Establishment and evaluation of a rat model of cervical spondylosis with Yin deficiency syndrome.

Am J Transl Res 2025;17(1):75-86 www.ajtr.org /ISSN:1943-8141/AJTR0161628 Original Article Establishment and evaluation of a rat model of cervical spondylosis with Yin deficiency syndrome Wenlong Yang1, Muqing Liu2, Fangming Liu1 Department of Pain Medicine, The First Affiliated Hospital of Shandong First Medical University and Shandong Provincial Qianfoshan Hospital, Jinan, Shandong, China; 2School of Acupuncture-Tuina, Shandong University of Traditional Chinese Medicine, Jinan, Shandong, China 1 Received October 31, 2024; Accepted December 26, 2024; Epub January 15, 2025; Published January 30, 2025 Abstract: Objective: To establish and evaluate a rat model of cervical spondylosis (CS) with yin deficiency syndrome. Methods: Thirty-six male Sprague-Dawley rats were randomly assigned to the control group, CS group, and CS with Yin deficiency syndrome (YCS) group (n = 12 per group). The control group underwent daily routine care for 37 days. The CS group underwent induction of cervical static-dynamic imbalance, followed by 30 days of standard care. The YCS group underwent cervical static-dynamic imbalance induction, followed by 7 days of sleep deprivation to model Yin deficiency cervical spondylosis. Behavioral performance, heart rate, blood pressure, mechanical pain thresholds, serum cAMP, cGMP, cAMP/cGMP levels, and the expression of collagen-II, Bcl-2, Bax, and Bcl-2/Bax in cervical intervertebral discs were analyzed at various time points. Results: Following CS induction, modeled rats exhibited significant changes in intervertebral disc structure, including misalignment of the annulus fibrosus, atrophy of the nucleus pulposus, rough cartilaginous endplate boundaries, and disc degeneration. Mechanical pain thresholds decreased. In the YCS group, compared with the CS and control groups, rats showed heightened excitability, dull fur, reddish mouth, lips, nose, paws, and tail, resistance to handling, slower weight gain, initial heart rate elevation followed by decline, and progressive blood pressure reduction. Serum cAMP and cAMP/cGMP ratios were significantly elevated, while cGMP levels were reduced. Collagen-II, Bcl-2, and Bcl-2/Bax levels decreased, and Bax levels increased. Conclusion: The established rat model of Yin deficiency syndrome aligns with clinical and traditional Chinese medicine characteristics, making it a promising model for studying Yin deficiency syndrome. Keywords: Yin deficiency syndrome, cervical spondylosis, male rats, model evaluation Introduction Cervical spondylosis (CS), characterized by cervical pain and activity limitation, is a degenerative disease of the cervical intervertebral discs. Its secondary pathological changes progressively affect the function of peripheral nerves, blood vessels, and surrounding tissues [1]. Based on clinical manifestations and pathological features, CS can be classified into cervical, nerve root, spinal cord, and other subtypes [2, 3]. The condition is common, primarily resulting from occupational factors and lifestyle-related issues [4]. With increasing work-related stress, the prevalence of CS has risen annually, affecting younger populations and imposing significant physical, psychological, and economic burdens on individuals, families, and society [5]. In traditional Chinese medicine (TCM), CS falls under the categories of “arthralgia”, “Xiang paralysis”, and “Xiang Qiang”. TCM recognizes the complexity and variability of CS symptoms. Clinical observations have noted that some patients with CS experience neck, shoulder, and arm pain that worsens at night, accompanied by yin deficiency symptoms such as hot flashes, night sweats, irritability, insomnia, and a fine pulse [6]. This indicates a strong correlation between CS and yin deficiency according to TCM theory. TCM treatments, including acupuncture, tuina, and the internal and external application of herbal medicines, have demonstrated unique advantages in managing CS by alleviating symptoms and restoring yin-yang balance. However, https://doi.org/10.62347/LEJV6241 Rat models for cervical spondylopathy with yin deficiency syndrome the absence of animal models tailored to the characteristics of TCM evidence has hindered in-depth research into the mechanisms underlying TCM therapies. This limitation has also restricted the broader clinical application of TCM in CS management [7-9]. In this context, the present study aims to establish an animal model of CS with TCM-specific characteristics of yin deficiency. This model will serve as an essential experimental platform to explore the TCM pathogenesis of CS, validate the efficacy and safety of TCM treatments, and provide a scientific basis for further studies. Materials and methods Animals and materials Animals: Thirty-six male Sprague-Dawley rats (200 ± 20 g) were obtained from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. (SCXKLu2019-0003, Shandong, China). The rats were housed in a clean room with controlled conditions: temperature of 23°C ± 2°C, relative humidity of 60% ± 10%, and a 12-hour lightdark cycle. All animals were ad libitum fed a standard solid diet, with three rats per cage. A 7-day acclimatization period preceded the experiment. Ethical statement: The experimental protocol was approved by the Ethics Committee of the First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital ([2019] no. S177). All animal procedures complied with the ARRIVE guidelines and relevant ethical regulations. Rats were euthanized by intraperitoneal injection of sodium pentobarbital (150 mg/kg) prior to autopsy. Reagents and equipment: Key materials included cAMP and cGMP kits (Wuhan Elabscience, E-EL-0056c, E-EL-0083c), 3% sodium pentobarbital, and hematoxylin-eosin (HE) stain (Beijing Dingguo Changsheng Biotechnology Co., Ltd.). Equipment used included a paraffin sectioning machine (LEICA RM2235, Germany), biological tissue spreading and baking machine (Beijing Dingguo Changsheng Biotechnology Co., Ltd.), biological tissue spreader (PHY-III, Wuhan Zhongtian Hongbo Medical Equipment Co., Ltd.), tabletop cryogenic centrifuge (Thermo Scientific Heraeus Multifuge X1R, USA), biochemical analyzer (HERA. Multiftige X1R), an ultra-low temperature freezer 76 (-80°C, Thermo Scientific FORMA700 SERIES), an optical microscope (OLYMPUS FSX 100), a sleep deprivation instrument (Beijing Xinxin XR-XS107), hair clippers (HC1066, Philips, Netherlands), a small animal noninvasive blood pressure detector (Beijing Softlung BP-2010A), an enzyme marker (BioTek Epoch, USA), BCA protein test kit (P0012, Beo Tianmei Biotechnology Co., Ltd., Shanghai, China). Antibodies used included anti-collagen-II (1:1000 dilution, ab188570, Abcam), anti-Bcl-2 (1:500 dilution, ab196495, Abcam), anti-Bax (1:1000 dilution, ab32503, Abcam), GAPDH (1:2000 dilution, E-AB-20059, Elabscience, Wuhan, China), and HRP-conjugated goat anti-rabbit IgG (1:5000 dilution, EF0002, Sparkjade, Shandong, China). Methods a (...truncated)