We greatly thank Direction des Usages du Num&#233;rique (DUN) from La R&#233;union University for editing the video (in particular, Jean-Fran&#231;ois F&#233;vrier, 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 R&#233;union University (Bonus Qualit&#233; Recherche, Dispositifs incitatifs), Conseil R&#233;gional de La R&#233;union, European Union (CPER/FEDER), and Philancia association. ACD is a recipient of a fellowship grant from the Minist&#232;re de l&#39;Education Nationale, de l&#39;Enseignement Sup&#233;rieur et de la Recherche, La R&#233;union University (Contrat Doctoral).

Adult wildtype zebrafish (Danio rerio) were maintained under standard photoperiod (14/10 h light/dark) and temperature (28 &#176;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.
1. Establishing a Model of Acute Hyperglycemia in Zebrafish
<ol>
	<li>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 &#176;C.</li>
	<li>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%).</li>
	<li>Transfer one zebrafish (3 to 6 months) from its tank to the anesthetic until it stops moving.</li>
	<li>Remove the fish using a cut pipette and quickly dry it on absorbent tissue paper.</li>
	<li>Weigh the fish.</li>
	<li>Prepare a syringe of D-glucose dissolved in 1X PBS and inject 50 &#181;L of D-glucose (2.5 g/kg bodyweight). 
	NOTE: For instance, a 0.6 g fish will receive 50 &#181;L of 3% D-glucose/PBS solution.</li>
	<li>Put the fish on its back and insert the needle of the syringe into the intraperitoneal cavity.</li>
	<li>Slowly inject the D-glucose/PBS solution and then place the fish back in the water.</li>
	<li>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.</li>
</ol>
2. Establishing a Model of Chronic Hyperglycemia in Zebrafish
<ol>
	<li>Prepare a 2 L tank of clean fish water.</li>
	<li>Dissolve 40 g of D-glucose into the 2 L of fish water for a final D-glucose concentration of 111 mM.</li>
	<li>Immerse 5 to 7 adult zebrafish in the D-glucose-containing water.</li>
	<li>Replace the D-glucose fish water every 2 days in order to avoid the growth of bacteria or other micro-organisms.</li>
	<li>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 glucose-level measurement.</li>
</ol>
3. Measuring Blood Glucose Levels in Zebrafish
<ol>
	<li>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.</li>
	<li>Wipe the fish with absorbent tissue paper to remove all water and to avoid any blood dilution during the blood glucose measurement.</li>
	<li>Remove an eye using dissecting forceps and wait until the eye cavity is filled with blood.</li>
	<li>Put a test strip on a glucometer and insert the strip into the eye cavity.</li>
	<li>Measure the blood glucose levels.</li>
</ol>
4. Analyzing Brain Cell Proliferation Following Hyperglycemia
<ol>
	<li>Solutions and buffers: requirements and preparation
	<ol>
		<li>Prepare 1 L of 1x PBS by adding 100 mL of 10x PBS to 900 mL of distilled H2O (dH2O) and mix.</li>
		<li>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 Materials Table) to 1 L of PBS.</li>
		<li>Prepare an ethanol series (100% x 2; 95%; 85%; 70%; 50%, and 30%) and 0.85% NaCl.</li>
		<li>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.</li>
		<li>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 dH20. Adjust the pH to 6 prior to filling to 1 L.</li>
		<li>Prepare a 4% paraformaldehyde-PBS buffer, adding 4 g of paraformaldehyde to 100 mL of 1x PBS. Warm it under agitation at 58-60 &#176;C until it dissolves completely.</li>
	</ol>
	</li>
	<li>Sample preparation for immunohistochemistry: fixation and dehydration
	<ol>
		<li>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.</li>
		<li>Fix the heads overnight at 4 &#176;C in 4% paraformaldehyde dissolved in PBS (PFA-PBS).</li>
		<li>The next day, briefly wash the heads in PBS.</li>
		<li>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.</li>
		<li>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 &#176;C before paraffin embedding.</li>
	</ol>
	</li>
	<li>Sample preparation for immunohistochemistry: tissue preparation for paraffin embedding
	<ol>
		<li>Place the brains in a small glass beaker. 
		NOTE: Do not use a plastic receptacle, as toluene can damage some plastics.</li>
		<li>Remove the ethanol and replace it with toluene, as the brains must be totally recovered by toluene. Perform two 30 min baths of toluene.</li>
		<li>Remove the toluene and place the brains in an embedding cassette. Put the closed cassette in melted paraffin series beakers at 58-60 &#176;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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Insert the paraffin block into the arm of the microtome. Cut 50 &#181;m-thick sections until the brain sample level is reached. Trim the paraffin block to obtain a trapeze and adjust the sectioning thickness to 7 &#181;m. Collect the paraffin ribbons using paintbrushes and place them on black paper. Cut them gently every 3 to 4 sections.</li>
		<li>Put a slide on a warming plate and cover it with dH2O water.</li>
		<li>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 &#176;C.</li>
	</ol>
	</li>
	<li>Immunohistochemistry procedure
	<ol>
		<li>Remove the paraffin wax by placing sections in three containers of xylene for 7 min each.</li>
		<li>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 dH2O and twice in PBS for 5 min each.</li>
		<li>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.</li>
		<li>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.&#160;proliferating cell nuclear antigen (PCNA) for proliferative cell staining; 1/100) diluted in blocking buffer (i.e.&#160;PBST, 1% milk).</li>
		<li>Wash three times in PBST, 5 min each, and incubate the sections for 90 min with appropriate secondary antibodies (e.g.&#160;goat anti-mouse Alexa Fluor 488; 1/200) diluted in blocking buffer and with DAPI (1/500).</li>
		<li>Wash three times in PBST, 5 min each, and mount the slides with fluorescent mounting medium (see the table of materials).</li>
		<li>Analyze the staining using an epifluorescence and/or confocal microscope.</li>
		<li>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 described24.</li>
	</ol>
	</li>
	<li>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 described24,25,26. Briefly:
	<ol>
		<li>Anesthetize adult zebrafish with tricaine.</li>
		<li>Place the fish under a dissecting microscope with light.</li>
		<li>Hold the fish with one hand.</li>
		<li>With the other hand, insert a 30 G syringe vertically through the skull into the medial region of the right telencephalic hemisphere.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
5. Imaging the Biodistribution of Radiolabeled Molecules by PET/CT in Zebrafish: Fluorodeoxyglucose ([18F]-FDG) to Analyze Glucose Metabolism
<ol>
	<li>Prepare a 50 &#181;L syringe containing 20 MBq of a saline solution of [18F]-FDG.</li>
	<li>Place the syringe behind the radiation protective screen.</li>
	<li>Anesthetize an adult zebrafish with tricaine.</li>
	<li>Place the fish behind a radiation protective screen.</li>
	<li>Inject [18F]-FDG into the intraperitoneal cavity. Wipe the injection site with a small piece of tissue paper to prevent the detection of residual [18F]-FDG that could leak out of the fish belly.</li>
	<li>Use a radioisotope calibrator to measure the remaining activity contained on the syringe and the tissue to calculate the exact injected dose.</li>
	<li>Place the fish on a new, small piece of absorbent tissue soaked with tricaine and gently wrap it up. Place the fish with the absorbent tissue on the bed of the PET/CT imager.</li>
	<li>Insert the bed into the PET/CT imaging system and start the acquisition.</li>
	<li>Proceed to conduct the PET/CT acquisition.</li>
	<li>At the end of the procedure, place the fish back into a small amount of fresh fish water. 
	NOTE: Alternatively, after the [18F]-FDG injection, the fish can be put back into a small amount of fresh water for 10 min to 1 h to facilitate the diffusion of [18F]-FDG before anesthetizing the fish once again for PET/CT imaging.</li>
</ol>

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<li>Connaughton, V. P., Baker, C., Fonde, L., Gerardi, E., Slack, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternate+Immersion+in+an+External+Glucose+Solution+Differentially+Affects+Blood+Sugar+Values+in+Older+Versus+Younger+Zebrafish+Adults%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">Alternate Immersion in an External Glucose Solution Differentially Affects Blood Sugar Values in Older Versus Younger Zebrafish Adults.</a> Zebrafish. , (2016).</li>
<li>Prasad, S., Sajja, R. K., Naik, P., Cucullo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diabetes+Mellitus+and+Blood-Brain+Barrier+Dysfunction%3A+An+Overview%5BTitle%5D)+AND+%22J+Pharmacovigil%22%5BJournal%5D)">Diabetes Mellitus and Blood-Brain Barrier Dysfunction: An Overview.</a> J Pharmacovigil. 2 (2), 125(2014).</li>
</ol>

No potential conflicts of interest were disclosed.

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, blood-brain 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 &#946;-cells34,35. 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 &#181;L intraperitoneal injection is designed for a medium-sized fish (0.5 g). Indeed, a small fish might not be able to receive a 50 &#181;L 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 permeability36.
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.

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 problem1. 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 depression2,3,4,5,6. In addition, the admission of patients with hyperglycemia has been associated with worse functional, neurological, and survival outcomes after ischemic stroke7,8,9,10,11. 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 survival2,12.
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 adulthood13,14,15,16. Notably, such capacities are possible due to the persistence of neural stem/progenitor cells, including radial glia and neuroblasts17,18,19. In addition, the zebrafish has recently emerged as a model for studying metabolic disorders, including obesity and hyperglycemia/diabetes20,21,22.
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 function12,23. 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-glucose12. 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, [18F]-FDG) using PET/CT.

<table><tbody><tr><td>1mL Luer-Lok Syringe</td><td>BD, USA</td><td>309628</td><td></td></tr><tr><td>4&#039;,6&#039;-diamidino-2-phenylindole (DAPI)</td><td>Sigma-Aldrich, Germany</td><td>D8417</td><td></td></tr><tr><td>7 mL bijou container plain lab</td><td>Dutscher, France</td><td>080171</td><td></td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich, Germany</td><td>67021</td><td></td></tr><tr><td>Digital camera</td><td>Life Sciences, Japan</td><td>Hamamatsu ORCA-ER</td><td></td></tr><tr><td>Disposable base molds&amp;nbsp;</td><td>Simport, Canada</td><td>M475-2</td><td></td></tr><tr><td>Donkey anti-rabbit Alexa fluor 488</td><td>Life Technologies, USA</td><td>A21206</td><td></td></tr><tr><td>Embedding center</td><td>Thermo Scientific, USA</td><td>Shandon Histocentre 3</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Nikon, Japan</td><td>Eclipse 80i</td><td></td></tr><tr><td>Fluorodeoxyglucose (&lt;sup&gt;18&lt;/sup&gt;F-FDG)</td><td>Cyclotron, France</td><td></td><td></td></tr><tr><td>Glucometer test strip</td><td>LifeScan, France</td><td>One-Touch 143 Ultra</td><td></td></tr><tr><td>Goat anti-mouse Alexa fluor 594</td><td>Life Technologies, USA</td><td>A11005</td><td></td></tr><tr><td>&lt;em&gt;In-Vivo&lt;/em&gt; Imaging System</td><td>TriFoil Imaging, Canada</td><td>Triumph Trimodality&amp;nbsp;</td><td></td></tr><tr><td>Microtome</td><td>Thermo Scientific, USA</td><td>Microm HM 355 S</td><td></td></tr><tr><td>Monoclonal mouse anti-PCNA</td><td>DAKO, USA</td><td>clone PC10</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich, Germany</td><td>P6148-500G</td><td></td></tr><tr><td>Polyclonal rabbit anti-GFAP</td><td>DAKO, USA</td><td>Z033429</td><td></td></tr><tr><td>Slide drying bench</td><td>Electrothermal, USA</td><td>MH6616</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich, Germany</td><td>S9888</td><td></td></tr><tr><td>Sodium citrate trisodium salt dehydrate&amp;nbsp;</td><td>Prolabo, France</td><td>27833.294</td><td></td></tr><tr><td>Sterile needle</td><td>BD Microlance 3</td><td>30 G 1/2 ; 0.3 mm&amp;times; 13 mm</td><td></td></tr><tr><td>Student Dumont #5 Forceps</td><td>Fine Science Tools</td><td>91150-20</td><td></td></tr><tr><td>Student surgical scissors</td><td>Fine Science Tools</td><td>91400-14</td><td></td></tr><tr><td>Superfros Plus Gold Slides</td><td>Thermo Scientific, USA</td><td>FT4981GLPLUS</td><td></td></tr><tr><td>Surgical microscope</td><td>Leica, France</td><td>M320-F12</td><td></td></tr><tr><td>Tissue embedding cassettes</td><td>Simport, Canada</td><td>M490-10</td><td></td></tr><tr><td>Tissue embedding medium</td><td>LeicaBiosystems, USA</td><td>39602004</td><td></td></tr><tr><td>Toluene</td><td>Sigma-Aldrich, Germany</td><td>244511</td><td></td></tr><tr><td>Tricaine MS-222</td><td>Sigma-Aldrich, Germany</td><td>A5040</td><td></td></tr><tr><td>Triton X100</td><td>Sigma-Aldrich, Germany</td><td>X100-500 mL</td><td></td></tr><tr><td>Vectashield medium&amp;nbsp;</td><td>Vector Laboratories, USA</td><td>H-1000</td><td></td></tr><tr><td>Xylene</td><td>Sigma-Aldrich, Germany</td><td>534056</td><td></td></tr><tr><td>Fish Strain</td><td>AB</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Saline phosphate buffer (10X PBS) pH 7.4 (for 1 liter)&lt;/strong&gt;</td><td></td><td></td><td>For preparing 10X PBS, add the following&amp;nbsp; salts and complete to 1 liter with distilled water</td></tr><tr><td>&lt;em&gt;Potassium chloride (MM : 74.55 g/mol): 2.00 g&lt;/em&gt;</td><td>&lt;em&gt;Sigma-Aldrich, Germany&lt;/em&gt;</td><td>&lt;em&gt;746436&lt;/em&gt;</td><td></td></tr><tr><td>&lt;em&gt;Potassium phosphate monobasic (MM: 136,09 g/mol): 2.40g&lt;/em&gt;</td><td>&lt;em&gt;Sigma-Aldrich, Germany&lt;/em&gt;</td><td>&lt;em&gt;795488&lt;/em&gt;</td><td></td></tr><tr><td>&lt;em&gt;Sodium chloride (MM : 58.44 g/mol): 80.00 g&amp;nbsp;&lt;/em&gt;</td><td>&lt;em&gt;Sigma-Aldrich, Germany&lt;/em&gt;</td><td>&lt;em&gt;S9888&lt;/em&gt;</td><td></td></tr><tr><td>&lt;em&gt;Sodium phosphate dibasic (MM: 141,96 g): 14,40 g&lt;/em&gt;</td><td>&lt;em&gt;Sigma-Aldrich, Germany&lt;/em&gt;</td><td>&lt;em&gt;795410&lt;/em&gt;</td><td></td></tr></tbody></table>

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

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 fish12. 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 shown12,22.
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 proliferation12, 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 injury25,27,28,29,30. 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55203/55203fig1.jpg" /> 
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). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55203/55203fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55203/55203fig2.jpg" /> 
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 &#181;m (A and B), 200 &#181;m (C). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55203/55203fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55203/55203fig3.jpg" /> 
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 atlas31. The red dots indicate proliferating cells32,33. 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 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55203/55203fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55203/55203fig4.jpg" /> 
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. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55203/55203fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled

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

This work describes methods to establish acute and chronic hyperglycemia models in zebrafish. The aim is to investigate the impact of hyperglycemia on physiological processes, such as constitutive and injury-induced neurogenesis. The work also highlights the use of zebrafish to follow radiolabeled molecules (here, [18F]-FDG) using PET/CT.

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological ...

acute-chronic-models-hyperglycemia-zebrafish-method-to-assess-impact

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

13.4K Views.  Université de La Réunion, INSERM.  Dit werk beschrijft methodes om acute en chronische hyperglycemie modellen in zebravis te bepalen. Het doel is om de invloed van hyperglycemie op fysiologische processen te onderzoeken, zoals constitutieve en schade-geïnduceerde neurogenese. Het werk benadrukt ook het gebruik van zebravis om radioactieve gelabelde moleculen te volgen (hier, [ 18 F] -FDG) met behulp van PET / CT. 

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

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

Medicine

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

Cancer Research

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

We present a behavioral chamber designed to assess cognitive performance. We provide data showing that once acquired, zebrafish remember the task 8 weeks later. We also show that hyperglycemic zebrafish have altered cognitive performance, indicating that this paradigm is applicable to studies assessing cognition and memory.

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or ...

Behavior

Simultaneous Calcium Imaging and Glucose Stimulation in Living Zebrafish to Investigate In Vivo &#946;-Cell Function

The pancreatic &#946;-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in &#946;-cell function are associated with hyperglycemia that can lead to diabetes. During the process of insulin secretion, &#946;-cells experience an influx of Ca2+. Thus, imaging the glucose-stimulated Ca2+ influx using genetically encoded calcium indicators (GECIs) provides an avenue to studying &#946;-cell function. Previously, studies showed that isolated zebrafish islets expressing GCaMP6s exhibit significant Ca2+ activity upon stimulation with defined glucose concentrations. However, it is paramount to study how &#946;-cells respond to glucose not in isolation, but in their native environment, where they are systemically connected, vascularized, and densely innervated. To this end, the study leveraged the optical transparency of the zebrafish larvae at early stages of development to illuminate &#946;-cell activity in vivo. Here, a detailed protocol for Ca2+ imaging and glucose stimulation to investigate &#946;-cell function in vivo is presented. This technique allows to monitor the coordinated Ca2+ dynamics in &#946;-cells with single-cell resolution. Additionally, this method can be applied to work with any injectable solution such as small molecules or peptides. Altogether, the protocol illustrates the potential of the zebrafish model to investigate islet coordination in vivo and to characterize how environmental and genetic components might affect &#946;-cell function.

Here, the study presents a protocol for calcium imaging and glucose stimulation of the pancreatic &#946;-cells of the zebrafish in vivo.

The pancreatic &#946;-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in ...

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Biology

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Neuroscience

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish

Zebrafish (Danio rerio) are an excellent model to investigate the effects of chronic hyperglycemia, a hallmark of Type II Diabetes Mellitus (T2DM). This alternate immersion protocol is a noninvasive, step-wise method of inducing hyperglycemia for up to eight weeks. Adult zebrafish are alternately exposed to sugar (glucose) and water for 24 hours each. The zebrafish begin treatment in a 1% glucose solution for 2 weeks, then a 2% solution for 2 weeks, and finally a 3% solution for the remaining 4 weeks. Compared to water-treated (stress) and mannitol-treated (osmotic) controls, glucose-treated zebrafish have significantly higher blood sugar levels. The glucose-treated zebrafish show blood sugar levels of 3-times that of controls, suggesting that after both four and eight weeks hyperglycemia can be achieved. Sustained hyperglycemia was associated with increased Glial Fibrillary Acidic Protein (GFAP) and increased nuclear factor Kappa B (NF-kB)&#160;levels in retina and decreased physiological responses, as well as cognitive deficits suggesting this protocol can be used to model disease complications.

This protocol noninvasively induces hyperglycemia in zebrafish for up to 8 weeks. Using this protocol, an in-depth study of the adverse effects of hyperglycemia can be made.

Zebrafish (Danio rerio) are an excellent model to investigate the effects of chronic hyperglycemia, a hallmark of Type II Diabetes Mellitus (T2DM). This ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

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

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