Name: The Creation of a Rat Model for Osteosarcopenia via Ovariectomy (Video) | JoVE Journal
Uploaded: 2025-02-21T12:40:00.000+00:00
Duration: 3 min 52 s
Description: Learn to create a rat model of osteosarcopenia using ovariectomy for aging research. This protocol enables study of bone and muscle loss mechanisms and supports therapeutic intervention testing.

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5 minutes

This work is supported by grants from (1) National Nature Science Foundation (82305275). (2) Liaoning Provincial Natural Science Foundation program (2022-YGJC-80 and 2022-YGJC-79). (3) High-level Chinese Medicine Key Discipline Construction Project of National Administration of TCM (zyyzdxk-2023040).

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.
<ol>
	<li>Keep the rats in the SPF animal room and follow all necessary procedures using sterilized equipment in a sterile environment.</li>
	<li>Administer the analgesic drug Tramadol Hydrochloride at a dose of 10 mg/kg (200&#160;&#181;L from 0.1mg/mL) via subcutaneous injection 30 min before surgery. Use 5% isoflurane dissolved in 2 L/min of oxygen for inhalation anaesthesia.</li>
	<li>Following the administration of anesthesia, carefully monitor the rat&#39;s breathing and pinch their toes to confirm the surgical plane of anesthesia. 
	NOTE: If there are any signs of spasms or convulsions, it is advisable to wait longer before proceeding.</li>
	<li>Position the rat on a resin workbench and provided a heating support.&#160;Secure its limbs, and remove the hair on both sides of its back using a trimmer&#160;ensuring that 150% of the area surrounding the surgical site is free of fur, and then drape the animal (Figure 2A). 
	NOTE: If the hair removal effect is not ideal, hair removal cream can be used for hair removal.</li>
	<li>Disinfect the area where the hair was removed using cotton balls soaked in both an iodine-based and alcohol. 
	NOTE: The process of surgical disinfection involves starting from the center and moving outwards in a circular pattern, typically repeated three times.</li>
	<li>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.</li>
	<li>Finding the ovary can be challenging at first. Start by locating the oviduct and tracing it to the ovary&#39;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.</li>
	<li>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&#39; 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.</li>
	<li>Release the hemostatic forceps and gently return the uterus to the abdominal cavity.</li>
	<li>Individually suture the skin and muscle layers using 5-0 absorbable surgical sutures made of polyethylene glycol (Figure 2D).</li>
	<li>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.</li>
	<li>Within 48 h postoperatively, administer Ketorolac Tromethamine at a dose of 1&#8211;2 mg/kg via subcutaneous injection twice a day.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Fully detach the femur by slicing the joint capsule upwards along the femur. Then, eliminate the nearby muscle and ligament tissue.</li>
</ol>
3. Pathological examination
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place the sample in paraffin wax. Section the samples for the routine histological examination27.</li>
</ol>
4. Statistical analysis
<ol>
	<li>Present the continuous variables as mean &#177;&#177; 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 &#60; 0.05. Use appropriate data analysis software to perform data analyses.</li>
</ol>

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</ol>

Each author declares no competing financial interests.

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...

Osteosarcopenia is a multifaceted degenerative condition that encapsulates the clinical manifestations of both osteoporosis and sarcopenia1,2,3,4. 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 mass5,6. Maryam&#39;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 society10,11. 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 interventions13,14. 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 necrosis17,18 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 osteoporosis19,20.
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 sarcopenia23,24, 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.

<table><tbody><tr><td>Double lion Irradiated Rodent Diet</td><td>Suzhou Shuangshi Experimental Animal Feed Technology Co., Ltd.</td><td>GB 14924.3</td><td>Animal feed</td></tr><tr><td>Disposable medication changing tray</td><td>Yangzhou Chenglin Medical Technology Co., Ltd.</td><td>RVnpFXLc</td><td></td></tr><tr><td>Dual Energy X-ray Bone Densitometer</td><td>Xuzhou PinyuanElectronic Technology Co., Ltd.</td><td>DXA-800E</td><td></td></tr><tr><td>Iodine</td><td>Shanghai Likang Sterilization Hi-Tech Co., Ltd.</td><td>LK-310512</td><td></td></tr><tr><td>IVCs rat cage</td><td>Suzhou Monkey King Animal Experimental Equipment Technology Co., Ltd.</td><td>HH-MMB-2</td><td>Animal barrier</td></tr><tr><td>Ketorolac tromethamine Injection</td><td>Yunnan Longhai Natural Plant Medicine Co., Ltd.</td><td>H20233312</td><td></td></tr><tr><td>Penicillin sodium</td><td>North China Pharmaceutical Group Limited Liability&amp;nbsp; Co., Ltd.</td><td>H13020654</td><td></td></tr><tr><td>Sterile cotton ball</td><td>Henan Piaoan Group Co., Ltd</td><td>20140017</td><td></td></tr><tr><td>Straight Mayo scissors</td><td>Shenzhen Huayang Biotechnology Co., Ltd.</td><td>18-0410</td><td></td></tr><tr><td>Straight needle holder</td><td>Shanghai Simplicity Biotechnology Co., Ltd.</td><td>32100-14</td><td></td></tr><tr><td>Suture line</td><td>Shenzhen Huayang Biotechnology Co., Ltd.</td><td>18-5902</td><td></td></tr><tr><td>Suture needle</td><td>Shenzhen Huayang Biotechnology Co., Ltd.</td><td>18-5036</td><td></td></tr><tr><td>Syringes</td><td>Shenzhen Huayang Biotechnology Co., Ltd.</td><td>21-3021</td><td></td></tr><tr><td>Tramadol hydrochloride</td><td>Grunenthal GmbH</td><td>H20140812</td><td></td></tr></tbody></table>

the creation of a rat model for osteosarcopenia via ovariectomy

The Creation of a Rat Model for Osteosarcopenia via Ovariectomy

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.

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...

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

Osteosarcopenia (OS), a complex degenerative disorder, is characterized by the concurrent decline in skeletal muscle mass and bone mineral density (BMD), ...

the-creation-of-a-rat-model-for-osteosarcopenia-via-ovariectomy

Research

JoVE Journal

Medicine

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Learn to create a rat model of osteosarcopenia using ovariectomy for aging research. This protocol enables study of bone and muscle loss mechanisms and supports therapeutic intervention testing.

The Creation of a Rat Model for Osteosarcopenia via Ovariectomy

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