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 damage⁴, including impaired bone structures^5,6, reduced bone mineral density, and even increased risk of osteoporosis⁷.

Mineralized tissue is of great importance to bone strength⁸, and bone mineralization matrix deposition is a critical index of bone formation⁹. Alizarin red has a high affinity for calcium ions, and alizarin red staining is a standard procedure for assessing bone formation¹⁰. According to this method, mineralized tissue is stained red, while all other tissue remains transparent. The stained area is then quantified by digital image analysis¹¹.

Zebrafish is an important model organism widely used in drug discovery and disease models. Genetic studies in zebrafish and humans have demonstrated similarities in the underlying mechanisms of skeletal morphogenesis at the molecular level¹². Moreover, high-throughput drug or biomolecule screening is more feasible in large clutches of zebrafish than murine models, facilitating the mechanistic study of proosteogenic or osteotoxic molecules¹³. Differential staining of the skeleton in toto¹⁰ is frequently used in studying skeletal dysplasia in small vertebrates and mammalian fetuses. Alizarin red staining was performed to investigate the bone developmental toxicity of chemicals in zebrafish larvae. Herein, we used lead as an example to describe a protocol for detecting lead acetate-induced bone defects in zebrafish larvae.

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 biochemical incubator maintained at 28.5 °C before the experiment.

2. Chemical exposure

1. Prepare the mother stock: weigh lead acetate trihydrate (5 g), and dissolve it in ultrapure water (50 mL) with stirring.
   CAUTION: PbAc is toxic. Wear suitable dust masks, protective clothing, and gloves. Perform the experiment under a fume hood.
2. Select and randomly distribute zebrafish embryos into clean 6-well plates (30 embryos per well in 3 mL of zebrafish breeding water; see Table 1 for its composition). Treat the embryos with PbAc (0, 5, 10, 20 mg/L) from 2 to 120 h post fertilization (hpf).
   NOTE: The concentrations of PbAc were chosen according to the dose-range-finding experiments. The results showed that the LC₅₀ of lead acetate on zebrafish embryos was 41.044 mg/L at 120 hpf. Hence, the highest concentration was set to half of the LC₅₀ and a series of solutions prepared by 2-fold dilution.
3. Feed the larvae twice a day from 5 days post fertilization (dpf). Maintain the zebrafish embryos at 28.5 ± 0.5 °C with a 14:10 h light/dark cycle.
4. Refresh the Pb-free medium (zebrafish breeding water) every 24 h until 9 dpf.
   ​NOTE: Upon completion of the experiments, all lead-containing solutions were poured into a designated liquid tank and treated by the experiment management center.

3. Alizarin red staining

NOTE: Wear suitable dust masks, protective clothing, and gloves during the entire staining process. The compositions of all solutions are shown in Table 1.

1. Specific staining steps
   NOTE: The dyeing process was carried out in a 24-well plate at room temperature. After each solution was added, place the 24-well plate on the shaking table set at a low speed.
   1. Remove 10 zebrafish larvae randomly from each group at 9 dpf and fix the fish in 1 mL of 2% paraformaldehyde/1x phosphate-buffered saline for 2 h.
   2. Decant the solution and wash the zebrafish larvae with 100 mM Tris-HCl (pH 7.5)/10 mM MgCl₂ for 10 min.
   3. Decant the solution and incubate the larvae in the following solutions for 5 min each for destaining: anhydrous ethanol (EtOH) solution (80% EtOH/100 mM Tris-HCl (pH 7.5)/10 mM MgCl₂, 50% EtOH/100 mM Tris-HCl (pH=7.5), and 25% EtOH/100mM Tris-HCl (pH=7.5).
   4. Decant the solution, remove the pigment by bleaching with a solution consisting of 3% H₂O₂ and 0.5% KOH, and observe once every 10 min until the pigment is completely removed.
   5. Rinse the zebrafish larvae several times with 25% glycerin/0.1% KOH for 10 min each until there are no bubbles.
      NOTE: It is important to avoid bubbles; otherwise, the bubbles in the zebrafish body will appear as a black field of view under the microscope, which will affect observations and quantitative analysis.
   6. Stain the larvae by soaking in 1 mL of 0.01% alizarin for 50 min.
   7. Decant the solution and add 1mL of 50% glycerin/0.1% KOH for 10 min to clear the background. Store the fishes in fresh 50% glycerin/0.1% KOH for subsequent observation.

4. Image acquisition

1. Transfer one larva onto the glass slide each time, keeping the larva in the middle of the liquid drop.
2. Observe the larvae under a stereo microscope.
3. Turn on the camera, open the software (see the Table of Materials), and keep the default settings.
4. Click on AE, and choose an appropriate exposure time (60 ms in this experiment) to obtain the best image.
5. Capture all the images under the same settings. Save the images in .tif format for later analysis.

5. Image analysis

NOTE: See the Supplemental file for an example with a set of sample graphs for image analysis.

1. Double-click the ImageJ icon and analyze the images saved in step 4.5.
   1. Click on File | Open to open the images saved in step 4.5.
   2. Click on Image | Type, select 8-bit.
   3. Click on Edit | Invert.
   4. Click on Analyze | Calibrate, select Uncalibrated OD in the popup interface, check Global calibration at the bottom left of the lower interface, and click OK.
   5. Click on Analyze | Set Scale | Click to Remove scale in the popup interface, check Global below, and click OK.
   6. Click on Analyze | Set Measurements, select the item area in the popup interface, check the Limit to threshold below (to measure only the selected range), and click OK.
   7. Click on Image | Adjust| Threshold, slide the slider in the middle of the popup interface to select the appropriate threshold (change the threshold for each image) so that all targets to be tested in one image are selected, and click Set.
   8. Click on Analyze | Measure. Record the dates of each group.
2. Use one-way ANOVA followed by Tukey's multiple comparison test to analyze the differences, and set the significance level at p < 0.001 (***).

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