Tension-Free Weight-Bearing Model of Steroid-Induced Osteonecrosis of Femoral Head in Rats

Summary

The protocol shows building a simple and cost-effective rat weight-bearing training model for steroid-induced osteonecrosis of the femoral head using elastic therapeutic tape.

Abstract

Unlike humans, rats are animals that walk on all fours, while humans are bipedal animals that stand, and the hips are subjected to tremendous pressure when walking and standing. In rat steroid-induced femoral head necrosis models, the biomechanical characteristics of the human hip under higher pressure often need to be simulated. Some scholars try to emulate the state of human hip pressure by making rats bear a certain weight, but fixing the weight-bearing object on the rat is tough. Rats can easily break free of immobilization, and sticking the weight on the rats with adhesive tape will cause the rats to suffocate or die from intestinal obstruction. Our research group used elastic therapeutic tape to perform tension-free immobilization of weight-bearing objects in rats so that the rats could breathe freely and not break away from the immobilization under weight-bearing conditions. Compared to the usual steroid-induced femoral head necrosis rat model, we found that this weight-bearing intervention can aggravate the progression of femoral head necrosis in rats.

Introduction

The administration of glucocorticoids is the most common risk factor for non-traumatic osteonecrosis of the femoral head (ONFH)¹. Numerous pieces of evidence suggest that, in addition to glucocorticoids, the pressure load on the hip joint in patients is also associated with the occurrence of ONFH. Factors such as body weight and physical labor intensity are recognized as risk factors for ONFH². Multiple clinical studies have demonstrated a close relationship between hip joint loading conditions and the timing and incidence of joint replacement^3,4,5,6. Therefore, establishing a model that reflects the relationship between load bearing and steroid-induced osteonecrosis of the femoral head (SONFH) is important for a comprehensive investigation of this condition.

Large bipedal birds, such as ostriches and emus, serve as good models to simulate hip joint stress, resembling human leg loads^7,8. However, maintaining large bird species is challenging, and the associated research costs are high. Spontaneously hypertensive rat models^9,10 can exhibit higher rates of ONFH, but the compartment pressure load in the marrow generated by spontaneous hypertension differs significantly from mechanical pressure and is unsuitable for studying the impact of mechanical pressure on SONFH.

Small animal models are commonly used in research on SONFH. However, quadrupedal reptiles have lower hip joint stress, and their hip models cannot simulate the biomechanical environment of human hip joints during bipedal walking. Single limb immobilization models¹¹ and partial unloading models¹² are common, but both reduce limb loading. As humans are bipedal organisms with substantial lower limb loads while standing and walking, reducing the load in these models diminishes the connection between animal models and human diseases.

This study aims to establish a simple and cost-effective model for weight-bearing training to investigate the impact of weight-bearing training on steroid-induced osteonecrosis of the femoral head in rats. Currently, rat models have been used to study steroid-induced osteonecrosis of the femoral head^13,14, but there is still no model that can provide long-term, safe fixation with minimal disruption to movement, which is also relatively simple and inexpensive. In this study, a high-adhesive fixation material is applied, adopting tension-free fixation, which maintains the rats' mobility and reduces the suffering and even death caused by improper fixation.

Protocol

The protocol adheres to the ethical guidelines set by the Institutional Animal Care and Use Committee (IACUC) at Beijing University of Chinese Medicine, Protocol number BUCM-4-2022062001-2109. The protocol uses Sprague Dawley (SD) rats (SCXK(Jing)2019-0008), aged 8-10 weeks and weighing 200 g-250 g.

1. Adaptation training

1. Albino rats generally have a visual range of less than 60 cm¹⁵. During the modeling process, place the rats in opaque cages and keep them as far away as possible from non-modeled rats (at least, other rats should be outside the maximum visual range of the modeled rats) to prevent them from experiencing traumatic stress.
2. Turn on the power and set the treadmill to 10 m/min with a 0° incline. Place the rats on the treadmill and ensure the movements are as gentle as possible to avoid stress. Do not use additional stimulation; the rats will naturally crawl forward because they are unfamiliar with the environment.
3. The training lasts for 15 min. After the training, clean up the feces and urine left by the rats and spray alcohol to eliminate the animal odor. Then, prepare the next rat for training.
   NOTE: If a rat refuses to walk on the treadmill for 5 minutes, exclude it from the study.
4. Continue adaptive training until the end of the 1^st week.

2. Measuring the maximum weight-bearing capacity in rats

1. Prepare two test tubes, a hook, and a loop fastener approximately 80 cm long. Place steel balls into the test tubes, ensuring that the initial combined weight of the test tubes, the hook, and the loop fastener is 150 g. Prepare test tubes with different total weights in 10 g increments, with the maximum weight being 350 g.
2. A researcher uses both hands to fasten rats using gloves (Figure 1). Another researcher places the test tube on the back of the rat about 1 cm from the midline of the back. Since this step does not require movement of the rat, perform fixation by simply wrapping the hook and loop fastener around the rat without using additional measures to secure the tube.
3. Change the tube until the rat's maximum weight is reached. When the rat reaches maximum weight-bearing, it cannot stand when prodded or the experimenter leaves. Reduce the weight by 10 g to allow the rat to stand. This weight represents the rat's maximum weight-bearing capacity.

3. Preparation of weight load

1. Since secure fixation is required during training, secure the objects using 1-1.5 m elastic therapeutic tape based on rat size, which has strong adhesion. Use clay to adjust the weight of the test tubes to reduce the swinging of the load.
2. After weighing the weight of the test tubes and the fixation objects, add an appropriate amount of clay into the test tubes to adjust them to the target weight. Use a glass stirring rod to evenly spread the clay inside the test tubes. Clay concentrated at one end of the test tube may cause it to detach easily during training.
3. Adjust the total weight of the adjusted test tubes and clay at 50% of the rat's maximum weight-bearing capacity (Figure 1A). Exceeding this weight will make it difficult for the rat to complete the training. Do not adjust the weight of the load again until the end of the experiment.

4. Establishing steroid-induced osteonecrosis of the femoral head model

1. Use the methylprednisolone combined with lipopolysaccharide (LPS+MPS) method for SONFH model establishment^16,17. Perform intraperitoneal injection of LPS (20 µg/kg) using a standard needle at the midline of the rat's abdomen once every 24 h, for a total of 2 injections.
2. After the last LPS injection, inject MPS (40 mg/kg) into the unilateral gluteal muscle 24 h after regular feeding and continue injections every 24 h, alternating between the two gluteal areas, for a total of three injections.

5. Tension-free weight-bearing immobilization using elastic therapeutic tape and treadmill training

1. Mark the rat's back midline at 1-2 cm on both sides; this is the reference point for the weight load. Ensure the marks are appropriately positioned to prevent the rat from tipping or hindering movement during treadmill training.
2. With one investigator fixing the rat using gloves, place one hand under the rat's armpit to secure the forelimb and jaw while the other hand secures the hind limbs and tail. Avoid excessive force to prevent stress or suffocation (Figure 1B).
3. Adhere the elastic therapeutic tape to the test tube without applying tension to secure the load on the rat's back. In the process of winding, do not stretch the elastic therapeutic tape to achieve tension-free fixation.
   NOTE: The elastic therapeutic tape used in this model is designed for taping during intense physical activities, providing high adhesion and elasticity¹⁸. Due to its high elasticity, it greatly reduces the impact on the rat's movement while crawling.
4. Ask the second investigator to fix the test tube to the rat's back parallel to the rat's spine, with the fixation position based on the markings made, i.e., 1-2 cm away from the midline on both sides of the rat's back.
5. To minimize the suffering of the rat, reduce the impact on the rat's movement, and decrease mortality caused by fixation, perform fixation without applying tension. Stick the test tube with adjusted weight onto the elastic therapeutic tape, then wrap the test tube around the rat's body using a tension-free technique.
6. Ensure tension-free immobilization and maintain the original length of the elastic therapeutic tape without stretching in order to prevent any adverse effects on rat breathing and movement (Figure 1C).
7. After wrapping the test tube around the rat's body, place the rat in an open space or a single cage, monitoring its breathing and direction. If signs of rapid breathing or mouth opening appear, promptly release the rat to prevent suffocation.
   1. Ideal immobilization allows the rat to move freely and perform activities like drinking, lifting forelimbs, and feeding (Figure 1D). If immobilization is unsatisfactory, release temporarily and re-immobilize after 20 min to minimize stress on the rat due to repeated immobilization stimuli.
8. Turn on the power and set the treadmill to 1 m/min with a 0° incline. The treadmill training time is 30 min. Gently transfer the rat onto the treadmill and begin the timer.
9. Do not provide additional stimulation to the rat. If the rat cannot complete the 30 min training, place it in a cage for a 10 min rest without removing the fixation.
   NOTE: The elastic therapeutic tape used in this study has excellent stretchability and adhesiveness. When properly secured without tension, long-term fixation will not cause the test tube to detach or result in suffocation and death of the rat.
10. After the training, clean up the feces and urine left by the rats and spray alcohol to eliminate the animal odor.
11. Avoid the above steps being seen by other rats and handle the animals as gently as possible to prevent animals from vocalizing, thus causing traumatic stress to other rats.
12. Immediately release the rat from fixation after completing training (Figure 1E). During release, use fingers to press down on the rat's fur to protect it, minimizing fur loss during release (Figure 1F).

6. Animal grouping

1. To study the impact of weight-bearing training on SONFH, randomly divide 60 rats into four groups.
2. In the Control group, do not establish the common steroid-induced osteonecrosis of the femoral head (SONFH) model or perform weight-bearing training. In the Control+Load group, carry out weight-bearing training only, without establishing the steroid-induced osteonecrosis of the femoral head model. In the Model group, establish the steroid-induced osteonecrosis of the femoral head model only. In the Model+Load group, perform weight-bearing training after establishing the steroid-induced osteonecrosis of the femoral head model.

7. Euthanasia and specimen collection

1. Place the rats into a small animal euthanasia chamber and inject enough carbon dioxide to achieve a concentration higher than 5%. Once the rats lose consciousness, administer a dose of 0.3 mL/kg of body weight of a 20% potassium chloride solution, totaling 20 mL, via an intracardiac injection.
2. Monitor the rats' breathing and heartbeat to ensure they experience no pain or discomfort during euthanasia. Confirm death by checking for the absence of breathing and heartbeat. Spray alcohol to clean away any odor.
3. Post-euthanasia, dissect the rat's hip region and extract the femoral head while keeping the femur intact. Perform a microCT scan following pretreatment, followed by Hematoxylin-Eosin (HE) staining.

8. Hematoxylin-Eosin staining

1. After the modeling is completed, euthanize the rats. Remove the femurs for HE staining.
2. Soak the rat femurs in 4% paraformaldehyde for 24 h to preserve biological tissues as specimens. Soak the rat femur specimens in 5% formic acid for decalcification for 5 days.
3. Section the specimens into 5 µm slices for HE staining.
   NOTE: Empty lacunae refer to the situation in bone tissue¹⁹ where the lacunae, which should normally contain osteocytes, are devoid of these cells. It is an important indicator for assessing the health of bone tissue. In the pathological process of bone necrosis, insufficient blood supply may cause osteocyte death, leading to empty lacunae. In the diagnosis and evaluation of bone necrosis, the number and distribution of empty lacunae are important parameters for measuring the severity of the disease^20,21.
   1. Place rat femoral specimens into melted paraffin (56-60 °C), first soaking in low-melting-point paraffin, if necessary, then gradually transfer to higher melting point paraffin, each time for about 1 h, to ensure thorough infiltration of the tissue with paraffin.
   2. Prepare embedding molds and pour melted paraffin into the molds to the appropriate depth. Retrieve the tissue samples from the paraffin using forceps after soaking, remove excess paraffin, and place them in molds. Cool to room temperature to solidify the paraffin, forming paraffin blocks.
   3. Using a paraffin microtome, cut the embedded paraffin blocks into 5 µm thick sections.
4. Bake the rat femoral specimens with attached slices in a slide warmer for 1 h to enhance the adhesion between the slices and coverslip and reduce floating during subsequent staining processes.
5. Immerse the baked slices sequentially in pure xylene to remove paraffin, followed by a series of dehydration through absolute ethanol, gradient ethanol (100%, 95%, 80%, 70%), and water, for 2 min each, completing the deparaffinization and rehydration process.
6. Immerse the deparaffinized slices in a hematoxylin staining solution and stain for 10 min. Rinse with running water to remove unbound hematoxylin.
7. Immerse the hematoxylin-stained sections in eosin staining solution and stain for 2 min to color the cytoplasm and connective tissue. Rinsed with a 1% acetic acid solution to fix the staining effect.
8. Immerse the rat femoral bone specimen slices sequentially in 70%, 80%, 90%, 95%, and 100% ethanol, with each gradient for 2 min, gradually removing moisture.
9. Immerse the rat femoral bone specimen slices in pure xylene for transparency, followed by rinsing with running water to remove the xylene. Apply neutral mounting resin, covering the slides, gently tapping to remove air bubbles, and allowing the mounting medium to solidify to complete the mounting process.
10. Using the randomizr package in R, select 30 HE-stainedslides for each group and allocate 15 slides each to two researchers. Without informing them of the groupings, ask the researchers to calculate the empty lacunae rate of the slidesand collect the results for statistical analysis.

9. MicroCT analysis

1. After the modeling is completed, euthanize the rats. Remove the femurs for Hematoxylin-Eosin staining.
2. Soak the rat femurs in 4% paraformaldehyde for 24 h to preserve biological tissues as specimens. Place the rat femurs in the small animal-specific microCT device.
3. Set the microCT scanning conditions as follows: X-ray voltage: 80 kV, Current: 100 µA, Single exposure time: 50 ms, scanning resolution: 25 µm. Perform scans at intervals of 0.5°, focusing on the trabecular bone from beneath the cartilage to the upper part of the epiphysis as the region of interest.
4. Define the area above the rat femoral neck as the region of interest.
5. Perform coronal plane reconstruction of the scanning results using microCT device and collect bone morphometric measurements generated by the built-in software of the microCT device. Record the following observed indices: Total Volume (TV), Percent Bone Volume (BV/TV), Bone Surface/Volume Ratio (BS/BV), Structure Thickness (Tb.Th), Structure Separation (Tb.Sp), Number of Bone (Tb.N), Bone Density (BD).

10. Statistical analysis

1. Present the data as the mean ± standard deviation (SD). Conduct statistical analyses for micro-CT and quantitative histologic evaluations using the independent sample t-test. Consider a p-value < 0.05 as statistically significant.

Results

Histopathology analysis
Hematoxylin and eosin staining revealed that in the Control group and Control+Load group, bone trabeculae were intact and arranged regularly. Blood vessel endothelial cells were present in bone dimples, and the cell morphology appeared plump. In contrast, the Model group and Model+Load group exhibited fractured and disordered bone trabeculae, along with a significantly higher number of empty lacunae. The Model+Load group had more empty lacunae compared to the Model group. In...

Discussion

Currently, various animals, such as rabbits²⁵, rats²⁶, mice²⁷, pigs²⁸, broilers²⁹, ostriches⁸, and emus³⁰ can be used to establish models of femoral head necrosis. Among them, rats, mice, and rabbits are the most commonly used species. The rat, as a model for femoral head necrosis, offers numerous advantages. Rats are easy to feed and breed, grow rapidly, ...

Materials

Name	Company	Catalog Number	Comments
15ml centrifuge tube	Corning,USA	430791
5mm stainless steel bead	Gelisen,China	5mm
Acetic acid	Merck KGaA, Germany	64-19-7
Anhydrous alcohol	Merck KGaA, Germany	64-17-5
clay	Mincai stationery,China	102
Coverslip	Servicebio,China	WMWD-1818
Flat pressure bottle 10ml	BEHNCKE,China	MD10ml
Formic acid	Macklin Biochemical ,China	64-18-6
HE staining kit	Solarbio,China	G1120
HistoCore AUTOCUT	Leica, Germany	149AUTO00C1
Kinesio tape (elastic therapeutic tape)	Fuluo medicine,China	CL1819
Lipopolysaccharide	Solarbio,China	L8880
Lipopolysaccharides (LPS)	Selleck,USA	S7850
Manual carbon dioxide euthanasia box	Yuyan,China	LC-500-S1
Methylprednisolone sodium succinate,MPS	AbMole,China	M25573
MicroCT	Hiscan,China	Hiscan VM Pro
Neutral resin	Beijing Zhongshan Golden Bridge Biotechnology l ,China	ZLI-9555
Paraffin	Servicebio,China	WGHB-319213129
Paraformaldehyde	Servicebio,China	G1101-500ML
Potassium chloride	Macklin Biochemical ,China	7447-40-7
Slide	Servicebio,China	WG6012
Treadmill for Rats and  mice	Litc Life Science,USA	801
Xylene	Macklin Biochemical ,China	1330-20-7