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

Summary

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.

Abstract

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.

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 problem¹. Indeed, uncontrolled diabetes leads to several physiological disorders affecting the cardiovascular system, kidneys, and the peripheral and central nervous systems.

Interestingly, acute and chr....

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.

1. 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 storag....

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 fish¹². For chronic treatment, zebrafish were immersed in D-glucose water (111 mM) and became hyperglycemi.......

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 d.......

Materials

List of materials used in this article

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