Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Summary

This protocol describes a canonical method to understand the critical genes controlling osteoclast activity in vivo. This method uses a transgenic mouse model and some canonical techniques to analyze skeletal phenotype.

Abstract

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms and pharmaceutical treatments of osteoporosis. Cathepsin K (Ctsk)-Cre mice have been widely used for functional studies of osteoclasts. The signal transducer and activator of transcription 3 (STAT3) is relevant in bone homeostasis, but its role in osteoclasts in vivo remains poorly defined. To provide the in vivo evidence that STAT3 participates in osteoclast differentiation and bone metabolism, we generated an osteoclast-specific Stat3 deletion mouse model (Stat3 ^fl/fl; Ctsk-Cre) and analyzed its skeletal phenotype. Micro-CT scanning and 3D reconstruction implied increased bone mass in the conditional knockout mice. H&E staining, calcein and alizarin red double staining, and tartrate-resistant acid phosphatase (TRAP) staining were performed to detect bone metabolism. In short, this protocol describes some canonical methods and techniques to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.

Introduction

Skeletal bone is the main load-bearing organ of the human body and is under pressure from both the internal and external environment during walking and exercise¹. Throughout one’s life, bones continuously go through self-renewal, which is balanced by osteoblasts and osteoclasts. The process of osteoclasts clearing old bones and osteoblasts forming new bone maintains the homeostasis and mechanical function of the skeletal system². Disturbance in the balance may induce bone metabolic diseases, such as osteoporosis. Osteoporosis, which is caused by excess osteoclastic activity, is globally prevalent and causes substantial economic losses to society^2,3,4. According to the limited number of drugs available for osteoporosis treatment and their risk of adverse effects⁴, it is important to unveil the details of osteoclast formation and activity.

Osteoclasts derived from the monocyte/macrophage hematopoietic lineage have multiple nuclei (may have 2 to 50 nuclei) and are large (usually greater than 100 μm in diameter)². Although the exploration of mechanisms and the screening of drugs for osteoclastic disorders have been widely improved via in vitro osteoclast culture, the complicated organic reactions make in vivo evidence indispensable for the targeted therapy. Due to genetic and pathophysiological similarities between mice and humans, genetically engineered mouse models are commonly used for studying the mechanisms and the pharmaceutical treatments of human disease in vivo⁶. The Cre-loxP system is a widely-used technology for mouse gene editing and has enabled researchers to investigate gene functions in a tissue-/cell-specific manner⁵. Cathepsin K (CSTK) is a cysteine protease secreted by osteoclasts that can degrade bone collagen⁸. It is well accepted that CTSK is selectively expressed in mature osteoclasts; therefore, Ctsk-Cre mice are considered to be a useful tool for functional studies of osteoclasts and has been used⁶.

The signal transducer and activator of transcription (STAT) family is classical and highly significant in immunity and cancer progression and development^7,8. Among seven STATs, STAT3 is reported to be the most relevant to bone homeostasis^9,10. Several in vivo studies have reported that specific inactivation of STAT3 in osteoblasts decreases bone formation^9,10. Nevertheless, solid evidence regarding the participation of STAT3 in osteoclast formation and bone metabolism in vivo is still limited. Recently, we provided in vivo evidence with an osteoclast-specific Stat3 deletion mouse model (Stat3^fl/fl; Ctsk-Cre, hereafter called Stat3^Ctsk) that STAT3 participates in osteoclast differentiation and bone metabolism¹¹. In the present study, we describe the methods and protocols that we used to analyze the changes in bone mass, bone histomorphology, and bone anabolism and catabolism of the Stat3^Ctsk mice in order to study the influence of osteoclast-specific STAT3 deletion on bone homeostasis.

Protocol

All methods relating to the animals described here were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiaotong University School of Medicine.

1. Breeding of osteoclast specific Stat3 deletion mice

NOTE: Stat3^fl/fl mice were obtained commercially. Ctsk-Cre mice were provided by S. Kato (University of Tokyo, Tokyo, Japan¹²). The mice were bred and maintained under specific pathogen-free (SPF) conditions in the institutional animal facility under standardized conditions.

1. Pair a sexually mature male mouse with two female mice of the same age. After 18 days, check daily checks for newborns. Separate the pregnant female mice and keep it alone if needed. Change male mice among different breeding cages if the female mice were not pregnant within 1 month of the pairing.
2. Cross Stat3^fl/fl mice to Ctsk-Cre mice (F0). Clip the tails for genotyping and keep the male Stat3^fl/+; Ctsk-Cre mice until they are sexually mature, which is around 6 weeks of age (F1). Use the following primers: Stat3 F-TTGACCTGTGCTCCTACAAAAA; Stat3 R-CCCTAGATTAGGCCAGCACA; Ctsk-cre F-GAACGCACTGATTTCGACCA; Ctsk-cre R-GCTAACCAGCGTTTTCGTTC.
3. Cross 6-week-old male Stat3^fl/+; Ctsk-Cre mice with female Stat3^fl/fl mice. Clip the tails for genotyping and keep the male Stat3 ^fl/fl; Ctsk-Cre mice until they are sexually mature, which is around 6 weeks of age (F2).
4. Cross 6-week-old male Stat3 ^fl/fl; Ctsk-Cre mice with female Stat3^fl/fl mice (F3). Clip the tails for genotyping and replace the old breeding mice with younger mice in time (F3+N).

2. Specimen collection

1. Euthanize six pairs of 8-week old male Stat3^fl/fl and Stat3^Ctsk littermate mice separately with carbon dioxide asphyxiation.
   NOTE: The CO₂ flow rate displaces 30% of the cage volume per minute (e.g., for 45 cm x 30 cm x 30 cm cage the CO₂ flow rate is 40 L/min).
2. Place the mice in a supine position. Dislocate the bilateral hip joints gently by hand. Use ophthalmic scissors to cut off the skin vertically from the distal tibia and then dissect the entire skin from the hind limb.
3. Cut off the articular ligament of the right hip joint and knee joint with scissors to separate the hind limb. Cut the trochanter and the junction of the fibula and then immerse the hind limb in 4% paraformaldehyde. Keep the right hind limbs for step 3. Appropriately cut the bone at both ends to fully immerse and fix the bone marrow with 4% paraformaldehyde.
4. Cut the articular ligament of the left hip joint and knee joint with scissors, gently remove the soft tissue, and carefully separate the tibia and femur. Immerse the tibia and femur separately in 75% ethanol. Keep the femora for step 4 and tibiae for step 6. Ensure to keep the trochanter intact.

3. Paraffin section preparation

1. Fix the right hind limb in 4% paraformaldehyde at 4 °C for 48 h.
2. Decalcify: Gently wash the specimens with 1x PBS for 10 min 3 times. Decalcify the specimens in 15% EDTA (150 g EDTA in 800 mL of ddH₂O and 100 mL of 10x PBS) with an ultrasonic decalcifier for 3 to 4 weeks until the bones can be bent. Replace with fresh decalcifying fluid every other day.
3. Gently wash the specimens 3x with 1x PBS and then immerse them in 75% ethanol at 4 °C overnight.
4. Dehydrate: On the second day, sequentially immerse specimens in 95% ethanol, 100% ethanol, and xylene, each for 1 h twice.
5. Immerse specimens in 1/2 xylene 1/2 paraffin for 30 min. Immerse the specimens in paraffin at 65 °C overnight.
6. Embed: Select a suitable embedding tank for embedding. Place the tibia uniformly underneath. Place the femur and tibia at a 90° angle. After the paraffin has fully cooled and solidified, remove it from the embedding tank. Number the specimens and store them at -20 °C overnight.
7. Cut 5 μm thick sections continuously using the microtome. Cut 20–40 sections. Spread the sections on 37 °C water, adhere them to microscope slides, and bake at 42 °C overnight.

4. Micro-CT scanning and analysis

1. Scan the left femora with a micro-CT scanner. Resolution: 10 μm; Voltage: 70 kV; Current: 114 μA; Fliter: 0.5 mm Al; Rotation step: 0.5°.
2. Reconstruct 3D images of the cortical bone and trabecular bone using the scanner’s supporting software following manufacturer’s instruction. ROIs are in a total 1 mm width of trabecular bone close to the distal growth plate and in a total 1 mm wide section of cortical bone in the middle of the femora.
3. Calculate the quantitative microarchitecture parameters: bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th.), trabecular number (Tb.N.), trabecular separation (Tb.Sp.), and cortical bone thickness (Ct.Th.).

5. TRAP staining

1. Bake the paraffin sections at 65 °C for 30 min.
2. Dewax: Immerse the sections in xylene for 10 min. Perform this step 3x with fresh xylene.
3. Rehydrate: Immerse the sections sequentially in 100% ethanol, 95% ethanol, 70% ethanol, and ddH₂O, each for 5 min twice.
4. Prepare the staining solution using the TRAP staining kit following the manufacturer’s instructions and warm to 37 °C.
   NOTE: TRAP staining solution should be freshly prepared immediately before every assay.
5. Add 50–100 μL staining solution to each sample and incubate in a 37 °C humid chamber for 20–30 min. Check the staining status of the osteoclasts under a light microscope every 5 min until red multinucleated osteoclasts can be seen. End the reaction with ddH₂O.
6. Counterstain in hematoxylin solution for 30 s. Create a stable blue color by immersion in 1% ammonia solution for 1 min. Rinse in slowly running tap water.
7. Mount the sections with coverslips using neutral balsam and dry overnight.
8. Capture 3–5 fields of interest by a microscope. Analyze the trabecular perimeter by Image J: measure the length of scale bar (Ls) using the ‘straight line’ tool as L1, then measure the length of trabecular perimeter using the ‘segmented line’ tool as L2, the physical length (Lp)= Ls*L2 /L1). Count the number of TRAP-positive cells with more than three nuclei.

6. Calcein and alizarin red double labeling

1. Specimen preparation: Intraperitoneally inject 20 mg/kg calcein (1 mg/mL in 2% NaHCO₃ solution) on day 0, and 25 mg/kg alizarin red S (AL, 2 mg/mL in H₂O) on day 4. Sacrifice mice on day 7. Carefully disassociate the tibiae and fix in 4% paraformaldehyde at 4 °C for 48 h.
2. Dehydrate: After fixation, gently wash the tibiae 3x with 1x PBS. Sequentially immerse the specimens in 95% ethanol, 100% ethanol, and xylene for 5 min twice separately.
3. Immerse the specimens in acetone for 12 h, in 1/2 acetone 1/2 resin for 2 h, and in pure resin in a drying oven overnight.
   NOTE: The resin was prepared by commercially available resin (e.g., Embed 812 Resin) according to the manufacturer’s instructions.
4. Embed: Add pure resin into a suitable silica gel embedding tank and place the specimens gently to avoid bubbles. Polymerize the resin in a drying oven at 60 °C for 48 h.
5. Cut the specimens into 5 μm thick sections continuously with a rotary microtome. Store the rest of the samples with desiccant at room temperature.
6. Adhere the sections with tweezers in a drop of 75% alcohol. Mount the sections with coverslips using neutral balsam. Capture red and green fluorescence labeling with a fluorescence microscope.
7. Measure the width between two labeling lines (Ir.L.Wi), single-labeled trabecular perimeter (sL.Pm), double-labeled trabecular perimeter (dL.Pm), and total trabecular perimeter (Tb.Pm). Calculate the mineral apposition rate (MAR) and bone formation rate (BFR/BS). MAR = Ir.L.Wi/interval days. BFR/BS= (dL.Pm±sL.Pm/2)*MAR/Tb.Pm*100 %.

Results

Using the present protocol, osteoclast specific Stat3 deletion mice were generated to study the influence of STAT3 deletion on osteoclast differentiation. Stat3^Ctsk mice and their wildtype (WT) littermates were bred and kept after genotyping. Bone marrow macrophages were isolated and cultured into osteoclasts, and STAT3 deletion in Stat3^Ctsk mice was demonstrated (Figure 1).

Femora reconstruction a...

Discussion

Genetically engineered mouse models are commonly used for studying the mechanism and pharmaceutical treatment of human disease¹³. Ctsk-Cre mice have been widely used for functional studies of osteoclasts⁶. The present study described the protocols of the methods to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.

Histological analysis is the best intuitive method to detect bone metabolis...

Materials

Name	Company	Catalog Number	Comments
4% Paraformaldehyde solution	Sangon biotech Co., Ltd.	E672002
Acetone	Shanghai Experimental Reagent Co., Ltd.	80000360
Alizarin	Sigma-Aldrich	A5533
Ammonia solution	Shanghai Experimental Reagent Co., Ltd.
Calcein	Sigma-Aldrich	C0875
Ctsk-Cre mice			a gift from S. Kato, University of Tokyo, Tokyo, Japan
DDSA	Electron Microscopy Sciences	13710
DeCa RapidlyDecalcifier	Pro-Cure	DX1100
DMP-30	Electron Microscopy Sciences	13600
EDTA	Shanghai Experimental Reagent Co., Ltd.	60-00-4
EMBED 812 RESIN	Electron Microscopy Sciences	14900
fluorescence microscope	Olympus	IX73
Hematoxylin solution	Beyotime Biotechanology	C0107
Micro-CT	Scanco Medical AG	μCT 80
NaHCO3	Shanghai Experimental Reagent Co., Ltd.	10018918
Neutral balsam	Sangon biotech Co., Ltd.	E675007
NMA	Electron Microscopy Sciences	19000
Paraffin	Sangon biotech Co., Ltd.	A601889
rotary microtome	Leica	RM2265
Stat3fl/fl mice	GemPharmatech Co., Ltd	D000527
TRAP staining kit	Sigma-Aldrich	387A
xylene	Shanghai Experimental Reagent Co., Ltd.	1330-20-7