Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae

Summary

Here, we have used alizarin red staining to show that lead acetate exposure causes a bone mass change in zebrafish larvae. This staining method can be adapted to the investigation of bone loss in zebrafish larvae loss induced by other hazardous toxicants.

Abstract

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the specific effects and mechanisms in zebrafish remain unclear. Alizarin red has a high affinity for calcium ions and can help visualize the bone and illustrate skeletal mineral mass. In this study, we aimed to detect lead acetate (PbAc)-induced bone loss in zebrafish larvae by using alizarin red staining. Zebrafish embryos were treated with a series of PbAc concentrations (0, 5, 10, 20 mg/L) between 2 and 120 h post fertilization. Whole-mount skeletal staining was conducted on larvae at 9 days post fertilization, and the total stained area was quantified using ImageJ software. The results indicated that the mineralized tissues were stained in red, and the stained area decreased significantly in the PbAc-exposure group, with a dose-dependent change in bone mineralization. This paper presents a staining protocol for investigating skeletal changes in PbAc-induced bone defects. The method can also be used in zebrafish larvae for the detection of bone loss induced by other chemicals.

Introduction

Recent studies have confirmed that osteoporosis due to glucocorticoids, aromatase inhibitors, and excessive alcohol consumption is common^1,2. Lead (Pb) is a toxic metal found in plants, soil, and aquatic environments³. Although the adverse effects of Pb on the human body have attracted much attention, its irreversible impact on bone needs to be investigated further. Lead intoxication causes a diverse array of pathological changes in both the developing and adult skeleton, affecting normal life activities. Studies have found an association between chronic Pb exposure and bone damage

Protocol

All animal procedures outlined here have been reviewed and approved by the Animal Care Institute of The Ethics Committee of Soochow University.

1. Fish husbandry and embryo collection ¹⁴

1. Feed fish three times every day; ensure the zebrafish are maintained at 28.5 ± 0.5 °C with a 14:10 h light/dark cycle.
2. Separate the male and female adult fish by isolation boards in spawning tanks at a 2:1 male to female ratio in the evening.
3. The next morning, remove the isolation boards at 9:00 AM and collect the embryos 2 h later.
4. Place the embryos in a biochemica....

Results

Alizarin red staining is a sensitive and specific method for measuring changes in bone mineralization in zebrafish larvae. In this study, we have observed that PbAc had adverse effects on zebrafish larvae, including death, malformation, decreased heart rate, and body length shortening. Moreover, the mineral skeleton areas of zebrafish larvae were evaluated to examine PbAc-induced bone loss. At 9 dpf (Figure 1A), many bones of the head skeleton are mineralized and hence stained in red, such a.......

Discussion

The zebrafish is a suitable model for studying bone metabolic disease. Compared to rodent models, zebrafish models are relatively fast to establish, and measurement of the severity of disease is easier. In wild-type zebrafish larvae, mineralization of the head skeleton occurs at 5 dpf and the axial skeleton at 7 dpf¹⁵. Thus, cranial bones such as PS, OP, CB, and NC are well developed at 9 dpf. After the larvae were completely destained and bleached, the soft tissues were cleared, resulting in a tr.......

Materials

Name	Company	Catalog Number	Comments
1 M Tris-HCl (pH=7.5)	Solarbio,Beijing,China	21	for detaining
4% Paraformaldehyde Fix Solution	BBI,Shanghai,China	14	fixing tissues
10x PBS buffer	BBI,Shanghai,China	15	for fixing
35% H2O2	Yonghua,Jiangsu,China	8	removing pigment
50 mL Centrifuge tube	AKX,Jiangsu,China	4
95% Anhydrous ethanol	Enox,Jiangsu,China	2	destaining
Alizarin red (Purity 99.5%)	Solarbio,Beijing,China	1	staining
Biochemical incubator	Yiheng,Shanghai,China	3	raising zebrafish embryos
Electronic scale	Sartorius,Germany	5	weighing the solid raw materials
Glycerin (Purity 99.5%)	BBI,Shanghai,China	7	storing the stained fish
ImageJ (software)	USA	9	digital analysis
KOH (Purity 99.9%)	Sigma,America	10	bleaching solution
Lead acetate trihydrate (Purity 99.5%)	Aladdin,Shanghai,China	11
MgCl2 (Purity 99.9%)	Aladdin,Shanghai,China	12	cleaning solution
NIS-Elements F (software)	Nikon, Japan	13	observing and taking photos
Pipe	AKX, Jiangsu, China	18	removal of embryos and solution
plates (24-well)	Corning,America	17	container for staining embryos
plates (6-well)	Corning,America	16	container for breeding embryos
Shaking table	Beyotime, China	19	mixing the solution
Stereo microscope	Nikon,Japan	20	observing and taking photos
Zebrafish	Zebrafish Experiment Center of Soochow University,Suzhou,China	22	experimental animal