The Creation of a Rat Model for Osteosarcopenia via Ovariectomy

Summary

This current protocol outlines a procedure for creating a rat model of osteosarcopenia using ovariectomy.

Abstract

Osteosarcopenia (OS), a complex degenerative disorder, is characterized by the concurrent decline in skeletal muscle mass and bone mineral density (BMD), posing an enormous health hazard for the elderly population. Despite its clinical relevance, the pathophysiological mechanisms underlying OS are not fully understood, underscoring the necessity for a deeper comprehension of its etiology to facilitate effective treatment strategies. The development of a reliable animal model is pivotal in this endeavor. This study presents a refined protocol for the induction of postmenopausal osteosarcopenia in rats through bilateral ovariectomy, a method known to accelerate the onset of age-related muscle and bone loss. In this study, rats aged 12 weeks were stratified by body weight and randomly assigned to either a sham operation group or an ovariectomized (OVX) group. Tissue samples from the quadriceps and triceps muscles of the left hind limb, as well as the left femur, were systematically collected at 4, 8, and 12 weeks post-surgery. This methodical approach ensures a comprehensive evaluation of the effects of ovariectomy on muscle and bone health. Histological evaluation of muscle fiber atrophy and femoral morphology was conducted using hematoxylin and eosin (HE) staining, while bone mineral density was quantified using dual-energy X-ray absorptiometry (DXA). The temporal progression of OS was meticulously monitored at the aforementioned intervals, providing insights into the dynamic interplay between muscle and bone degeneration. This model not only accurately reflects the clinical manifestations of OS but also serves as a robust platform for investigating novel therapeutic approaches and their underlying mechanisms.

Introduction

Osteosarcopenia is a multifaceted degenerative condition that encapsulates the clinical manifestations of both osteoporosis and sarcopenia^1,2,3,4. Osteoporosis, a prevalent skeletal disorder, is characterized by diminished bone mass, compromised microarchitecture, and heightened susceptibility to fractures. Sarcopenia, often referred to as muscle wasting syndrome, is typified by a reduction in muscle strength and mass^5,6. Maryam's⁷ findings revealed that osteosarcopenia increased the risk of death by 30% over sarcopenia alone and by 8% over low BMD alone. Research has shown that 16.4% of community-dwelling individuals aged 60 and above are affected by osteosarcopenia⁸. In South Korea, the incidence of osteosarcopenia among elderly individuals aged 60 and above who have sustained hip fractures is reported to be 27.2%⁹. Individuals with OS face higher risks of falls, fractures, hospitalization, and institutionalization, which burdens the healthcare system and society^10,11. Given the gravity of these consequences, it is crucial to develop and implement efficient measures for the prevention and treatment of OS. Despite the urgency, research in this field remains nascent, with ongoing debates surrounding diagnostic criteria and the efficacy of various treatment modalities. The development of reliable animal models is thus essential for dissecting the pathogenesis of OS and uncovering the molecular underpinnings that could inform more efficacious treatment approaches.

Currently, commonly used models for preclinical studies on osteosarcopenia include the aging model, which simulates the human aging process without drug intervention. This approach is closer to the natural process and is cost-effective; however, it demands a significant investment of time for maturation¹². The chemical drug injection method offers certain benefits, such as a short modeling cycle, stable results, and low cost. However, it also presents challenges, including the precise determination of hormone dosage, the technical skill required for injection, and the variable effects of hormonal interventions^13,14. Genetic engineering models may involve genetically modified organisms that can be both genetically defective and costly. Although these models are highly specific, they are notably more complex and expensive to produce¹⁵. Disuse models simulate the effects of prolonged bed rest on clinical patients¹⁶. Disuse models are effective and cost-efficient for addressing muscle loss but are associated with complications such as blood clots and pressure sores. These models are routinely monitored to prevent limb necrosis^17,18 and hormone-deficient models; there is a prevailing agreement within the scientific community that bilateral ovariectomy serves as an effective method for establishing an animal model of osteoporosis^19,20.

Research indicates that bone and muscle tissues can also interact with each other through autocrine, endocrine, and paracrine mechanisms²¹. The accumulation of adipose tissue in muscle and bone marrow serves as an indicator of reduced bone and muscle mass in the context of osteosarcopenia². Sarcopenia in older adults is directly associated with a reduction in bone density and the deterioration of bone microarchitecture. Additionally, diminished muscle mass serves as an independent risk factor for the degradation of bone microstructure²². This methodology has been recognized as a viable strategy for the modeling of sarcopenia^23,24, which could potentially serve as a combined model for both conditions²⁵. Despite the limited body of research concerning the application of ovariectomy as a means to induce osteosarcopenia, this approach demonstrates potential efficacy. The benefits of utilizing ovariectomy in preclinical studies encompass a swift modeling process, the elimination of pharmacological interventions, the creation of a stable experimental model, straightforward implementation, and cost-effectiveness.

The present study aims to delineate the procedure for creating a preclinical model in female rats through the removal of a segment of both fallopian tubes and ovaries in non-pregnant individuals. This approach serves as a valuable tool for investigating the molecular underpinnings of OS and for assessing the therapeutic benefits of interventions in a controlled experimental setting.

Protocol

Female Sprague Dawley rats (n = 36), aged 12 weeks and weighing approximately 200-240 g, were housed individually in ventilated cages in a specific-pathogen-free (SPF) animal room with a 12-h light/dark cycle. They had free access to SPF feed and sterile water. The rats were allowed to acclimate to the environment for a week before the experiments. Using random allocation, the rats were divided into ovariectomized (OVX) groups (each with 6 rats) and sham groups (each with 6 rats) for 4, 8, and 12 weeks post-surgery. All animal procedures were carried out following the approved guidelines of the animal welfare committee at Liaoning University of Traditional Chinese Medicine (No. 21000042021040).

1. Ovariectomy in rats

NOTE: The surgical apparatus used in this protocol is in Figure 1.

1. Keep the rats in the SPF animal room and follow all necessary procedures using sterilized equipment in a sterile environment.
2. Mix sodium pentobarbital, a white powder, with distilled water or a 0.9% normal saline solution to create an anesthetic solution. The standard dose is 30 mg/kg; fill the syringe accordingly.
   NOTE: It is important to note that the solution is unstable and should be used immediately. Prepare the required amount for one experiment at a time.
3. Elevate the rat's abdomen above its head to shift the viscera to the upper abdomen. Using the dominant hand, position a syringe 1-1.5 cm from the left (or right) side of the midline of the abdomen and insert it at a 45° angle into the rat's body. After the drug solution is administered, rotate the needle and then withdraw.
4. Following the administration of anesthesia, carefully monitor the rat's breathing and pinch their toes to confirm that it is completely anesthetized.
   NOTE: If there are any signs of spasms or convulsions, it is advisable to wait longer before proceeding.
5. Position the rat on the operating table, secure its limbs, and remove the hair on both sides of its back using a trimmer (Figure 2A).
   NOTE: If the hair removal effect is not ideal, hair removal cream can be used for hair removal.
6. Disinfect the area where the hair was removed using cotton balls soaked in Iodine.
   NOTE: The process of surgical disinfection involves starting from the center and moving outwards in a circular pattern, typically repeated three times.
7. Make an incision on the back, about 1.0 cm away from the centerline. Make the incision close to the juncture between the rib cage curvature and the border of the spinal column, slightly lower by 0.5-1 cm by separating the skin, fascia, and muscle on both sides (Figure 2B).
   NOTE: For accessing the abdominal cavity through the weaker muscle layer of the posterior abdominal wall, the incision is kept as minimal as feasible.
8. Finding the ovary can be challenging at first. Start by locating the oviduct and tracing it to the ovary's terminal end, which is encased in a layer of loose adipose tissue.
   NOTE: The right ovary is positioned on the side of the 4th to 5th lumbar vertebrae, 7-12 mm behind the kidney and 15 mm away from the center line. The left ovary is situated on the side of the 5th to 6th lumbar vertebrae, 3-5 mm behind the kidney and 11 mm from the center line.
9. Carefully lift the ovary and the end of the oviduct out of the body (Figure 2C). Apply the hemostatic forceps at the most constricted region between the uterine end and the ovary. Use a surgical thread to tie it off, and then excise the ovary completely with scissors.
   NOTE: It is crucial to be gentle when handling the oviduct and the uterus during the procedure, avoiding excessive pulling. The ligature used prior to ovariectomy must be firmly secured, as the soft lipid tissue around the ovary can easily cause it to come loose. This precaution is necessary to prevent postoperative bleeding, which could result in the rats' death. In the sham group, adipose tissue of equal volume and size adjacent to the ovary was excised, followed by suturing of the muscle and skin.
10. Release the hemostatic forceps and gently return the uterus to the abdominal cavity.
11. Administer penicillin to the abdominal wounds where the ovaries and fallopian tubes are ligatured to avoid infection.
    NOTE: Administer penicillin 80,000 units/rat once a day for 3 consecutive days.
12. Individually suture (size 3-0) the skin and muscle layers (Figure 2D).
    NOTE: Sterilization should be conducted 24-48 h post-surgery, spaced 1-2 days apart.
13. Place the rat back in a sanitized cage and monitor it until it completely regains consciousness from the anesthesia.
    NOTE: Continue to provide heat support during the procedure until the animal has fully recovered from anesthesia.
14. To avoid wound infection, administer the rats in each group with an intramuscular injection of penicillin sodium 80,000 units/rat once a day for 3 consecutive days²⁶.

2. Collection of bone tissue and muscle tissues

NOTE: Rats were euthanized with an overdose of sodium pentobarbital (100-200 mg/kg) at 4, 8, and 12 weeks after the modeling surgery. A total of 36 samples were collected.

1. Expose the triceps brachii and quadriceps muscles of the left calf. Carefully identify and dissect these muscles at their points of origin and endpoint to preserve their integrity. Following this, record and calculate the average of the wet weights of the muscles to determine the wet weight coefficients of the muscles.
   NOTE: Animal body weight and skeletal muscle wet weight coefficient = rat muscle wet weight/body weight.
2. Fully detach the femur by slicing the joint capsule upwards along the femur. Then, eliminate the nearby muscle and ligament tissue.

3. Pathological examination

1. Immerse the muscle tissues in a receptacle containing a 10% neutral buffered formalin solution for a duration of 24 h. Following this, rinse the muscle tissues extensively under flowing water to remove the fixative.
2. Place the left femur in a 4% paraformaldehyde solution for 1 week, then soak it in a sufficient amount of ethylenediaminetetraacetic acid (EDTA) decalcification solution to remove calcium deposits, with the buffer being changed daily.
3. Measure the bone mineral density values using a dual-energy X-ray absorptiometry (DXA) bone densitometer. Place the femur in a dual-energy X-ray. Set the measurement accuracy to Fine, adjusting the mode to Small Animal-Specific Mode, and analyze the BMD of rat femurs using the accompanying BMD analysis software.
4. Place the sample in paraffin wax. Section the samples for the routine histological examination²⁷.

4. Statistical analysis

1. Present the continuous variables as mean ±± standard deviation (SD) and compare between the two groups using the independent sample t-test. All statistical analyses followed a two-sided approach, with statistical significance set at P < 0.05. Use appropriate data analysis software to perform data analyses.

Results

This protocol provides a detailed description of the bilateral ovariectomy procedure for establishing a rat model of Osteosarcopenia. Figure 3 demonstrates a decrease in the wet weight coefficient of the quadriceps muscle in the OVX group in comparison to the sham group. Although there was no statistically significant variance in BMD between the two groups 4 weeks after surgery, the BMD in the OVX group was significantly lower than that in the sham-operated g...

Discussion

The bilateral ovariectomized animal model is instrumental in elucidating the mechanisms underlying osteosarcopenia and assessing potential therapeutic interventions. Ovariectomy-induced osteoporosis in rats, which mirrors the abrupt decrease in estrogen levels seen in postmenopausal women, is commonly employed as a model for osteoporosis research. Furthermore, research has highlighted a significant association between osteoporosis and sarcopenia in older individuals, with concurrent muscle and bone loss frequently observ...

Materials

Name	Company	Catalog Number	Comments
Double lion Irradiated Rodent Diet	Suzhou Shuangshi Experimental Animal Feed Technology Co., Ltd.	GB 14924.3	Animal feed
Disposable medication changing tray	Yangzhou Chenglin Medical Technology Co., Ltd.	RVnpFXLc
Dual Energy X-ray Bone Densitometer	Xuzhou PinyuanElectronic Technology Co., Ltd.	DXA-800E
Iodine	Shanghai Likang Sterilization Hi-Tech Co., Ltd.	LK-310512
IVCs rat cage	Suzhou Monkey King Animal Experimental Equipment Technology Co., Ltd.	HH-MMB-2	Animal barrier
Penicillin sodium	North China Pharmaceutical Group Limited Liability  Co., Ltd.	H13020654
sodium pentobarbital	Sigma-Aldrich, St. Louis, MO	P3761-5G
Sterile cotton ball	Henan Piaoan Group Co., Ltd	20140017
Straight Mayo scissors	Shenzhen Huayang Biotechnology Co., Ltd.	18-0410
Straight needle holder	Shanghai Simplicity Biotechnology Co., Ltd.	32100-14
Suture line	Shenzhen Huayang Biotechnology Co., Ltd.	18-5902
Suture needle	Shenzhen Huayang Biotechnology Co., Ltd.	18-5036
Syringes	Shenzhen Huayang Biotechnology Co., Ltd.	21-3021