Tobias Sahr

Carmen Buchrieser

Co-Immunoprecipitation: protein-RNA and protein-DNA interaction

Keywords: Legionella pneumophila, next generation sequencing, co-immunopreciptitation, protein-RNA, protein-DNA interaction

Transcriptional and post-transcriptional regulators play a critical role in allowing a bacterium to adapt to the diverse environments and conditions it encounters. In order to characterize the role of these regulators the identification of their specific interaction partners is of utmost importance. Co-Immunoprecipitation (IP) is based on antigen/antibody complex formation to purify a protein of interest from the rest of the samples together with its interaction partner. This method allows us to study direct interaction of a regulator with its specific binding partners like protein-RNA, protein-DNA or protein-protein interactions. IP typically requires careful optimization and troubleshooting depending on the varying physicochemical characteristics of the protein of interest. In the following chapter we present a starting point and the basic guidelines to obtain the best possible results from an IP experiment with subsequent use of new generation sequencing (NGS) techniques to detect mRNA or ncRNAtargets (RIPseq) and protein-DNA interactions (ChIPseq).

Introduction

Bacteria have developed a variety of strategies with which they adapt their genetic expression to meet the challenges of their ever-changing surrounding environment. These include specific sigma factors, two-component systems, peptide or chemical-based quorum sensing systems, repressors, positive regulators, as well as small regulatory RNAs. Alone or in combination, these mechanisms enable bacterial cells to communicate with their environment, their hosts, and with each other, allowing the bacteria to adopt specific responses, express specific proteins (toxins, adhesins, invasins, siderophores) or develop specialized structures such as biofilms or spores to ensure survival, colonization of their ecological niches and dissemination. Expression profiles of Legionella lacking transcriptional regulators have been used successfully to broadly define genes impacted by the deleted regulator [START_REF] Albert-Weissenberger | Control of flagellar gene regulation in Legionella pneumophila and its relation to growth phase[END_REF][START_REF] Bruggemann | Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila[END_REF][START_REF] Hovel-Miner | ArgRregulated genes are derepressed in the Legionella-containing vacuole[END_REF][START_REF] Hovel-Miner | SigmaS controls multiple pathways associated with intracellular multiplication of Legionella pneumophila[END_REF][START_REF] Sahr | Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila[END_REF][START_REF] Tiaden | Synergistic contribution of the Legionella pneumophila lqs genes to pathogen-host interactions[END_REF][START_REF] Tiaden | The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA[END_REF]. Although providing informative data, transcriptional analysis alone cannot distinguish between direct and indirect regulatory effects or identify regulated genes that may be transcriptionally silent under the test conditions. Furthermore, in these experiments additional non-specific results like stress response or activation of subsequent regulatory cascades cannot be avoided and sometimes these secondary effects even overwhelm and hide original regulator-specific effects and only vague conclusions about primary/direct responses can be drawn from the experiments.

Co-Immunoprecipitation (IP) enables the purification of a protein based on antigen/antibody complex formation. Therefore, isolates of the protein purified from the rest of the samples can be achieved to study direct interaction between the protein and specific binding partners like nucleotides or other proteins. The most critical factor for the success of these experiments is the quality of the antibody used, but also many other factors during the different steps from lysis to elution are influencing the outcome of the IP [START_REF] Waldminghaus | ChIP on Chip: surprising results are often artifacts[END_REF]. ChIP coupled with next-generation sequencing (ChIP-seq) has revolutionized whole-genome mapping of DNA-binding protein sites. Although ChIP-seq rapidly gained support in eukaryotic systems [START_REF] Park | ChIP-seq: advantages and challenges of a maturing technology[END_REF], it remains underused in the mapping of bacterial transcriptional regulator-binding sites, and only three studies have been published to date [START_REF] Davies | Mapping the regulon of Vibrio cholerae ferric uptake regulator expands its known network of gene regulation[END_REF][START_REF] Kahramanoglou | Direct and indirect effects of H-NS and Fis on global gene expression control in Escherichia coli[END_REF][START_REF] Lun | A blind deconvolution approach to high-resolution mapping of transcription factor binding sites from ChIP-seq data[END_REF]. However, no study using L. pneumophila as model has been reported yet.

The following protocols should be seen only as starting points for conducting these experiments, as protocol optimization for each protein studied and each antibody used is indispensible. Here we describe two different methods i) RIPseq to characterize protein-RNA interactions and ii) ChIPseq to characterize protein-Chromatin/DNA interactions. Both of them lead to the construction of DNA-libraries optimized for Next Generation Sequencing (NGS) techniques. Alternatively, the library screening can be performed with hybridization to Microarrays (on Chip) for which additional steps for DNA dye labeling after PCR amplification are necessary.

Materials

Strains and growth media

1. Legionella pneumophila e.g. strains Paris, JR32 or Philadelphia-1 2. AYE medium. For 1 liter dissolve 12 g yeast extract and 10 g ACES, adjust pH to 6.9 with 1 M KOH. Add 10 mL of cysteine 40 g/L and 10 mL of iron pyrophosphate 30 g/L. Fill volume to 1 L with distilled water and filter sterilize.

3. CYE plates. For 1 liter dissolve 10 g yeast extract and 10 g ACES, adjust pH to 6.9 with 1M KOH, add 15 g of agar, 2 g of activated charcoal and autoclave. Add 10mL of filter sterilized cysteine 40 g/L and 10 mL of filter sterilized ferric nitrate 25 g/L. Poor plates.

RIP library construction

1. Formaldehyde 37% 1. Inoculate 50 mL fresh AYE media to OD600 = 0.1 from an overnight culture and allow bacteria to grow to an optimal OD (depending on the expression of desired protein).

2. Transfer each culture to separate disposable 50 mL polypropylene tubes and add 1.4 mL of 37% Formaldehyde (Note 2).

3. Incubate the formaldehyde-treated cells for 20 minutes at room temperature on a rotating platform, and transfer to a rotating platform overnight at 4°C. 4. Spin the 50 mL polypropylene tubes containing the formaldehyde-treated cells for 5min at 5000g (4°C), pour off the supernatant and wash the cells 3 times in cold 1X PBS (if not using the cells immediately, store cell pellet at -80°C). 2. Formaldehyde step is optional, but usually necessary as the protein-nucleotide interactions are weak and unstable resulting in too small quantities. Alternative to wild type cultures, cultures overexpressing the protein of interest can be used to increase quantity.

Resuspend the cell pellets using 1ml Lysis Buffer + protease inhibitor

3.

For IP experiments an amount of at least 3-5mg of total protein is recommended (depending on abundance of the protein of interest).

2 .

 2 Disposable 50 mL polypropylene tubes 3. Rotating wheel 4. Protease inhibitor cocktail (e.g. SigmaFast, Sigma, S8830-20TAB) 5. Lysis buffer: 50 mM HEPES-KOH pH7.5, 150mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, protease inhibitor 6. Sonicator 7. Specific antibody against protein of interest or alternatively antibody against Tag (e.g. Flag-or Strep-Tag) 8. Dynabeads® Protein A or G (Invitrogen, 100-01D or 100-03D), 9. Magnet for separation 10. Wash buffer RIPseq: 10 mM Tris-HCl pH 7.5, 250 mM NaCl, 0.5% NP40, 0.5% Nadeoxycholate, 1 mM EDTA 11. TE buffer : 10mM Tris, 1mM EDTA, pH 7.5 12. Elution buffer: 50mM Tris, 1mM EDTA, 1% SDS, pH8.0 13. Proteinase K 20µg/µl 14. DNase I recombinant, RNase free (10U/µl) 15. RNA fragmentation kit (Ambion, AM8740) 16. Alkaline Phosphatase 17. 3'-RNA adapter P-UCGUAUGCCGUCUUCYGCUUGUidT (idT = inverted deoxyT) 18. T4 Polynucleotide Kinase 19. 5'-RNA adapter GUUCAGAGUUCUACAGUCCGACGAU 20. Reverse Transcription Specific primer CAAGCAGAAGACGGCATACGA 21. Phenol solution, pH4.3 22. Chloroform:Isoamyl alcohol 24:1 23. RNase inhibitor 24. 3M Sodium Acetate, pH5.2 25. Ethanol p.a.; Etanol 75% 26. Glycogen (20mg/ml) 27. Tobacco acid pyrophosphatase 10U/µl, TAP 28. 5'-RNA adapter (25µM) GUUCAGAGUUCUACAGUCCGACGAU 29. T4 RNA Ligase 5U/µl 30. Superscript II Reverse Transcriptase Kit (Invitrogen, 18064014) 31. 10mM dNTP mix 32. Certified Low Range Ultra Agarose 33. Agarose gel electrophoresis equipment 34. 0.5M EDTA pH8.0 35. Distilled water DNase/RNase free 36. Thermocycler 37. High Fidelity DNA Polymerase 38. Primer 1 (25µM) CAAGCAGAAGACGGCATACGA 39. Primer 2 (25µM) AATGATACGGCGACCACCGACAGGTTCAG AGTTCTACAGTCCGA 40. PCR Clean-up kit 41. Bionalyzer 2100 (Agilent); 42. DNA 1000 Chips (Agilent, 5067-1504) 43. Bradford reagent 2.2 ChIPseq library construction 1. Formaldehyde 37% 2. Disposable 50 mL polypropylene tubes 3. Protease inhibitor cocktail 4. Lysis buffer: 50 mM HEPES-KOH pH7.5, 150mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, protease inhibitor 5. Sonicator 6. Specific antibody against protein of interest or alternatively antibody against Tag (e.g. Flag-or Strep-Tag) 7. Dynabeads® Protein A or G (Invitrogen, 100-01D or 100-03D), 8. Magnet for separation 9. Wash buffer ChIPseq: 10 mM Tris-HCl pH 7.5, 250 mM LiCl, 0.5% NP40, 0.5% Nadeoxycholate, 1 mM EDTA 10. TE buffer : 10mM Tris, 1mM EDTA, pH 7.5 11. Elution buffer: 50mM Tris, 1mM EDTA, 1% SDS, pH8.0 12. Proteinase K 20µg/µl 13. RNase A, DNase free (0.35µg/µl in PBS) 14. Genomic DNA Sample Prep Kit (Illumina, FC-102-1001) 3. RIPseq and ChIp-seq library construction (common steps; Note 1)

6 . 3 . 1 .

 631 And pulse-sonicate them with around 60 watt five times for 20secFor ChIP-seq additional DNA fragmentation: sonication on ice for 15 min 30sec ON/ 30sec OFF (avoid raising temperature!!) or nebulization necessary Centrifuge at max speed for 20min and transfer supernatant in a new tube, quantify the total protein with Bradford and adjust it to 1mg protein/ml (Note 3) 7. Preparation of magnetic beads -Aliquot 100µL of Dynabeads ProteinG for each sample to be immunoprecipitated and wash them 3 times in 1XPBS + 1%BSA solution, resuspend them in 250µl PBS/BSA + add 5-10µg of antibody and incubate them over night at 4°C on a rotating platform (Note 4) -do the same with 200µl of beads, but incubate them over night without antibody or with an unspecific antibody) -Wash the beads 3 times in cold PBS/BSA and resuspend them in 100µl PBS/BSA per sample 8. Co-Immunoprecipitation -Add 100µl of magnetic beads (without antibody or with an unspecific antibody) to the protein solution (prepared in step 6) and incubate for 4h at 4°C on a rotating platform (pre-clearing of solution from unspecific protein-beads interaction); place them on a magnet and transfer the supernatant into a fresh tube -Divide the fraction in two equal aliquots. Add in one 100µl of magnetic beads with specific antibody. For the second part, add 100µl of magnetic beads without antibody or with an unspecific antibody (negative control). Incubate samples over night at 4°C on a rotating platform 9. Place samples on a magnet and wash the beads twice with cold Lysis buffer containing additional 350mM NaCl 10. Wash the beads five times in cold Wash buffer (important for RIPseq use NaCl instead of LiCl!!!) 11. Wash once with cold TE buffer 12. Resuspend beads in 150µl Elution buffer and incubate them for 15min at 65°C with brief vortexing every 2min 13. Place the solution on a magnet and transfer supernant into a new tube (discard the beads!) 14. Reverse cross-linking (by incubation over night at 65°C) 15. P/C/I extraction and NaAc precipitation (Note 5 and 6), resuspend pellet in 50µl distilled water 16. Add 140µl TE buffer + 3µl Glycogen + 7µl Proteinase K (20µg/µl) and incubate 2h at 37°C 17. Extract again with P/C/I and resuspend after NaAc precipitation in 44µl distilled H2O. Continue with step 3.1 for RNA-Immunoprecipitation or with 3.2 for DNA/Chromatin-IP RIPseq (continuation) 1. DNase I digestion: add 5µl of DNase 10xbuffer and 1µl of DNase I and incubate reaction for 30min at 25°C. Exract with P/C/I and precipitate samples with NaAc. Resuspend the dry pellet in 10µl distilled RNase-free water 2. Fragmentation: the RNA was metal-catalyzed heat fragmented to sizes of around 100-200nt using the RNA fragmentation kit 1.1µl 10x fragmentation reagent (buffered zinc solution) 10µl RNA Incubation at 70°C for 5min Terminate reaction by putting on ice and add 1.1µl stop solution After NaAc precipitation, resuspend the dry pellet in 26µl RNase-free distilled H2O 3. RNA dephosphorylation: a) TAP treatment Denature the RNA from step 2 for 10min at 65°C After 1min on ice, add 3µl TAP buffer and 1µl TAP (10U) and incubate for 1h at 37°C b) Alkaline Phosphatase treatment Add 14µl distilled H2O, 5µl 10x Dephosphorylation buffer and 1µl Alkaline Phosphatase (10U) and incubate for 30min at 37°C Phenol/Chloroform/IAA extraction, NaAc precipitation; resuspend the dry pellet in 5.9 µl distilled water 4. 3'-RNA adapter ligation 5.9µl RNA 0.6µl 3'adapter (100µM) Incubate mix for 10min at 65°C, put on ice for 1min and add 1µl T4 RNA ligase buffer 1µl ATP solution 0.5µl RNase inhibitor 1µl RNA ligase Incubate for 6h at 20°C, following 4°C over night, purify with columns (e.g. NucleoSpin) and resuspend RNA after NaAc precipitation in 10µl distilled water 5. Re-phosphorylation of 5RNase free H2O P/C/I extraction and precipitation with NaAc; resuspend pellet in 5.3µl distilled H2O 6. 5'-RNA adapter ligation: 5.3µl RNA 1.2µl 5'-RNAadapter (25µM) Incubate 10min at 65°C, on ice for 1min and add 1µl T4 RNA ligase buffer 1µl ATP solution 0.5µl RNase inhibitor 1µl RNA ligase And incubate for 6h at 20°C, following 4°C over night 7. Reverse Transcription using RT Specific primer 10µl RNA +0.5µl RT Specific primer (100µM) Denature at 65°C for 10min, on ice 1min and add 6µl first strand buffer 1.5µl DTT (100mM) 1.5µl dNTP 1µl RNase inhibitor 8µl RNase-free H2O 1.5µl Superscript II RT Incubate 1h at 42°C and purify on columns (e.g. NucleoSpin), recover with 30µl of distilled water 8. PCR amplification (see also Note 7) fractionation Run samples of step 8 on a low range ultra 2% agarose gel. Cut the zones between 100-170nt and 170-250nt and purify independently with columns (e.g. Nucleospin). Combine the samples of the two "zones", precipitate and resuspend pellet in 30µldistilled water Purify with columns (e.g. Nucleospin), precipitate and resuspend pellets in 10µldistilled water 10. Check quality and quantity with the Bioanalyzer (DNA 1000) (Note 8) 11. Ready for Sequencing with Illumina 3.2. ChIP-seq library construction (continuation) 1. RNase digestion: add add 56µl of RNase A (final concentration 0.2 µg/µl) and incubate reaction for 2h at 37°C. Extract with P/C/I and precipitate samples with NaAc. Resuspend the dry pellet in 30µl distilled RNase-free water 2. blunt end-DNA by incubation with T4 DNA Polymerase 30µl DNA sample 45µl water 10µl T4 DNA Ligase Buffer (with 10mM ATP) 4µl dNTP (10mM) 5µl T4 DNA Polymerase 1µl Klenow Enzyme 5µl T4 Polynuleotide Kinase Incubate 30min at 20°C and purify with columns (e.g. NucleoSpin) 20°C, purification and resuspend in 30µl distilled water 5. PCR amplification (as decribed in 3.1. step 8) 6. Size fractionation on a low range ultra 2% agarose gel (see also 3.1. step 9) 7. Check quality and quantity with the Bioanalyzer (DNA 1000) (Note 8) 4. Notes 1. Keep aliquots of the different stages and test them by western blot to monitor the efficiency of your lysis, binding, washing and eluation steps.

4 .

 4 Specific antibodies or antibodies against Tags can be used similarly; test quality of antibodies in western blot experiments before use. 5. Phenol/Chloroform/IAA (P/C/I) extraction: Add 50% of Phenol to the sample and mix vigorously by vortexing. Add the same amount of Chloroform/Isoamyl alcohol and mix well. Centrifuge at max speed for 5 min, transfer the supernatant to a new tube and add 1 vol of Chloroform/Isoamyl alcohol, mix vigorously, centrifuge and transfer supernatant into a new tube. 6. NaAc precipitation: Add 10% 3M sodium acetate (pH5.2), 2% glycogen and 2.5vol of ethanol p.a. to the sample and store it min 2h (better over night) at-20°C. Centrifuge at 4°C for 15min at max speed and discard supernatant. Wash RNA pellet with 500µl icecold 75% ethanol centrifuge again for 5min, discard supernatant and let the pellet dry for 10min at RT. Resuspend the dry pellet RNase-free distilled H2O. 7. Number of cycles during PCR amplification can differ between experiments depending on the amount of cDNA obtained in previous step. Typically are 12 to maximal 17 cycles. More cycles are not recommended as additional PCR steps may introduce a significant amplification bias in cDNA representation. 8. DNA amounts are typically in nM range; anyhow, for the hybridization on the Cluster Station concentrations of 1-10 pM are recommended.

Acknowledgements

This work received support from the Institut Pasteur, the Centre national de la recherché scientifique (CNRS) and the Institut Carnot-Pasteur MI and from the ANR-10-PATH-004 project, in the frame of ERA-Net PathoGenoMics


Anne-Claire Dorsemans

St Ephanie Soul

Meltem Weger

Emmanuel Bourdon

Christian Lefebvre D'hellencourt

Olivier Meilhac

Dr Nicolas Diotel
email: nicolas.diotel@univ-reunion.fr

Impaired Constitutive and Regenerative Neurogenesis in Adult Hyperglycemic Zebrafish ABSTRACT

A growing body of evidence supports hyperglycemia as a putative contributor to several brain dysfunctions observed in diabetes patients, such as impaired memory capacity, neural plasticity, and neurogenic processes. Thanks to the persistence of radial glial cells acting as neural stem cells, the brain of the adult zebrafish constitutes a relevant model to investigate constitutive and injury-induced neurogenesis in adult vertebrates. However, there is limited understanding of the impact of hyperglycemia on brain dysfunction in the zebrafish model. This work aimed at exploring the impact of acute and chronic hyperglycemia on brain homeostasis and neurogenesis. Acute hyperglycemia was shown to promote gene expression of proinflammatory cytokines (il1b, il6, il8, and tnfa) in the brain and chronic hyperglycemia to impair expression of genes involved in the establishment of the blood-brain barrier (claudin 5a, zona occludens 1a and b). Chronic hyperglycemia also decreased brain cell proliferation in most neurogenic niches throughout the forebrain and the midbrain. By using a stab wound telencephalic injury model, the impact of hyperglycemia on brain repair mechanisms was investigated. Whereas the initial step of parenchymal cell proliferation was not affected by acute hyperglycemia, later proliferation of neural progenitors was significantly decreased by chronic hyperglycemia in the injured brain of fish. Taken together, these data offer new evidence highlighting the evolutionary conserved adverse effects of hyperglycemia on neurogenesis and brain healing in zebrafish. In addition, our study reinforces the utility of zebrafish as a robust model for studying the effects of metabolic disorders on the central nervous system. J. Comp. Neurol. 000:000-000, 2016.

Hyperglycemia corresponds to abnormally high blood glucose levels, which may reflect a response to situations of acute stress or to a chronic disorder of insulin secretion and/or resistance (diabetes). The prevalence of type 2 diabetes (characterized by insulin resistance) has increased rapidly in recent decades in association with drastic changes in lifestyle [START_REF] Lee | Effect of physical inactivity on major non-communicable diseases worldwide: an analysis of burden of disease and life expectancy[END_REF]World Health Organisation, 2015a,b). Today, 382 million people suffer from type 2 diabetes, and 14,000 people die daily from this disease or its related complications. Thus, type 2 diabetes is a major health problem and is projected to affect 592 million people by 2035 (IDF, 2015).

Hyperglycemia is a complex and multifactorial state caused by both genetic and nongenetic factors. It is associated with an increased risk of mortality related to cerebrovascular and renal dysfunctions, cancers, sepsis, and also neuropathies [START_REF] Koh | The impact of diabetes on the pathogenesis of sepsis[END_REF][START_REF] Storey | Impact of malglycemia on clinical outcomes in hospitalized patients with cancer: a review of the literature[END_REF][START_REF] Hafez | Hyperglycemia, acute ischemic stroke, and thrombolytic therapy[END_REF][START_REF] Lee | Epidemiology of diabetic retinopathy, diabetic macular edema and related vision loss[END_REF]. Furthermore, hyperglycemia is an independent risk factor for ischemic stroke and is a predictor of adverse neurological and survival outcomes [START_REF] Kagansky | The role of hyperglycemia in acute stroke[END_REF][START_REF] Gilmore | The role of hyperglycemia in acute ischemic stroke[END_REF][START_REF] Desilles | Diabetes mellitus, admission glucose, and outcomes after stroke thrombolysis: a registry and systematic review[END_REF]. Hyperglycemia has been shown to sensitize the brain against endogenous aggressions [START_REF] Sander | Reducing the risk of stroke in type 2 diabetes: pathophysiological and therapeutic perspectives[END_REF]. Both acute and chronic hyperglycemia increase oxidative stress and stimulate the production of proinflammatory cytokines that display deleterious effects on different organs (e.g., eyes, kidney, heart, brain) and that alter various physiological processes [START_REF] Kodl | Cognitive dysfunction and diabetes mellitus[END_REF][START_REF] Kayama | Diabetic cardiovascular disease induced by oxidative stress[END_REF][START_REF] Tangvarasittichai | Oxidative stress, insulin resistance, dyslipidemia and type 2 diabetes mellitus[END_REF]. In the central nervous system, chronic and acute hyperglycemia disrupt energy homeostasis, neural activity, and neural functions, which may alter cognition and promote dementia and depression [START_REF] Dejgaard | Evidence for diabetic encephalopathy[END_REF][START_REF] Cukierman | Cognitive decline and dementia in diabetes-systematic overview of prospective observational studies[END_REF][START_REF] Brismar | Predictors of cognitive impairment in type 1 diabetes[END_REF][START_REF] Gaudieri | Cognitive function in children with type 1 diabetes: a meta-analysis[END_REF][START_REF] Lang | Impaired neurogenesis in adult type-2 diabetic rats[END_REF][START_REF] Lu | Diabetes and the risk of multisystem aging phenotypes: a systematic review and metaanalysis[END_REF][START_REF] Ho | Effects of diabetes on hippocampal neurogenesis: links to cognition and depression[END_REF][START_REF] Ojo | Evaluating the association between diabetes, cognitive decline and dementia[END_REF]. In addition, diabetes may impact adult neurogenesis [START_REF] Ho | Effects of diabetes on hippocampal neurogenesis: links to cognition and depression[END_REF], a physiological process leading to the generation of functional neurons from neural stem/progenitor cell proliferation.

In mammals, adult neurogenesis occurs throughout life in two main neurogenic regions: the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricle [START_REF] Lindsey | A comparative framework for understanding the biological principles of adult neurogenesis[END_REF][START_REF] Grandel | Comparative aspects of adult neural stem cell activity in vertebrates[END_REF]. Additional neurogenic niches have also been described such as in the hypothalamus [START_REF] Migaud | Emerging new sites for adult neurogenesis in the mammalian brain: a comparative study between the hypothalamus and the classical neurogenic zones[END_REF][START_REF] Batailler | DCX-expressing cells in the vicinity of the hypothalamic neurogenic niche: a comparative study between mouse, sheep, and human tissues[END_REF]. Adult neurogenesis is regulated by intrinsic and extrinsic factors [START_REF] Hsieh | Orchestrating transcriptional control of adult neurogenesis[END_REF] and is markedly blunted under pathological conditions such as epilepsy and stroke (particularly in cases involving increased blood-brain barrier [BBB] permeability), in the contexts of increased oxidative stress and of inflammation [START_REF] Zhao | Mechanisms and functional implications of adult neurogenesis[END_REF][START_REF] Lin | Neurogenesis is enhanced by stroke in multiple new stem cell niches along the ventricular system at sites of high BBB permeability[END_REF]. Unlike mammals, adult teleost fish exhibit intense and widespread neurogenic activity throughout life and maintain a greater capacity for central nervous system repair after injury [START_REF] Kizil | Adult neurogenesis and brain regeneration in zebrafish[END_REF][START_REF] Schmidt | Neurogenesis in zebrafish -from embryo to adult[END_REF]. In teleosts, such capacities are notably due to the persistence of radial glial cells acting as neural progenitors, as well as to further committed progenitors corresponding to neuroblasts [START_REF] Chapouton | Adult neurogenesis in non-mammalian vertebrates[END_REF][START_REF] M€ Arz | Heterogeneity in progenitor cell subtypes in the ventricular zone of the zebrafish adult telencephalon[END_REF][START_REF] Lindsey | The cellular composition of neurogenic periventricular zones in the adult zebrafish forebrain[END_REF]. In the zebrafish telencephalon, the ventricular zone is described as comprising quiescent radial glial cells (type 1 progenitors), actively divided radial glial cells (type 2 progenitors), and neuroblasts (type 3 progenitors), mainly clustered in the subpallium (M€ arz et al., Abbreviations Anatomical terms used in this study are according to the Wullimann atlas [START_REF] Wullimann | Neuroanatomy of the zebrafish brain: a topological atlas[END_REF] [START_REF] Sarras | Use of zebrafish as a disease model provides a unique window for understanding the molecular basis of diabetic metabolic memory[END_REF]Capiotti et al., 2014a;[START_REF] Connaughton | Alternate immersion in an external glucose solution differentially affects blood sugar values in older versus younger zebrafish adults[END_REF], which may be useful for analyzing the impact of both acute and chronic hyperglycemia on brain plasticity and neurogenic processes.

In the current study, we set up an acute model of hyperglycemia and investigated the effects of both acute and chronic hyperglycemia on brain cell proliferation under homeostatic and regenerative conditions in zebrafish. We show that hyperglycemia upregulates the expression of proinflammatory cytokines in the brain, disturbs the expression of genes involved in the establishment of the BBB, and impairs both constitutive and regenerative neurogenesis.

MATERIALS AND METHODS Animals and ethics

Adult male and female wildtype zebrafish (Danio rerio) of 3-6 months in age were purchased from commercial suppliers and maintained under standard conditions of photoperiod (14/10 hours light/dark) and temperature (28 6 18C). The zebrafish were fed daily with commercially available dry food (TetraMin, Germany). Animals were euthanized by rapid chilling on ice to induce a lethal shock before spinal cord sectioning. For all "surgery" procedures and/or injections, fish were deeply anesthetized with 0.02%

Hyperglycemia models

To assess the impact of hyperglycemia in zebrafish, animals were submitted to acute or chronic exposure to D-glucose (Sigma-Aldrich). Acute hyperglycemia (AHG) was induced by an intraperitoneal (i.p.) injection of D-glucose (2.5 g/kg of body weight) dissolved in a 50 lL saline phosphate buffer (PBS; pH 7.4). The respective control fish were injected with 50 lL of PBS. Chronic hyperglycemia (CHG) was induced by fish water supplementation with 111 mM D-glucose over a period of 14 days, as previously described by Capiotti et al. (2014a). Fish water was changed every 2 days.

For acute HG and chronic HG models, control fish were rigorously maintained in the same conditions (light, temperature, and environment) as treated fish.

Measurement of blood glucose levels

Determination of blood glucose levels was performed after a 12-hour fasting period. Fish were euthanized at the same time of day and gently washed and dried prior to blood glucose level measurement. One eye was removed, and a glucometer test strip (One-Touch Ultra, LifeScan, France) was directly inserted in the ocular cavity. Fish were considered hyperglycemic when glycemia was found to be higher than 100 mg/dl. Fish following hyperglycemic treatment and displaying blood glucose levels <100 mg/dl were excluded from the study.

Stab wound of adult zebrafish telencephalon

Prior to stab wounding, zebrafish were deeply anesthetized with 0.02% tricaine. Mechanical injury to the telencephalon was performed by inserting a sterile needle (BD Microlance 3; 30 G 1 = 2 00 ; 0.3 3 13 mm) in the right telencephalic hemisphere, following a dorsoventral axis and guided by landmarks on the head, as previously described [START_REF] Diotel | Effects of estradiol in adult neurogenesis and brain repair in zebrafish[END_REF]. After the procedure fish were put back into their respective tanks and maintained until recovery.

Tissue preparation

For paraffin immunohistochemistry, fish were euthanized before being decapitated. Zebrafish heads were immersed overnight at 48C in 4% paraformaldehyde (PFA) dissolved in PBS. The next day, brains were extracted from the skull and postfixed in 4% PFA-PBS overnight at 48C before paraffin embedding. For freefloating vibratome section immunohistochemistry, fixed brains were stepwise dehydrated in a methanol/PBS concentration series and stored at -208C, as previously described [START_REF] Adolf | Conserved and acquired features of adult neurogenesis in the zebrafish telencephalon[END_REF].

Immunohistochemistry

For paraffin immunohistochemistry, zebrafish brains were cut into 7-lm-thick coronal sections using a microtome (Microm HM 355 S, Thermo Scientific, Pittsburgh, PA), and double glial fibrillary acidic protein (GFAP)/proliferating cell nuclear antigen (PCNA) immunostainings were performed as follows. The sections were dewaxed in xylene, progressively rehydrated through graded ethanol (100 to 50%), and then rinsed in PBS containing 0.2% Triton X-100 (PBS-T). Antigen retrieval was performed in a sodium citrate buffer (pH 6.0) in a microwave oven (750W, 2 minutes). After washing in PBS-T, the sections were saturated in PBS-T containing 0.5% milk powder to avoid nonspecific binding. The sections were then incubated overnight at room temperature with the following primary antibodies: polyclonal rabbit anti-GFAP (1:500; REF: Z033429, Dako, Carpinteria, CA; RRID: AB_2314535) and monoclonal mouse anti-PCNA (1:100; clone PC10, Dako; RRID: AB_2160651). The next day the sections were washed three times in PBS-T and incubated for 90 minutes at room temperature with 4 0 ,6 0 -diamidino-2phenylindole (DAPI) for cell nuclei counterstaining and with the respective secondary antibodies: donkey antirabbit Alexa Fluor 488 (1:200; REF: A21206; Life Technologies, Bethesda, MD; RRID: AB_10049650) and goat anti-mouse Alexa Fluor 594 (1:200; REF: A11005; Life Technologies; RRID: AB_141372). Finally, the sections were washed three times in PBS-T and the slides were mounted with the antifading medium Vectashield (H-1000, Vector Laboratories, Burlingame, CA).

For free-floating section immunohistochemistry using a vibratome, PCNA immunostaining was performed as follows. Zebrafish brains were progressively rehydrated through graded methanol (75 to 25%) and rinsed in PBS. After washing in PBS containing 0.1% Tween-20 (PTW), the brains were embed in 2% agarose in PBS. Next, the brains were cut into 50-lm-thick free-floating sections on a vibratome (Vibratome Line VT1000S, Leica, Germany) in a blocking buffer (BB; PBS containing 0.1% Tween-10, 0.2% bovine serum albumin, 1% dimethyl sulfoxide). The sections were then incubated overnight at 48C with the monoclonal mouse anti-PCNA (1:500; clone PC10, Dako) in BB. The next day, the sections were washed three times in PTW and incubated for 2 hours at room temperature with DAPI for cell nuclei counterstaining and with a donkey anti-mouse Alexa Fluor 488 antibody (1:1000; REF: A21202; Life Technologies; RRID: AB_141607). Finally, the sections were washed three times in PTW and the slides were then mounted with the antifading medium Aqua Polymount (REF: 18606, Polysciences, Warrington, PA). No staining was observed after omission of the primary antibodies (data not shown). Table 1 summarizes the antibodies used in this study.

Antibodies specificity

PCNA and GFAP antibodies have been previously documented for their use and their specificity in zebrafish [START_REF] Pellegrini | Identification of aromatase-positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish[END_REF][START_REF] Lam | gfap and nestin reporter lines reveal characteristics of neural progenitors in the adult zebrafish brain[END_REF]Diotel et al., 2010b[START_REF] Diotel | Mapping of brain lipid binding protein (Blbp) in the brain of adult zebrafish, co-expression with aromatase B and links with proliferation[END_REF][START_REF] Schmidt | Regenerative response following stab injury in the adult zebrafish telencephalon[END_REF][START_REF] Baumgart | Stab wound injury of the zebrafish telencephalon: a model for comparative analysis of reactive gliosis[END_REF].

Consistent with previous studies, PCNA-labeling was observed in cell nuclei across the neurogenic niches lining the ventricles as shown by [START_REF] Pellegrini | Identification of aromatase-positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish[END_REF]. In addition, its distribution is similar to BrdU proliferative assay [START_REF] Zupanc | Proliferation, migration, neuronal differentiation, and long-term survival of new cells in the adult zebrafish brain[END_REF]. GFAP-positive cells correspond to radial glial cell, as they display a soma localized along the ventricular layer and a long radial process that crosses the brain parenchyma and reaches the pial surface. Its expression and its distribution along the ventricles correspond to previous data obtained with the same antibody in zebrafish [START_REF] Baumgart | Stab wound injury of the zebrafish telencephalon: a model for comparative analysis of reactive gliosis[END_REF] or with GFAP:GFP transgenic line [START_REF] Lam | gfap and nestin reporter lines reveal characteristics of neural progenitors in the adult zebrafish brain[END_REF][START_REF] M€ Arz | Heterogeneity in progenitor cell subtypes in the ventricular zone of the zebrafish adult telencephalon[END_REF].

Microscopy

Sections were analyzed with an Eclipse 80i Nikon fluorescence microscope equipped with a Hamamatsu ORCA-ER digital camera (Life Sciences, Japan) or with a confocal microscope (Nikon D Eclipse C1), both of which were connected to a computerized image analysis system (NIS-Elements software; RRID: SCR_014329).

Cell counting

For analyzing constitutive neurogenesis, proliferative activity was determined by quantification of PCNAlabeled cells on images captured from at least three consecutive paraffin sections representative of a region of interest.

For analyzing injury-induced neurogenesis, counting was performed on 3-4 vibratome sections of 50 lm thickness each in the injured versus uninjured telencephalon. Images were analyzed for detection of PCNApositive nuclei using ImageJ software (National Institutes of Health, Bethesda, MD; RRID: SCR_003070) by adjusting parameters (threshold, binary, and watershed) and running an analysis on "particles" taking into consideration the size and the circularity. Briefly, the parameters were set up as follows for each picture: threshold 5 30-100, particle size 5 20-1,000, circularity 5 0.3-1.00. Minor modifications in these parameters could be slightly adjusted according to the experiments. In addition, ImageJ automated selection of PCNA-positive nuclei was manually double-checked and adjusted if necessary for each picture. No significant difference was observed concerning the average size of counted particles in control versus hyperglycemic conditions (data not shown).

Neuroanatomical structures were identified with DAPI counterstaining according to the zebrafish brain atlas [START_REF] Wullimann | Neuroanatomy of the zebrafish brain: a topological atlas[END_REF]. Cell counting was performed in blind conditions by two different people on at least 2-4 independent experiments on a total of n fish.

Brain RNA extraction and reverse transcription

Two whole mixed zebrafish (from a mixed-sex sampling zebrafish) brains were immediately removed from the skull, pooled, and stored at -808C prior to RNA extraction. Pools of brains were next grinded with TissueLyser II (Qiagen, Chatsworth, CA) for RNA extraction using TRIzol reagent (Invitrogen, France). A total of 2 lg of RNA was reverse transcribed to cDNA using random hexamer primers (Life Technologies) and M-MLV reverse transcriptase (Invitrogen).

Gene expression analysis by quantitative real-time polymerase chain reaction (qPCR)

PCR experiments were performed with the AB 7500 real-time PCR system (Applied Biosystems, Foster City, CA) using the SYBR green master-mix (Eurogentec) and specific zebrafish primers (Table 2). Each PCR cycle was conducted for 15 seconds at 958C and 1 minute at 608C. Melting curve analyses were performed to confirm correct amplification. Results were analyzed using the 7500 system SDS software (Applied Biosystems), and the relative expression of the claudin 5a, zo1a, zo1b, il1b, il6, il8, tnfa, neurod4, and nestin genes was normalized against the expression level of the ef1a gene.

Statistical analysis

Comparisons between groups were performed using a statistical Student's t-test. If more than two groups were analyzed, multiple testing was corrected by Benjamini-Hochberg. Error bars correspond to the standard error of the mean (SEM), and n values correspond to the number of animals for all experiments. P < 0.05 was considered statistically significant; *P 0.05, **P 0.01, and ***P 0.001.

RESULTS

Development of a new model of acute hyperglycemia in adult zebrafish

We first investigated the effect of acute hyperglycemia (acute HG) on brain cell proliferation. For this purpose, we established a model of acute HG by i.p. injection of D-glucose (2.5 g/kg of body weight). At 1 hour 30 minutes (1h30) post-injection (p.i.), zebrafish injected with D-glucose exhibited significantly higher

Brain cell proliferation is not impaired by acute hyperglycemia in adult zebrafish

To test our hypothesis that hyperglycemia may impact neural stem cell activity, we assessed brain proliferation after 24-hour acute HG p.i. by performing PCNA immunohistochemistry (Fig. 1). We thus focused our analysis on the ventricular regions that contain neural progenitors. We counted the number of PCNApositive cells at the junction between the olfactory bulbs (OB) and the telencephalon (TEL) (Fig. 1A, OB/ TEL); in the ventral nucleus of the ventral telencephalic area (Fig. 1B, Vv); in the dorsomedian telencephalon (Fig. 1C, Dm); in the periventricular pretectal nucleus (Fig. 1D, PPv); in the mediobasal hypothalamus, where the lateral recess starts to open (Fig. 1E, Hv LR); and in the caudal hypothalamus around the lateral and posterior recess of the diencephalic ventricle (Fig. 1F, LR PR). Acute HG did not impact neural stem cell proliferation in our experimental conditions, as further evidenced by PCNA and GFAP labeling at the OB/TEL junction, the Vv/Vd, and also in the PPv (Fig. 1G-L), even though a slight tendency for increased cell proliferation was observed in these regions except in the PPv (Fig. 1). In line with these observations, the gene expression of the neuronal marker neurod4 and the progenitors marker nestin was not altered in the brain of control (PBS) vs. acute HG fish at 24 hours p.i. (Fig. 2A).

Acute hyperglycemia induces brain inflammation

Hyperglycemia has been reported to induce the expression of proinflammatory cytokines and to increase BBB permeability in mammals (humans and rodents) [START_REF] Esposito | Inflammatory cytokine concentrations are acutely increased by hyperglycemia in humans: role of oxidative stress[END_REF][START_REF] Gyurko | Chronic hyperglycemia predisposes to exaggerated inflammatory response and leukocyte dysfunction in Akita mice[END_REF][START_REF] Hawkins | Increased blood-brain barrier permeability and altered tight junctions in experimental diabetes in the rat: contribution of hyperglycaemia and matrix metalloproteinases[END_REF][START_REF] Prasad | Diabetes mellitus and blood-brain barrier dysfunction: an overview[END_REF]. We thus tested the effects of acute HG on gene expression of inflammatory cytokines and proteins involved in the maintenance of BBB integrity. Our transcriptional analysis shows that acute HG resulted in a significant increased expression of il1b (P 5 0.0042), il8 (P 5 0.019), and tnfa (P 5 0.015) (Fig. 2B). However, we did not observe any transcriptional change in the expression of genes encoding for proteins involved in BBB integrity, namely, claudin 5a or zona occludens 1a and 1b, under acute HG conditions (Fig. 2C).

Chronic hyperglycemia impairs brain cell proliferation in adult zebrafish

We next investigated the impact of chronic hyperglycemia on brain cell proliferation. For this purpose, zebrafish were kept in fish water supplemented with Dglucose (111 mM) for 14 days. This protocol was previously shown to increase blood glucose levels from a baseline of about 3 mM (54 mg/dl) to about 12 mM (216 mg/dl) and to induce a wide variety of metabolic changes such as insulin resistance (Capiotti et al., 2014a). In our experimental conditions, after 14 days of this protocol, a significant proportion (17%) of fish displayed normal glycemia (<100 mg/dl) and were consequently excluded, while most zebrafish (83%) exhibited For acute HG experiments, adult zebrafish were intraperitoneally injected with PBS or with D-glucose. Glycemia was significantly increased in Dglucose injected fish at 1h30 p.i. compared to controls (P-value 5 0.0002), while no significant differences were observed between PBS and Dglucose-injected fish at 24h p.i. For chronic HG experiments, zebrafish were maintained in normal fish water (CTRL) while other zebrafish (chronic HG) were maintained in fish water supplemented with D-glucose (111mM) for 14 days. After the treatment, blood glucose levels are significantly higher in chronic HG fish. ***P 0.0001. n refers to the total number of fish.

Figure 1. Acute hyperglycemia does not impair brain cell proliferation. A-F: Statistical analysis of proliferative cells in control (PBS) and acute HG (AHG) zebrafish at 24 hours p.i. by performing PCNA immunohistochemistry. Schematic representations taken from the zebrafish brain atlas [START_REF] Wullimann | Neuroanatomy of the zebrafish brain: a topological atlas[END_REF] show the different regions of interest and the proliferative cells (red dots) as previously described in the literature [START_REF] Zupanc | Proliferation, migration, neuronal differentiation, and long-term survival of new cells in the adult zebrafish brain[END_REF][START_REF] Pellegrini | Identification of aromatase-positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish[END_REF]. Blue boxes highlight regions where the number of proliferative cells (PCNA-positive) was counted. No significant difference in proliferative activity was observed between PBS-and D-glucose-injected zebrafish in the OB/TEL (A), Vv (B), Dm (C), PPv (D), mediobasal hypothalamus (E), and caudal hypothalamus (F). G-L: Representative digital pictures of PCNA (red) and GFAP (green, a radial glial marker) immunohistochemistry on paraffin brain sections of PBS-and D-glucose-injected zebrafish (24 hours p.i.) counterstained with DAPI. Pictures were taken with an epifluorescence microscope. Error bars correspond to the standard error of the mean (SEM) and n refers to the total number of fish. Scale bar 5 70 lm.

significantly higher blood glucose levels (>100 mg/dl) compared to control fish (Table 3; 281.6 mg/dl 6 30.1 [n 5 24] vs. 66. 7 mg/dl 6 6.6 [n 5 21]). We then examined the effect of chronic HG on brain cell proliferation by performing PCNA immunohistochemistry in the above-described regions (Fig. 3). In contrast to acute HG, chronic HG resulted in a significant decrease in ventricular proliferation in most of the examined regions (Fig. 3). Indeed, chronic HG zebrafish displayed an almost 50% reduction in PCNA-positive cells in the anterior part of the brain, at the OB/TEL junction (Fig. 3A, A1, P 5 0.0008), in the ventral nucleus of the ventral telencephalic area (Fig. 3B, B1, P 5 0.0009), and in the dorsomedian telencephalon (Fig. 3C, C1, P 5 0.001). In more posterior regions, such as in the periventricular pretectal nucleus (PPv) localized just below the posterior commissure (Cpost), we observed a 63% reduction in the number of PCNA-positive cells in chronic HG-treated fish relative to control (Fig. 3D, D1, P 5 0.0011). In the diencephalon, including the anterior part of the preoptic area (data not shown) and the mediobasal and caudal hypothalamus (Fig. 3E, E1, 3F, F1), we observed no significant difference of proliferation between the two groups (33% decrease for Hv LR, P 5 0.08; and 24% for LR PR, P 5 0.16). Similar to the acute HG condition, neurod4 and nestin gene expression was unaltered in chronic HG fish compared to control (Fig. 4A).

Chronic hyperglycemia increased zo1a and zo1b gene expression

Expression of il1b, il6, il8, and tnfa was not modulated after a 14-day chronic hyperglycemic treatment (Fig. 4B). However, chronic HG-treated fish displayed a significant upregulation of zona occludens 1a and 1b, suggesting that chronic hyperglycemia may have an impact on the physiology of the BBB (Fig. 4C).

Chronic hyperglycemia impairs the regenerative neurogenesis after stab wounding in the adult telencephalon

In the last part of our study, we aimed at investigating the impact of acute and chronic hyperglycemia on Figure 2. Gene expression of proinflammatory cytokines and tight junctions in the brain of fish following acute hyperglycemia. Semiquantitative PCR analysis in the brain of adult zebrafish following acute hyperglycemia (24 hours p.i.). A: The neuronal and progenitor marker expression, neurod4 and nestin, is not significantly modulated at 24 hours p.i. B: Acute hyperglycemia upregulates proinflammatory cytokines gene expression of il1b, il6, il8, and tnfa at 24 hours p.i. This increase is statistically significant for il1b, il8, and tnfa (P 5 0.0042 for il1b, 0.019 for il8, 0.015 for tnfa). C: Acute hyperglycemia does not significantly modulate gene expression of claudin 5a (cl5a) or zona occludens 1a and 1b (zo1a and zo1b), encoding for proteins involved in the maintenance of the blood-brain barrier. *P 0.05, **P 0.01 and ***P 0.001. Error bars correspond to the standard error of the mean (SEM) and n refers to the total number of two pooled zebrafish brains.

Figure 3. Chronic hyperglycemia results in a significant decrease in neural progenitor proliferation. A-F: Schematic representations taken from the zebrafish brain atlas [START_REF] Wullimann | Neuroanatomy of the zebrafish brain: a topological atlas[END_REF] show the different regions of interest and the proliferative cells (red dots) as previously described in the literature. Blue boxes highlight the regions where the number of proliferative cells (PCNA-positive) was counted. A1-F1: Statistical analysis of proliferative cells (PCNA-positive) in the forebrain of control (CTRL) and CHG (D-glucose; 111 mM; 14d) zebrafish. The number of proliferative cells was decreased by around 50% at the OB/TEL junction (A1, P 5 0.0008) and in the Vv (B1, P 5 0.0009), Dm (C1, P 5 0.001), and PPv (D1, P 5 0.0011). In the hypothalamus, brain cell proliferation was also decreased, but not significantly, at the opening of the lateral recess (E1, P 5 0.0861) and around the lateral and posterior recess (F1, P 5 0.1687). The representative digital pictures of PCNA (red) immunohistochemistry staining at the OB/TEL junction, the Vv, the Dm, the PPv, the Hv LR, and the LR PR regions with DAPI (blue) counterstaining show a decrease in proliferation under chronic HG. Error bars correspond to the standard error of the mean (SEM). These data were obtained from four independent experiments, with n referring to the total number of fish. Pictures of vibratome sections were taken with an epifluorescence microscope. **P 0.01 and ***P 0.001. Scale bar 5 70 lm (H); 100 lm (L); 140 lm (F); 220 lm (B,D,J). brain cell proliferation during brain repair. For this purpose, the telencephalon was submitted to a mechanical injury, following an established procedure [START_REF] Ayari | Prokineticin 2 expression is associated with neural repair of injured adult zebrafish telencephalon[END_REF]Diotel et al., 2010a). It has been reported that at 1 day postlesion (dpl), the first proliferating cells are microglial cells and oligodendrocytes, which are localized in the brain parenchyma (M€ arz et al., 2011), whereas from 3 to 7 dpl, proliferative cells are localized along the brain ventricles and can be identified as radial glia cells that give rise to new neurons [START_REF] Schmidt | Regenerative response following stab injury in the adult zebrafish telencephalon[END_REF][START_REF] Baumgart | Stab wound injury of the zebrafish telencephalon: a model for comparative analysis of reactive gliosis[END_REF]. We thus investigated the effects of acute hyperglycemia on proliferation in the brain parenchyma and the effects of chronic hyperglycemia on proliferation along the ventricular layer. In both normoglycemic and acute HG conditions (24 hours p.i. and 1 dpl), the number of PCNA-positive cells increased by almost two times in the injured telencephalon (Fig. 5A,C). This observation indicates that acute HG does not impair the initial step of parenchymal brain cell proliferation.

We next analyzed the effect of chronic HG on the proliferation of ventricular cells mainly corresponding to radial glial cells (type 1 and 2 progenitors; type 1 corresponding to quiescent radial glia and type 2 to actively dividing radial glia). Therefore, we stab wounded fish after 7 days of chronic HG and allowed them to survive for 1 week in chronic HG conditions. In control and chronic HG conditions, we observed a significant increase in cell proliferation in the ipsilateral hemisphere compared to the contralateral one (Fig. 5B,D). Interestingly, injury-induced ventricular proliferation was significantly reduced in chronic HG-treated fish compared to control fish (Fig. 5B,D; P 5 0.0127). Consequently, our results clearly show that hyperglycemia impairs recruitment and/or activation of radial glial cells following stab wound injury.

DISCUSSION

In the central nervous system, dysglycemia is known for impairing synaptic transmission, neural plasticity, and cognition (Mooradian, 1997a,b;[START_REF] Kodl | Cognitive dysfunction and diabetes mellitus[END_REF][START_REF] Wang | Effects of acute and chronic hyperglycemia on the neurochemical profiles in the rat brain with streptozotocin-induced diabetes detected using in vivo (1)H MR spectroscopy at 9.4 T[END_REF][START_REF] Ho | Effects of diabetes on hippocampal neurogenesis: links to cognition and depression[END_REF][START_REF] Prasad | Diabetes mellitus and blood-brain barrier dysfunction: an overview[END_REF]. It has been reported that more than 50% of patients admitted for ischemic stroke experience hyperglycemia [START_REF] Scott | Prevalence of admission hyperglycaemia across clinical subtypes of acute stroke[END_REF][START_REF] Capes | Stress hyperglycemia and prognosis of stroke in nondiabetic and diabetic patients: a systematic overview[END_REF], whether due to stress or type 2 diabetes, and that hyperglycemia is associated with poor clinical outcome after thrombolysis [START_REF] Desilles | Diabetes mellitus, admission glucose, and outcomes after stroke thrombolysis: a registry and systematic review[END_REF].

In the present study, we investigated the impact of acute and chronic hyperglycemia on adult zebrafish neurogenesis and brain homeostasis. Our results showed that brain homeostasis was differently affected in acute HG and chronic HG conditions. Indeed, acute HG led to brain Figure 4. Proinflammatory cytokines and BBB gene expression following chronic hyperglycemia. Semiquantitative PCR analysis in the brain of adult zebrafish following chronic hyperglycemia (Dglucose; 111 mM; 14d). A,B: Chronic hyperglycemia treatment did not modulate expression of the neuronal and progenitor markers, neurod4 and nestin, respectively, or those of the proinflammatory cytokines il1b, il6, il8, and tnfa. C: Chronic hyperglycemia treatment results in a significant upregulation of zona occludens 1a and 1b (zo1a and zo1b, P 5 0.006 and 0.007, respectively), involved in the maintenance of the BBB. Although claudin 5a gene expression is not significantly modulated, it shows an increasing trend. **P 0.01. Error bars correspond to the standard error of the mean (SEM) and n refers to the total number of two pooled zebrafish brains. inflammation without impairing adult neurogenesis, whereas chronic HG resulted in a significant decrease in cell proliferation along the brain ventricle, where neural stem cells and further committed progenitors are localized. Finally, we showed that chronic HG markedly reduced neurogenesis following stab wound injury in the brain of adult zebrafish.

Hyperglycemia

As shown in our experimental conditions, both acute i.p. injection and chronic exposure to D-glucose in the fish water induced hyperglycemia (Table 3). We observed significantly higher blood glucose levels for acute HG zebrafish 1h30 after D-glucose injection compared to control fish (Table 3). We also observed an increasing trend in blood sugar level in PBS-injected fish at 1h30 p.i. compared to baseline glycemia, which could be attributed to stress induced by the injection and/or the use of the anesthetic that is known to induce a wide variation of blood glucose levels [START_REF] Chavin | Factors in the determination of normal serum glucose levels of goldfish, Carassius auratus L[END_REF][START_REF] Eames | Blood sugar measurement in zebrafish reveals dynamics of glucose homeostasis[END_REF]. As expected, at 24 hours p.i., D-glucose-injected fish displayed normoglycemic values (Table 3). Therefore, blood glucose levels appear to be dynamically regulated in zebrafish, as previously shown [START_REF] Chavin | Factors in the determination of normal serum glucose levels of goldfish, Carassius auratus L[END_REF][START_REF] Eames | Blood sugar measurement in zebrafish reveals dynamics of glucose homeostasis[END_REF].

To our knowledge, this is the first description of an acute hyperglycemia model in zebrafish. We also validated the chronic HG model, as zebrafish immersed in Dglucose water (111 mM for 14 days) became hyperglycemic. This is consistent with previous studies from Capiotti et al. (2014) showing an increase in blood glucose level that correlates with the time of immersion in Dglucose supplemented water (Capiotti et al., 2014a). Under such conditions, it was found that hyperglycemia had already been induced starting from 3 days of treatment (Capiotti et al., 2014a). Interestingly, i.p. D-glucose injection induced a peak of hyperglycemia at 500 mg/ dL, whereas acute exposure to D-glucose led to a glycemia of 280 mg/dl. Taken together, these data further strengthen the utility of our procedures as relevant models for studying acute HG and chronic HG. Importantly, we noticed that, in some experiments, not all the fish displayed hyperglycemia after the chronic immersion in D-glucose (data not shown; 17% nonhyperglycemic vs. 83% hyperglycemic). This might be due to different susceptibilities of the individual fish to hyperglycemia according to age or compensatory mechanisms [START_REF] Sarras | Use of zebrafish as a disease model provides a unique window for understanding the molecular basis of diabetic metabolic memory[END_REF][START_REF] Connaughton | Alternate immersion in an external glucose solution differentially affects blood sugar values in older versus younger zebrafish adults[END_REF].

Inflammation and BBB

In the brain of acute HG fish, we noticed an increase in proinflammatory cytokine expression (il1b, il6, il8, and tnfa), reaching statistical significance for il1b, il8, and tnfa (Fig. 2B). Such proinflammatory effects of hyperglycemia are already well-characterized in mammals in a wide variety of tissues, including tissues of the central nervous system [START_REF] Esposito | Inflammatory cytokine concentrations are acutely increased by hyperglycemia in humans: role of oxidative stress[END_REF][START_REF] Lin | The hyperglycemia-induced inflammatory response in adipocytes: the role of reactive oxygen species[END_REF][START_REF] Gyurko | Chronic hyperglycemia predisposes to exaggerated inflammatory response and leukocyte dysfunction in Akita mice[END_REF]. Proinflammatory cytokines are important mediators of the early immune response and display pleiotropic effects such as promoting immune cell proliferation [START_REF] Randelli | Cell markers and determinants in fish immunology[END_REF] and modulating neuronal survival and neurogenesis [START_REF] Fuster-Matanzo | Role of neuroinflammation in adult neurogenesis and Alzheimer disease: therapeutic approaches[END_REF][START_REF] Tobin | Neurogenesis and inflammation after ischemic stroke: what is known and where we go from here[END_REF][START_REF] Borsini | The role of inflammatory cytokines as key modulators of neurogenesis[END_REF]. In our experimental conditions, proinflammatory cytokine gene expression was unchanged in the brain of chronic HG zebrafish after 14 days (Fig. 4A). This finding does not preclude the possibility that the expression of these genes is modulated within the first days of chronic HG, but that an adaptive response may take place to counteract chronic inflammation, particularly in light of the zebrafish's high capacity for adaptation and regeneration [START_REF] Randelli | Cell markers and determinants in fish immunology[END_REF]. Such presumed compensatory mechanisms will require further investigation.

Regarding the BBB, claudin 5a and zona occludens 1a or 1b gene expression was not modulated under acute HG at 24 hours p.i. (Fig. 2B), but zona occludens 1a and 1b gene expression was significantly upregulated under chronic HG (Fig. 4B). Our results show that chronic hyperglycemia affects the expression of genes involved in BBB establishment. However, our attempts at testing for BBB leakage using Evans blue dye were not conclusive due to a wide variability in staining (data not shown). Numerous in vitro and in vivo studies have demonstrated that hyperglycemia increases BBB leakage by downregulating tight junction protein expression (e.g., occludin and zona occludens-1) [START_REF] Chehade | Diabetes-related changes in rat cerebral occludin and zonula occludens-1 (ZO-1) expression[END_REF][START_REF] Hawkins | Increased blood-brain barrier permeability and altered tight junctions in experimental diabetes in the rat: contribution of hyperglycaemia and matrix metalloproteinases[END_REF][START_REF] Ergul | Hyperglycemia, diabetes and stroke: focus on the cerebrovasculature[END_REF][START_REF] Chao | Hyperglycemia increases the production of amyloid beta-peptide leading to decreased endothelial tight junction[END_REF]. Indeed, type 2 diabetes rats were reported to display an impaired hippocampal BBB [START_REF] Yoo | Chronic type 2 diabetes reduces the integrity of the blood-brain barrier by reducing tight junction proteins in the hippocampus[END_REF]. In zebrafish, the blood-retinal barrier was equally impacted by hyperglycemia during development, as shown by abnormalities in blood vessels morphology and decreased levels of ZO-1 protein expression [START_REF] Alvarez | Predominant cone photoreceptor dysfunction in a hyperglycaemic model of nonproliferative diabetic retinopathy[END_REF][START_REF] Jung | High glucose-induced changes in hyaloid-retinal vessels during early ocular development of zebrafish: a short-term animal model of diabetic retinopathy[END_REF]. We hypothesized that the zona occludens 1a and 1b upregulation could be due to compensatory mechanisms in response to BBB aggression.

Neurogenesis

In our experiments, acute HG did not impair neural stem cell proliferation in the different brain regions or nuclei studied (Fig. 1); however, chronic HG-treated fish exhibited a significant decrease in ventricular proliferation in all regions studied (OB/TEL, Vv, Dm, PPv) except the mediobasal (Hv LR) and caudal hypothalamus (LR PR). Nevertheless, the hypothalamus displays a consistent decrease in cell proliferation (Fig. 3). Our observations are in line with previous reports in mammals showing that type 2 diabetes-associated oxidative stress and/or inflammation inhibit hippocampal proliferation and neuronal migration and promote neuronal death [START_REF] Beauquis | Reduced hippocampal neurogenesis and number of hilar neurones in streptozotocin-induced diabetic mice: reversion by antidepressant treatment[END_REF][START_REF] Wiltrout | Repairing brain after stroke: a review on post-ischemic neurogenesis[END_REF][START_REF] Jakubs | Inflammation regulates functional integration of neurons born in adult brain[END_REF][START_REF] Stranahan | Diabetes impairs hippocampal function through glucocorticoid-mediated effects on new and mature neurons[END_REF][START_REF] Yi | Effects of treadmill exercise on cell proliferation and differentiation in the subgranular zone of the dentate gyrus in a rat model of type II diabetes[END_REF][START_REF] Ho | Effects of diabetes on hippocampal neurogenesis: links to cognition and depression[END_REF]. High glucose levels were also shown to induce deleterious effects on embryonic quail neurogenesis, by decreasing neurite length and neuronal migration, and by increasing reactive oxidative species generation [START_REF] Chen | The negative influence of high-glucose ambience on neurogenesis in developing quail embryos[END_REF]. In both acute HG and chronic HG conditions, neurod4 and nestin gene expression remained unchanged, suggesting no influence of either acute or chronic hyperglycemia on neuronal differentiation and neural progenitor levels (Figs. 2A,4A). However, further investigations should elucidate how hyperglycemia impacts neurogenic processes.

Brain regeneration in zebrafish has been well characterized. It starts with an initial parenchymal proliferation of microglial cells and oligodendrocytes (day 1 to 3) that is followed by a highly sustained proliferation of radial glial cells in the ventricular layer from 3 days post-injury, allowing the production of new neurons [START_REF] Kaslin | Proliferation, neurogenesis and regeneration in the non-mammalian vertebrate brain[END_REF][START_REF] Kroehne | Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors[END_REF][START_REF] Schmidt | Regenerative response following stab injury in the adult zebrafish telencephalon[END_REF][START_REF] Diotel | Effects of estradiol in adult neurogenesis and brain repair in zebrafish[END_REF][START_REF] Alunni | A comparative view of regenerative neurogenesis in vertebrates[END_REF]. By performing stab wound injury of the telencephalon, we showed that acute HG did not impair the initial step of parenchymal brain cell proliferation but that chronic HG disrupted the ventricular cell proliferation at 7 days post-injury. Such a deleterious effect of hyperglycemia in regenerative mechanisms is consistent with the wellknown complications of type 2 diabetes relating to impaired wound healing [START_REF] Rosenberg | Wound healing in the patient with diabetes mellitus[END_REF][START_REF] Lioupis | Effects of diabetes mellitus on wound healing: an update[END_REF]. In zebrafish, hyperglycemia was also shown to affect the healing process during fin regeneration [START_REF] Olsen | Limb regeneration is impaired in an adult zebrafish model of diabetes mellitus[END_REF]. Additionally, numerous studies have documented the adverse effects of hyperglycemia/diabetes on brain stoke and traumatic brain injury [START_REF] Cherian | Hyperglycemia increases brain injury caused by secondary ischemia after cortical impact injury in rats[END_REF][START_REF] Huang | Acute hyperglycemia worsens ischemic stroke-induced brain damage via high mobility group box-1 in rats[END_REF][START_REF] Jauch-Chara | Glycemic control after brain injury: boon and bane for the brain[END_REF].

It is interesting to note that diabetes is also known for impairing brain memory [START_REF] Whitmer | Type 2 diabetes and risk of cognitive impairment and dementia[END_REF][START_REF] Ho | Effects of diabetes on hippocampal neurogenesis: links to cognition and depression[END_REF]. Recently, using the same experimental conditions, Capiotti and colleagues demonstrated that hyperglycemia induces memory impairment in adult zebrafish (Capiotti et al., 2014b), similar to the mammalian situation. Taken together, these data demonstrate that hyperglycemia in zebrafish impairs brain functions such as neurogenesis and memory.

Such adverse effects of hyperglycemia on brain cell proliferation have probably multiple direct and/or indirect origins. By increasing brain inflammation and by disrupting the BBB, hyperglycemia could favor the central effects of peripheral factors such as proinflammatory cytokines and adipokines that have been documented to disrupt neural stem cell activity [START_REF] Arnoldussen | Obesity and dementia: adipokines interact with the brain[END_REF][START_REF] Letra | Obesity as a risk factor for Alzheimer's disease: the role of adipocytokines[END_REF][START_REF] Parimisetty | Secret talk between adipose tissue and central nervous system via secreted factors-an emerging frontier in the neurodegenerative research[END_REF]. Hyperglycemia is also known for inducing an increase in advanced glycation endproducts that is correlated with decreased hippocampal neurogenesis in STZ-induced diabetic rats [START_REF] Ho | Effects of diabetes on hippocampal neurogenesis: links to cognition and depression[END_REF]. Variation of insulin and/or IGF-1 levels resulting from hyperglycemia could also be factors modulating the activity of neural stem cells [START_REF] Rafalski | Energy metabolism in adult neural stem cell fate[END_REF][START_REF] Ziegler | Insulin and IGF receptor signalling in neural-stem-cell homeostasis[END_REF]. Taken together, these data highlight the necessity of further investigations in order to understand the mechanistic behind the decreased proliferation observed in our model.

CONCLUSION

In the present study, we demonstrate that hyperglycemia disturbs brain homeostasis, influencing brain inflammation and neurogenesis in homeostatic and in injury-induced conditions. Taken together, our data and those reported by Capiotti and colleagues highlight an evolutionary-conserved impact of hyperglycemia on brain homeostasis and plasticity. Finally, this work confirms the value of the zebrafish model in studying the effects of hyperglycemia on the central nervous system, particularly in pathological situations such as stroke.

Figure 3 .

 3 Figure 3. Continued.

Figure 5 .

 5 Figure5. Chronic hyperglycemia impairs brain cell proliferation after stab wounding injury of the telencephalon. A: Fold induction of PCNApositive cells (ipsilateral vs. contralateral) in PBS-and D-glucose-(2.5 g/kg of body weight) injected fish at 24 hours post-injection (p.i.) and 1 day post-lesion (dpl). B: Fold induction of PCNA-positive cells (ipsilateral vs. contralateral) in control fish and chronic HG-treated fish (111 mM D-glucose; 14 days) with stab wounding at day 7. Ventricular cell proliferation induction after stab wounding is significantly decreased in chronic HG-treated fish (P 5 0.0127). C,D: PCNA immunohistochemistry (green) and DAPI counterstaining (blue) on vibratome brain sections of 24 hours stab-wounded telencephalon of PBS-and D-glucose-(2.5 g/kg of body weight) injected fish (C), and of chronic HG-treated fish (111 mM D-glucose; 14 days) stab-wounded at 7 days of treatment and allowed to survive for 7 days in chronic HG conditions. A decrease in ventricular cell proliferation is observed under chronic HG conditions. Pictures were taken with an epifluorescence microscope. *P 0.05. Error bars correspond to the standard error of the mean (SEM) and n refers to the total number of fish. Scale bar 5 220 lm.

TABLE 1 .

 1 Primary and Secondary Antibodies

	Primary antibodies

TABLE 2

 2

	.
	qRT-PCR Primers

blood glucose levels compared to the controls injected with the PBS vehicle only (Table

3

; 502.8 mg/dl 6 33.8 vs. 149.8 mg/dl 6 27.8; n 5 4; P < 0.0002; n 5 4). Importantly, blood glucose levels were similar between the two groups at 24 hours p.i. (Table

3

; 97.8 mg/dl 6 16.3 vs. 94.2 mg/dl 6 13.1; n 5 21 for PBS and n 5 26 for acute HG), showing that hyperglycemia was transient.

TABLE 3 .

 3 Blood Glucose Measurement in the Acute and Chronic Hyperglycemic Models

		Blood glucose levels (mg/dl)
		1h 30 p.i.	24h p.i.
	Acute HG	(2.5g D-glucose/kg of body weight)	(2.5g D-glucose/kg of body weight)
	PBS	149.8 6 27.8 (n 5 4)	97.8 6 16.3 (n 5 21)
	AHG	502.8 6 33.8 (n 5 4)***	94.2 6 13.1 (n 5 26)
	Chronic HG	Blood glucose levels (mg/dl) (14 days in 111mM D-glucose)
	CTRL		66.7 6 6.6 (n 5 21)
	CHG		281.6 6 30.1 (n 5 24)***

ACKNOWLEDGMENTS

We thank Dr. Wildriss Viranaicken for help in providing reagents and advice, and Janice Tail e for technical support. We thank Dr. Benjamin Weger for scientific discussion and critically reading the article.

Grant sponsor: La R eunion University (Bonus Qualit e Recherche, Dispositifs incitatifs); Grant sponsor: Conseil R egional de La R eunion, European Union (CPER/FEDER); Grant sponsor: Philancia Association; Grant sponsor: Ministe `re de l'Education Nationale, de l'Enseignement Sup erieur et de la Recherche, La R eunion University (Contrat Doctoral) (fellowship grant to A.C.D.).

CONFLICT OF INTEREST

The authors declare that they have no competing interest.

AUTHOR CONTRIBUTIONS

Conception and design of the experiments: ND, OM, CLDH, ACD, MW, EB. Acquisition of data: ACD, ND, SS. Analysis of the data: ND, ACD, SS, CLDH, OM, MW, EB.


Anne-Claire Dorsemans

Christian Lefebvre D'hellencourt

Imade Ait-Arsa

Emmanuelle Jestin

Olivier Meilhac

Nicolas Diotel

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological problems after stroke and is shown to impair neurogenic processes. Interestingly, the adult zebrafish has recently emerged as a relevant and useful model to mimic hyperglycemia/ diabetes and to investigate constitutive and regenerative neurogenesis. This work provides methods to develop zebrafish models of hyperglycemia to explore the impact of hyperglycemia on brain cell proliferation under homeostatic and brain repair conditions. Acute hyperglycemia is established using the intraperitoneal injection of D-glucose (2.5 g/kg bodyweight) into adult zebrafish. Chronic hyperglycemia is induced by immersing adult zebrafish in D-glucose (111 mM) containing water for 14 days. Blood-glucose-level measurements are described for these different approaches. Methods to investigate the impact of hyperglycemia on constitutive and regenerative neurogenesis, by describing the mechanical injury of the telencephalon, dissecting the brain, paraffin embedding and sectioning with a microtome, and performing immunohistochemistry procedures, are demonstrated. Finally, the method of using zebrafish as a relevant model for studying the biodistribution of radiolabeled molecules (here,[

18 F]-FDG) using PET/CT is also described.

Introduction

Hyperglycemia is defined as excessive blood glucose levels. Although it could reflect a situation of acute stress, hyperglycemia is also a condition that often leads to a diagnosis of diabetes, a chronic disorder of insulin secretion and/or resistance. In 2016, the number of adults living with diabetes has reached 422 million worldwide, and each year, 1.5 million people die from this disease, making it a major health problem 1 . Indeed, uncontrolled diabetes leads to several physiological disorders affecting the cardiovascular system, kidneys, and the peripheral and central nervous systems.

Interestingly, acute and chronic hyperglycemia may alter cognition and contribute to both dementia and depression 2,3,4,5,6

. In addition, the admission of patients with hyperglycemia has been associated with worse functional, neurological, and survival outcomes after ischemic stroke [START_REF] Capes | Stress hyperglycemia and prognosis of stroke in nondiabetic and diabetic patients: a systematic overview[END_REF][START_REF] Stead | Hyperglycemia as an independent predictor of worse outcome in non-diabetic patients presenting with acute ischemic stroke[END_REF][START_REF] Kagansky | The role of hyperglycemia in acute stroke[END_REF][START_REF] Gilmore | The role of hyperglycemia in acute ischemic stroke[END_REF][START_REF] Desilles | Diabetes mellitus, admission glucose, and outcomes after stroke thrombolysis: a registry and systematic review[END_REF] . It was also shown that hyperglycemia/diabetes affect adult neurogenesis, a process leading to the generation of new neurons, by impacting neural stem cell activity and neuronal differentiation, migration, and survival 2,[START_REF] Dorsemans | Impaired constitutive and regenerative neurogenesis in adult hyperglycemic zebrafish[END_REF] .

In contrast to mammals, teleost fish, like zebrafish, display intense neurogenic activity throughout the whole brain and exhibit an outstanding capacity for brain repair during adulthood [START_REF] Schmidt | Neurogenesis in zebrafish -from embryo to adult[END_REF][START_REF] Kizil | Adult neurogenesis and brain regeneration in zebrafish[END_REF][START_REF] Grandel | Comparative aspects of adult neural stem cell activity in vertebrates[END_REF][START_REF] Lindsey | A comparative framework for understanding the biological principles of adult neurogenesis[END_REF] . Notably, such capacities are possible due to the persistence of neural stem/progenitor cells, including radial glia and neuroblasts [START_REF]Heterogeneity in progenitor cell subtypes in the ventricular zone of the zebrafish adult telencephalon[END_REF][START_REF] Chapouton | Adult neurogenesis in non-mammalian vertebrates[END_REF][START_REF] Lindsey | The cellular composition of neurogenic periventricular zones in the adult zebrafish forebrain[END_REF] . In addition, the zebrafish has recently emerged as a model for studying metabolic disorders, including obesity and hyperglycemia/diabetes [START_REF] Sarras | Use of Zebrafish as a Disease Model Provides a Unique Window For Understanding the Molecular Basis of Diabetic Metabolic Memory[END_REF][START_REF] Oka | Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity[END_REF][START_REF] Capiotti | Persistent impaired glucose metabolism in a zebrafish hyperglycemia model[END_REF] .

Although the zebrafish is a well-recognized model of hyperglycemia and neurogenesis, few studies have investigated the impact of hyperglycemia on brain homeostasis and cognitive function [START_REF] Dorsemans | Impaired constitutive and regenerative neurogenesis in adult hyperglycemic zebrafish[END_REF][START_REF] Capiotti | Hyperglycemia induces memory impairment linked to increased acetylcholinesterase activity in zebrafish (Danio rerio)[END_REF] . To determine the impact of hyperglycemia on constitutive and injury-induced brain cell proliferation, a model of acute hyperglycemia was created through the intraperitoneal injection of D-glucose. In addition, a model of chronic hyperglycemia was reproduced through the immersion of fish in water supplemented with D-glucose 12 . Zebrafish exhibit many advantages in research. They are cheap, easy to raise, and transparent during the first stages of development, and their genome has been sequenced. In the context of this work, they also display several additional advantages: (1) they share similar physiological processes with humans, making them a critical tool for biomedical research; (2) they allow for the quick investigation of the impact of hyperglycemia on brain homeostasis and neurogenesis, given their widespread and strong neurogenic activity; and (3) they are an alternative model, allowing for the reduction of the number of mammals used in research. Finally, zebrafish can be used as a model for testing the biodistribution of radiolabeled molecules and potential therapeutic agents using PET/CT. The overall goal of the following procedure is to visually document how to set up models of acute and chronic hyperglycemia in zebrafish, use zebrafish to assess brain remodeling in hyperglycemic conditions, and monitor radiolabeled molecules (here, [ 18 F]-FDG) using PET/CT.

Protocol

Adult wildtype zebrafish (Danio rerio) were maintained under standard photoperiod (14/10 h light/dark) and temperature (28 °C) conditions. All experiments were conducted in accordance with the French and European Community Guidelines for the Use of Animals in Research (86/609/ EEC and 2010/63/EU) and were approved by the local Ethics Committee for animal experimentation.

Establishing a model of acute hyperglycemia in zebrafish

1. Prepare a stock solution of tricaine (MS-222) by dissolving 400 mg of tricaine powder in 97.9 mL of water and 2.1 mL of 1 M Tris/HCl buffer (pH 9). Adjust the pH to 7 and aliquot for storage at -20 °C. 2. Prepare an anesthetic for the adult zebrafish by putting 5 mL of tricaine (stock solution) into 100 mL of fish water (final tricaine concentration: 0.02%). 3. Transfer one zebrafish (3 to 6 months) from its tank to the anesthetic until it stops moving. 4. Remove the fish using a cut pipette and quickly dry it on absorbent tissue paper. 5. Weigh the fish. 6. Prepare a syringe of D-glucose dissolved in 1X PBS and inject 50 µL of D-glucose (2.5 g/kg bodyweight). NOTE: For instance, a 0.6 g fish will receive 50 µL of 3% D-glucose/PBS solution. 7. Put the fish on its back and insert the needle of the syringe into the intraperitoneal cavity. 8. Slowly inject the D-glucose/PBS solution and then place the fish back in the water. 9. Check on the fish until it recovers completely. Return it to its tank until the required time for performing the blood glucose measurement is reached. NOTE: The blood glucose is measured 1.5 h after the injection.

Establishing a model of chronic hyperglycemia in zebrafish

1. Prepare a 2-L tank of clean fish water. 2. Dissolve 40 g of D-glucose into the 2 L of fish water for a final D-glucose concentration of 111 mM. 3. Immerse 5 to 7 adult zebrafish in the D-glucose-containing water. 4. Replace the D-glucose fish water every 2 days in order to avoid the growth of bacteria or other micro-organisms. 5. After 14 days of treatment, place the fish briefly in fresh water to remove the D-glucose outside the body prior to taking the blood glucoselevel measurement.

Measuring blood glucose levels in zebrafish

1. Cover the fish with ice for rapid euthanasia. NOTE: Do not use an overdose of tricaine, as this induces wide variations in blood glucose levels. Do not leave the fish in the ice for too long, as this will cause the blood to clot. 2. Wipe the fish with absorbent tissue paper to remove all water and to avoid any blood dilution during the blood glucose measurement. 3. Remove an eye using dissecting forceps and wait until the eye cavity is filled with blood. 4. Put a test strip on a glucometer and insert the strip into the eye cavity. 5. Measure the blood glucose levels. 3. Sample preparation for immunohistochemistry: tissue preparation for paraffin embedding 1. Place the brains in a small glass beaker. NOTE: Do not use a plastic receptacle, as toluene can damage some plastics. 2. Remove the ethanol and replace it with toluene, as the brains must be totally recovered by toluene. Perform two 30-min baths of toluene. 3. Remove the toluene and place the brains in an embedding cassette. Put the closed cassette in melted paraffin series beakers at 58-60 °C (30 min in each paraffin beaker). Turn on the warming inclusion forceps. At the end of the paraffin baths, take out the cassette and pour some liquid paraffin into a mold. 4. Pour the melted paraffin into a mold and place the brain inside. Orient the brain using the warming inclusion forceps, using the anteroposterior orientation of the brain as a guide. Let the paraffin harden on the cooling part of the embedding machine. 5. For technical reasons, position the brains along the anteroposterior axis. After unmolding the paraffin block, crop it and fix it on a cassette, with the melted paraffin in the proper orientation to allow for transversal sectioning. 6. Insert the paraffin block into the arm of the microtome. Cut 50 µm-thick sections until the brain sample level is reached. Trim the paraffin block to obtain a trapeze and adjust the sectioning thickness to 7 µm. Collect the paraffin ribbons using paintbrushes and place them on black paper. Cut them gently every 3 to 4 sections. 7. Put a slide on a warming plate and cover it with dH 2 O water. 8. Gently position the cut ribbons on the water. Remove the water with absorbent tissue paper when the paraffin ribbons are sufficiently spread out/unfolded. Remove the last drops mechanically. Remove the water from the slides and let them dry for at least 3 h on the warming plate at around 30-40 °C.

Analyzing brain cell proliferation following hyperglycemia

4. Immunohistochemistry procedure 1. Remove the paraffin wax by placing sections in three containers of xylene for 7 min each.

2. Rehydrate the sections by putting the slides in two containers of 100% EtOH for 2 min each, followed by baths of 95% EtOH, 85% EtOH, 70% EtOH, and 30% EtOH for 30 s each. Finally, briefly put the slides in dH 2 O and twice in PBS for 5 min each. 3. Perform antigen retrieval by incubating the sections in citrate buffer in a microwave (2 min at 500 W) until the buffer starts to boil. Let the slides recover at room temperature for 15 min. 4. Wash three times in PBST, 5 min each, and block the sections for 45 min in PBST containing 1% milk. Incubate overnight with primary antibodies (e.g. proliferating cell nuclear antigen (PCNA) for proliferative cell staining; 1/100) diluted in blocking buffer (i.e. PBST, 1% milk). 5. Wash three times in PBST, 5 min each, and incubate the sections for 90 min with appropriate secondary antibodies (e.g. goat antimouse Alexa Fluor 488; 1/200) diluted in blocking buffer and with DAPI (1/500). 6. Wash three times in PBST, 5 min each, and mount the slides with fluorescent mounting medium (see the table of materials). 7. Analyze the staining using an epifluorescence and/or confocal microscope. 8. Quantify the brain cell proliferation on at least three successive brain sections of a region of interest in at least three distinct animals. NOTE: Alternatively, brain embedding can be done in agarose to proceed to vibratome sectioning and free-floating immunohistochemistry, as previously described 24 .

5. Studying the impact of hyperglycemia on brain repair mechanisms NOTE: Alternatively, the investigation of brain repair under acute and chronic hyperglycemia can be performed after stab wound injury of the telencephalon, as previously described [START_REF] Schmidt | Stab wound injury of the zebrafish adult telencephalon: a method to investigate vertebrate brain neurogenesis and regeneration[END_REF][START_REF] Diotel | Effects of estradiol in adult neurogenesis and brain repair in zebrafish[END_REF][START_REF] Rodriguez Viales | The helix-loop-helix protein id1 controls stem cell proliferation during regenerative neurogenesis in the adult zebrafish telencephalon[END_REF] . Briefly: 1. Anesthetize adult zebrafish with tricaine. 2. Place the fish under a dissecting microscope with light. 3. Hold the fish with one hand. 4. With the other hand, insert a 30G syringe vertically through the skull into the medial region of the right telencephalic hemisphere. 5. Place the fish back into fresh fish water, D-glucose-supplemented water (111 mM), or control fish water. Allow the fish to survive for 7 days post-injury. NOTE: Refer to step 4 for the rest of the procedure.

Imaging the biodistribution of radiolabeled molecules by PET/CT in zebrafish: Fluorodeoxyglucose ([18F]-FDG) to analyze glucose metabolism

, chronic hyperglycemia induced a significant decrease in neural stem cell proliferation along the ventricle, as previously shown by Dorsemans and colleagues (2016). Indeed, the number of PCNA-positive cells was reduced in the subpallium (Vv/Vd), the pallium (Dm), and the regions surrounding the lateral and posterior recess of the caudal hypothalamus (LR/PR) (Figure 2).

Injury-induced neurogenesis was also studied after the mechanical injury of the telencephalon under acute and chronic hyperglycemia. As previously described after brain injury in zebrafish, a first parenchymal proliferation of microglial cells and oligodendrocytes occurred, followed by a strong up-regulation of proliferation at the ventricular layer 7 days after the injury [START_REF] Diotel | Effects of estradiol in adult neurogenesis and brain repair in zebrafish[END_REF][START_REF] Kaslin | Proliferation, neurogenesis and regeneration in the non-mammalian vertebrate brain[END_REF][START_REF] Kroehne | Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors[END_REF][START_REF] Alunni | A comparative view of regenerative neurogenesis in vertebrates[END_REF][START_REF] Schmidt | Regenerative response following stab injury in the adult zebrafish telencephalon[END_REF] . Acute hyperglycemia did not modulate the initial step of proliferation in the brain parenchyma. In contrast, chronic hyperglycemia impaired brain cell proliferation along the telencephalic ventricles 7 days after injury (Figure 3).

The zebrafish model is also interesting for monitoring the biodistribution of radiolabeled molecules using PET/CT imaging. Here, [18F]-FDG was intraperitoneally injected into adult zebrafish. After 30 min, PET/CT acquisition shows that the glucose is distributed not only at the site of injection, but also in the head of the fish, including the brain, and along the spinal cord (Figure 4).

Discussion

This work describes various methods to establish acute and chronic models of hyperglycemia in zebrafish. The main advantages of these procedures are that: (1) they allow for a reduction in the number of mammals used for research, (2) they are simple to set up and quick to implement, and (3) they are economical. Therefore, such models allow for the investigation of the impact of hyperglycemia on a large number of animals to study its impact on different physiological processes, including atherothrombosis, cardiovascular dysfunctions, retinopathies, bloodbrain barrier leakage, and constitutive and regenerative neurogenesis. This work describes how to proceed with investigations on the effects of hyperglycemia on brain cell proliferation under normal or injury-induced conditions.

One critical limitation of the chronic hyperglycemia procedure is that, in some experiments, some fish do not display hyperglycemia after the chronic immersion in D-glucose water (111 mM for 14 days). The percentage of responsive and non-responsive fish has been previously estimated by Dorsemans and colleagues (2016) to be 83% versus 17%, respectively. It is possible that fish display individual susceptibilities according to their age, sex, and capacity to compensate for hyperglycemia by making more pancreatic β-cells [START_REF] Sarras | Use of Zebrafish as a Disease Model Provides a Unique Window For Understanding the Molecular Basis of Diabetic Metabolic Memory[END_REF][START_REF] Connaughton | Alternate Immersion in an External Glucose Solution Differentially Affects Blood Sugar Values in Older Versus Younger Zebrafish Adults[END_REF] . For acute hyperglycemia, the blood glucose levels are quite homogeneous 1.5 h after the injection, demonstrating the robustness of the method.

A critical step of this procedure concerns blood-glucose-level measurements. The quantity of blood that fills the eye cavity is, in rare cases, too low to allow for the loading of the glucometer test strip. In addition, the fish should not stay on ice for too long in order to avoid blood coagulation. However, they should remain on ice for a time sufficient to ensure the induction of anesthesia and the death of the animals. It is also important to mention that, for acute hyperglycemia, the volume of D-glucose injected should be altered to account for the size of the fish. The 50-µL intraperitoneal injection is designed for a medium-sized fish (0.5 g). Indeed, a small fish might not be able to receive a 50-µL intraperitoneal injection and the volume of injection must be reduced to prevent animal suffering and to avoid solution being pushed straight back out by pressure.

Another critical step is the reproducibility of the stab wound injury of the adult telencephalon; which requires some technical experience. Additionally, counting should be performed on three successive sections of a region of interest and in at least three animals. Automated counting in larger brain areas can reveal important information concerning the global effect of hyperglycemia on the process of neurogenesis.

Another reason to use zebrafish is for the ability monitor the biodistribution of radiolabeled molecules using PET/CT. Here, [18F]-FDG was used, and its distribution throughout the zebrafish body was demonstrated, notably including the brain and the spinal cord. Such techniques are of particular interest when determining the delivery and bioaccumulation of potential therapeutic agents in in vivo models. This technique also represents an alternative method to investigate the ability of some molecules to cross through the blood-brain barrier and to determine their potential effects on the central nervous system under physiological or pathophysiological conditions. Indeed, hyperglycemia and hypoglycemia are known to modulate blood-brain barrier permeability 36 .

One critical limitation of PET/CT imaging in zebrafish after intraperitoneal injection is the necessity to anesthetize the fish in order to avoid any movement during the acquisition. Such anesthesia could strongly reduce the heart rate and therefore the radiotracer biodistribution. To solve this problem, fish can be injected and allowed to recover in fresh water for a few minutes or hours, depending the imaging protocol and the half-life of the radioisotope used. In addition, the intraperitoneal injection could result in the strong accumulation of signal in the peritoneal cavity.

To conclude, this work described efficient methods to establish models of hyperglycemia in zebrafish and to monitor radiolabeled-molecule distribution. Such approaches could open a field of research relating to the investigation of the impact of metabolic disorders on brain homeostasis and on the biodistribution of potential therapeutic agents.

Disclosures

No potential conflicts of interest were disclosed.

Figure 1 :

 1 Figure 1: Acute and chronic models of hyperglycemia in zebrafish A: The intraperitoneal injection of D-glucose (2.5 g/kg bodyweight) results in a significant increase in blood glucose levels 1.5 h after the injection (n = 3). B: The immersion of zebrafish in D-glucose water (111 mM) for 14 days results in a significant increase in blood glucose levels (n = 15). Please click here to view a larger version of this figure.

Figure 2 :

 2 Figure 2: Chronic hyperglycemia impairs brain cell proliferation after 14 days of treatment Proliferative cells are labeled in green with a PCNA antibody. Cell nuclei are counterstained with DAPI (blue). Chronic hyperglycemia decreases brain cell proliferation after 14 days of treatment in the subpallium (A), in the pallium (B), and in the caudal hypothalamus around the lateral and posterior recess of the ventricle (C). Scale bar: 120 µm (A and B), 200 µm (C). Please click here to view a larger version of this figure.

Figure 3 : 31 . 33 .

 33133 Figure 3: Stab wound injury of the telencephalon upregulates brain proliferation at 7 days post-lesion A: Schematic overview of a transversal section of the zebrafish telencephalon at the level indicated in the upper sagittal. Schema have been taken from the zebrafish brain atlas 31 . The red dots indicate proliferating cells 32,33 . The needle indicates the site of the lesion. B: PCNA (green) immunohistochemistry 7 days after brain injury shows a strong upregulation of proliferation along the brain ventricle in the injured telencephalon. Scale bar: 200 µm. Please click here to view a larger version of this figure.

Figure 4 :

 4 Figure 4: PET/CT imaging of [18F]-FDG (20 MBq injected) 30 min after the intraperitoneal injection Representative images of PET/CT imaging show a wide distribution of [18F]-FDG in the body of the zebrafish, including the head, the brain, and the spinal cord. Please click here to view a larger version of this figure.

1 .

 1 Solutions and buffers: requirements and preparation 1. Prepare 1 L of 1X PBS by adding 100 mL of 10X PBS to 900 mL of distilled H 2 O (dH 2 O) and mix. 2. Prepare 1X PBS with 0.2% detergent (PBS-T; see the table of materials). NOTE: To prepare 1 L of PBS-T, add 2 mL of detergent (see the table of materials) to 1 L of PBS. 3. Prepare an ethanol series (100% x 2; 95%; 85%; 70%; 50%, and 30%) and 0.85% NaCl. 4. Prepare blocking buffer: PBS-T containing 0.5% to 1% milk powder. NOTE: To prepare 200 mL of blocking buffer, add 2 g of milk powder to 200 mL of PBST. 5. Prepare antigen retrieval buffer, sodium citrate. NOTE: To prepare 1 L of sodium citrate buffer, add 2.94 g of sodium citrate trisodium salt dehydrate to 1 L of dH 2 0. Adjust the pH to 6 prior to filling to 1 L. 6. Prepare a 4% paraformaldehyde-PBS buffer, adding 4 g of paraformaldehyde to 100 mL of 1X PBS. Warm it under agitation at 58-60 °C until it dissolves completely. . The next day, briefly wash the heads in PBS. 4. Dissect the brains carefully using a microscope. Rinse the fixed heads with PBS and use a needle to secure a head under a dissecting microscope. Remove the eyes and the top of the skull with forceps. Carefully extract the brain and place it in 1X PBS. 5. Successively wash the fixed brains with 1X PBS for 30 min, 0.85% NaCl for 30 min, 70% EtOH/0.85% NaCl (v/v) for 15 min, 70% EtOH for 15 min (twice), 85% EtOH for 20 min, 95% EtOH for 20 min, and 100% EtOH for 20 min (twice). Incubate overnight in a final 100% EtOH solution. NOTE: Dehydrated brains can remain for several months in 100% EtOH at 4 °C before paraffin embedding.

2. Sample preparation for immunohistochemistry: fixation and dehydration

1. After measuring the blood glucose level, separate the head from the body by cutting the head behind the gills. Note: Alternatively, the brain can be directly extracted and frozen for other experiments, such as mRNA extraction. 2. Fix the heads overnight at 4 °C in 4% paraformaldehyde dissolved in PBS (PFA-PBS). 3

Acknowledgements

We greatly thank Direction des Usages du Numérique (DUN) from La Réunion University for editing the video (in particular, Jean-François Février, Eric Esnault, and Sylvain Ducasse), Lynda-Rose Mottagan for the voiceover, Mary Osborne-Pellegrin for proofreading the voice-over, and the CYROI platform. This work was supported by grants from La Réunion University (Bonus Qualité Recherche, Dispositifs incitatifs), Conseil Régional de La Réunion, European Union (CPER/FEDER), and Philancia association. ACD is a recipient of a fellowship grant from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche, La Réunion University (Contrat Doctoral).

Representative Results

Using the procedures described in this article, the intraperitoneal injection of D-glucose (2.5 g/kg bodyweight) was performed on adult zebrafish and led to a significant increase in blood glucose levels 1.5 h after injection (Figure 1A). 24 h post-injection, the blood glucose levels were similar between D-glucose and PBS-injected fish 12 . For chronic treatment, zebrafish were immersed in D-glucose water (111 mM) and became hyperglycemic at the end of their 14 days of treatment (Figure 1B), as was previously shown [START_REF] Dorsemans | Impaired constitutive and regenerative neurogenesis in adult hyperglycemic zebrafish[END_REF][START_REF] Capiotti | Persistent impaired glucose metabolism in a zebrafish hyperglycemia model[END_REF] .

To investigate the impact of hyperglycemia on brain cell proliferation, PCNA immunohistochemistry was performed on zebrafish brains following the induction of acute and chronic hyperglycemia. Although acute hyperglycemia did not impact brain cell proliferation