventive effects on body weight gain, hyperglycemia and liver steatosis in a diet-induced obesity model (DIO) performed in adults. It also limited the deleterious effects of overfeeding on the central nervous system (i.e., cerebral oxidative stress, blood-brain barrier breakdown, neuro-inflammation and blunted neurogenesis). Interestingly, adult DIO fish treated with P. mauritianum display normal feeding behavior but higher feces production. This indicates that the "anti-weight-gain" effect is probably due to the action of P. mauritianum on the intestinal lipid absorption and/or on the microbiota, leading to the increase in feces production. Therefore, in our experimental conditions, the aqueous extract of P. mauritianum exhibited "anti-weight-gain" properties, which prevented the development of obesity and its deleterious effects at the peripheral and central levels. These effects should be further investigated in preclinical models of obese/diabetic mice, as well as the impact of P. mauritianum on the gut microbiota.

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Hypericum lanceolatum Lam. (H. lanceolatum) is a traditional medicinal plant from Reunion Island used for its pleiotropic effects mainly related to its antioxidant activity. The present work aimed to 1) determine the potential toxicity of the plant aqueous extract in vivo and 2) investigate its putative biological properties using several zebrafish models of oxidative stress, regeneration, estrogenicity, neurogenesis and metabolic disorders. First, we characterized the polyphenolic composition by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified chlorogenic acid isomers, quercetin and kaempferol derivatives as the major compounds. We then evaluated for the first time the toxicity of an aqueous extract of H. lanceolatum and determined a maximum non-toxic concentration (MNTC) in zebrafish eleutheroembryos from 0 to 96 hpf following OECD (Organization for Economic Cooperation and Development) guidelines. This MNTC test was also determined on hatched eleutheroembryos after 2 days of treatment (from 3 to 5 dpf). In our study, the anti-estrogenic effects of H. lanceolatum are supported by the data from the EASZY assay. In a tail amputation model, we showed that H. lanceolatum at its MNTC displays antioxidant properties, favors immune cell recruitment and tissue regeneration. Our results also highlighted its beneficial effects in metabolic disorders. Indeed, H. lanceolatum efficiently reduces lipid accumulation and body mass index in overfed larva- and adult-models, respectively. In addition, we show that H. lanceolatum did not improve fasting blood glucose levels in a hyperglycemic zebrafish model but surprisingly inhibited neurogenesis impairment observed in diabetic conditions. In conclusion, our study highlights the antioxidant, pro-regenerative, anti-lipid accumulation and pro-neurogenic effects of H. lanceolatum in vivo and supports the use of this traditional medicinal plant as a potential alternative in the prevention and/or treatment of metabolic disorders.

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High density lipoproteins (HDLs) display pleiotropic functions such as anti-inflammatory, antioxidant, anti-protease, and anti-apoptotic properties. These effects are mediated by four main receptors: SCARB1 (SR-BI), ABCA1, ABCG1, and CD36. Recently, HDLs have emerged for their potential involvement in brain functions, considering their epidemiological links with cognition, depression, and brain plasticity. However, their role in the brain is not well understood. Given that the zebrafish is a well-recognized model for studying brain plasticity, metabolic disorders, and apolipoproteins, it could represent a good model for investigating the role of HDLs in brain homeostasis. By analyzing RNA sequencing data sets and performing in situ hybridization, we demonstrated the wide expression of scarb1, abca1a, abca1b, abcg1, and cd36 in the brain of adult zebrafish. Scarb1 gene expression was detected in neural stem cells (NSCs), suggesting a possible role of HDLs in NSC activity. Accordingly, intracerebroventricular injection of HDLs leads to their uptake by NSCs without modulating their proliferation. Next, we studied the biodistribution of HDLs in the zebrafish body. In homeostatic conditions, intraperitoneal injection of HDLs led to their accumulation in the liver, kidneys, and cerebral endothelial cells in zebrafish, similar to that observed in mice. After telencephalic injury, HDLs were diffused within the damaged parenchyma and were taken up by ventricular cells, including NSCs. However, they failed to modulate the recruitment of microglia cells at the injury site and the injury-induced proliferation of NSCs. In conclusion, our results clearly show a functional HDL uptake process involving several receptors that may impact brain homeostasis and suggest the use of HDLs as delivery vectors to target NSCs for drug delivery to boost their neurogenic activity.

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Citrus fruits are known for their beneficial health effects associated with the prevention of metabolic syndrome/type 2 diabetes that is mainly attributed to flavonoids. Few investigations have reported the potential anti-diabetic effects of retinoids from the bioconversion of ß-cryptoxanthin (bcx), a citrus carotenoid. Therefore, the present study explored the anti-diabetic effect of a citrus functional food, obtained by membrane eco-technology of a citrus clementina juice, especially enriched in bcx but also in flavonoids and pectin. We assessed the in vivo effect of citrus bcx absorption and its bioconversion into retinoids in metabolic syndrome/type 2 diabetic fructose rats. Fructose-fed rats were used as a prediabetic control, and a prediabetic group was treated with the citrus concentrate for 8 weeks. The citrus-based food treatment improved glucose tolerance, dyslipidemia and blood pressure, in prediabetic rats. Although these effects were in part due to the synergy between enriched phytonutrients (bcx, hesperidin, pectin) of the citrus matrix, the role of bcx and its bioconversion into retinoids were highlighted. We showed that prediabetic rats absorbed less bcx and the bioconversion was less efficient. Bcx from citrus-based food was able to restore vitamin A status in prediabetic rats suggesting that the absorption/bioconversion of bcx may have a key role in improvement of metabolic syndrome/type 2 diabetes.

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Citrus pectin is known to influence carotenoid bioaccessibility and absorption in humans, but limited attention has been given to the influence of pectin structure related to the particle size from differentially processed citrus food matrices. In this context, this study aims to investigate the nutritional health benefits of an innovative Citrus clementina concentrate, which is a new citrus-based food made by cross-flow microfiltration. This concentrated citrus-based food was selectively enriched 8-fold in ß-cryptoxanthin (43-55 µg g-1) and ß-carotene (6-9 µg g-1) as well as 6-fold in pectin (376-462 mg per 100 g). The bioaccessibility of pro-vitamin A carotenoids from commercial and fresh clementina juices versus their concentrates was assessed, including the intestinal carotenoid uptake by Caco-2 cells. Differences in particles size and pectin status resulted in a 7-fold increase in the bioaccessibility of carotenoids in industrial products versus fresh products while limiting their cellular uptake in correlation with larger micelle sizes (10.6 nm and 6.82 nm for industrial and fresh concentrates, respectively). Overall, the highest carotenoid bioaccessibility from industrial concentrate was sufficient to offset the lower carotenoid intestinal uptake related to micelle size. This study highlights that the structure of pectins, more specifically their degree of methoxylation, favors carotenoid bioaccessibility but impairs the intestinal absorption of carotenoids from citrus concentrates.

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