Continuous mechanical compression-induced intervertebral disc degeneration and pain in the caudal spine in rats

Abstract

Background

Chronic low back pain imposes substantial social and economic burdens due to its rising prevalence and associated disability. Intervertebral disc degeneration is the primary driver of discogenic back pain, though the mechanistic links between degenerative changes and persistent pain remain incompletely defined. Aberrant mechanical loading is recognised as a key trigger of disc degeneration. This work built a caudal spine compression rat model to explore how the P2Y2 receptor mediates load-induced disc degeneration and associated pain.

Methods

Fifty-six eight-week-old male laboratory rats were randomised into four cohorts: 2-week sham surgery, 2-week axial compression, 3-week sham surgery, and 3-week axial compression. An external fixator delivered 8 N continuous caudal spine compression to experimental animals, while sham rats received identical implantation without compressive force. Degenerative severity was evaluated via magnetic resonance imaging and two histological staining protocols. Von Frey filament testing measured mechanical pain sensitivity at baseline and multiple time points after device removal. Immunofluorescence and Western blotting detected target protein levels within degenerative disc tissue collected from both human surgical patients and experimental rats.

Results

Intervertebral disc degeneration was markedly aggravated after two weeks of compression and worsened further with three weeks of sustained loading. Sham-operated rats only had transient mechanical hypersensitivity at day 3 and fully recovered by days 9–15. By contrast, compressed rats maintained persistently lowered pain thresholds over the whole observation window. The P2Y2 receptor was elevated in degenerative disc tissue and co-localised with YAP and TAZ in human and rat disc core cells. In human samples, receptor levels rose alongside advancing degeneration; YAP and TAZ were elevated in moderately degenerated tissue and decreased in severely degenerated specimens.

Conclusions

Prolonged mechanical compression of the rat caudal spine produces disc degeneration and pain. The findings suggest that the P2Y2 receptor and downstream Hippo pathway mediators YAP and TAZ are involved in the degenerative process, offering a basis for future mechanistic studies.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12891-026-10252-x.

Introduction

Low back pain (LBP) is a prevalent health issue worldwide, and a majority of individuals experience at least one episode of acute LBP in their lifetime. The incidence of LBP has been increasing annually due to aging populations and sedentary lifestyles resulting from technological advancements [1]. LBP is now one of the major causes of disability [2]. Intervertebral disc degeneration (IVDD) is a critical contributor to LBP and is known as discogenic low back pain (DLBP) [3, 4]. There are many causes of IVDD, and abnormal mechanical loading is recognized as one of the main causes [5–7]. Excessive mechanical loading disrupts disc structure and triggers a series of cell-mediated responses that lead to further disc destruction [7]. However, the mechanisms underlying IVDD and DLBP remain unclear, and outcomes have been unsatisfactory.

Animal models of disc degeneration have been established through static compression of the rat tail caudal intervertebral disc (IVD) [8–10]. However, studies on pain behavior in this model are lacking in most of these models. Combining mechanical compression-induced disc degeneration with pain behavioral evaluation would have significant clinical and translational relevance. Nonphysiological loading is considered a crucial factor in the development and progression of disc degeneration, including aging and reduced nutrient supply [4]. Under mechanical loading, lactate and adenosine triphosphate (ATP) levels in the annulus fibrosus and nucleus pulposus were significantly increased [11]. ATP not only supplies energy to cells but also regulates various cellular biological activities by activating P2X or P2Y receptors (P2XRs or P2YRs) [11, 12]. Recent studies have shown increased expression of P2X7 receptors in degenerated intervertebral discs [13, 14]. P2Y2, another important ATP subtype, has been extensively studied and shown to play a role in cell proliferation, apoptosis, migration, differentiation, and pain [15–23]. However, the role of P2 receptors in IVDD and DLBP remains unclear. Further research is needed to determine whether P2Y2R play an important role in mechanical loading-induced disc degeneration and are involved in the development of DLBP.

The Hippo signaling pathway is known to play a crucial role in various cellular processes, such as cell proliferation, apoptosis, migration, and differentiation. This pathway is composed of several key tandem kinases, including YAP and TAZ, which are phosphorylated and remain in the cytoplasm without exerting any biological effects [24]. However, dephosphorylation of YAP/TAZ allows them to enter the nucleus and activate transcriptional processes, leading to cell proliferation, inhibiting apoptosis, or inducing gene expression. Recent studies have shown that dysregulation of the Hippo-YAP/TAZ pathway is associated with IVDD, which is a common cause of back pain [25]. For instance, ATP can activate P2Y2R in human macrophages to promote interleukin 6 (IL-6) transcription, which activates YAP1 through tyrosine phosphorylation in nucleus pulposus (NP) cells [26]. Overexpression of YAP1 decreases the expression of collagen type II and aggrecan while increasing the level of matrix metallopeptidase 13 (MMP13), leading to IVD degeneration [27]. Moreover, high hydrostatic pressure can trigger apoptosis in disc cells through the Hippo-YAP/TAZ pathway [28]. YAP was also shown to be associated with NPC senescence, and its interference through YAP accelerated cell senescence [29]. However, overexpression of YAP reversed the exogenously induced degeneration of nucleus pulposus cells [30]. In conclusion, the role and mechanism of YAP/TAZ activation in IVDD are not yet fully understood and require further investigation.

The objective of this study was to develop a reliable rat model of disc degeneration and pain induced by mechanical stress, specifically caudal disc degeneration, and assess the mechanical pain threshold in the rat tail. Additionally, this study aimed to investigate the alterations in P2Y2R, YAP, and TAZ protein expression in the model by analyzing rat and human degenerated disc nuclei.

Materials and methods

Experimental animals

Fifty-six 8-week-old male Sprague‒Dawley rats (SPF grade weighing 310 ± 10 g) were selected for this study and provided by Yunnan Commitment to Science and Technology Co., Ltd., with Experimental Animal Certificate No. SCXK [Beijing] 2019-0010. The rats were housed in cages with ad libitum access to water and food, and the temperature and relative humidity were controlled. The animal room was artificially lit from 7 a.m. to 7 p.m. The experimental procedures were conducted at the Experimental Zoology Department of the 920th Hospital of Joint Logistic Support Force, and the animal experiments were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health of China. All human tissue sampling and in vivo animal procedures described in this manuscript were reviewed and approved by the Ethics Committee of the 920th Hospital of Joint Logistic Support Force (Approval No. 2016002). A priori sample size calculation was performed via G*Power software (α = 0.05, power = 0.80, Cohen’s f = 0.5), with 14 rats per group (total n = 56) reserved to compensate for potential animal attrition.

Collection of nucleus pulposus tissue from human degenerated discs

This experiment included 39 patients with lumbar disc degeneration between 22 and 73 years of age, as shown in Table 1. During surgery, discs were collected from patients, and nucleus pulposus (NP) tissue was isolated for double-label fluorescent immunohistochemistry and Western blot experiments. Normal disc tissue was obtained from patients with thoracolumbar spinal fractures who required decompression and fusion surgery, and was confirmed as Pfirrmann grade I on preoperative MRI. Due to the limited sample size, this tissue was only used for immunofluorescence experiments. Disc degeneration was graded via preoperative MRI using the Pfirrmann classification system [31]. Nucleus pulposus tissues were categorized into four groups: Pfirrmann grade I as normal, grade II as mild degeneration (LIDD), grade III as moderate degeneration (MIDD), and grades IV–V as severe degeneration (SIDD).

Table 1.

Patients ID 19–24 were classified as MIDD

Patient ID	Gender	Age	Disc level	Classification
1	Female	57	L3-L4	LIDD
2	Female	57	L4-L5	LIDD
3	Male	22	L4-L5	LIDD
4	Female	24	L1-L2	LIDD
5	Male	23	L1-L2	LIDD
6	Female	55	L1-L2	LIDD
7	Female	33	L2-L3	LIDD
8	Female	31	L5-S1	LIDD
9	Male	29	L4-L5	LIDD
10	Female	36	L4-L5	LIDD
11	Female	39	L3-L4	LIDD
12	Male	42	L5-S1	LIDD
13	Male	73	L3-L4	MIDD
14	Female	46	L3-L4	MIDD
15	Female	58	L3-L4	MIDD
16	Male	53	L5-S1	MIDD
17	Female	58	L4-L5	MIDD
18	Male	73	L4-L5	MIDD
19	Male	52	L4-L5	LIDD
20	Male	47	L4-L5	LIDD
21	Female	45	L5-S1	LIDD
22	Female	38	L4-L5	LIDD
23	Female	62	L4-L5	LIDD
24	Female	59	L5-S1	LIDD
25	Male	70	L3-L4	SIDD
26	Female	54	L5-S1	SIDD
27	Female	61	L4-L5	SIDD
28	Male	61	L4-L5	SIDD
29	Female	52	L4-L5	SIDD
30	Male	52	L1-L2	SIDD
31	Female	63	L4-L5	SIDD
32	Female	65	L4-L5	SIDD
33	Female	58	L4-L5	SIDD
34	Male	60	L5-S1	SIDD
35	Female	67	L4-L5	SIDD
36	Male	63	L5-S1	SIDD
37	Male	26	L3-L4	Control
38	Female	29	L3-L4	Control
39	Male	25	L4-L5	Control

Experimental methods

Establishment of the rat IVDD model

Fifty-six male SD rats of SPF grade were randomly divided into four groups (n = 14/group): 2- or 3-week sham-operated (2WS, 3WS) or experimental (2WD, 3WD) groups. Under anesthesia (40 mg/kg pentobarbital sodium, i.p.), an Ilizarov device was fixed to the tail using Kirschner wires. The target intervertebral discs (Co8–Co9–Co10) were palpated and marked. After identifying the entry points on the 8th and 9th vertebrae, the Ilizarov device was affixed using 0.8 mm Kirschner wires drilled at a 45°angle into the vertebral bodies. Four springs delivered compressive force to each experimental rat; each spring exerted 2 N, creating a total load of 8 N. The 2WD group received compression for 2 weeks, while the 3WD group underwent loading for 3 weeks. Sham-operated animals underwent identical procedures without spring compression. Postoperatively, penicillin (40,000 U/kg/day) was administered intramuscularly for 3 days. Upon completion of the compression period, the device and K-wires were aseptically removed following disinfection with iodophor.

MRI examination

In each group, MRI was performed two days after the removal of the Ilizarov device and Kirschner wires. Using a 3.0T MRI scanner (Philips Ingenia 3.0T), rats were anesthetized by intraperitoneal injection of 2% pentobarbital sodium (40 mg/kg). Upon loss of pain response, each rat was placed prone on a plastic pallet with its tail straightened and secured parallel to the pallet using paper tape. Following localization of the 8th and 9th vertebrae, sagittal T2-weighted MRI of the caudal spine was acquired with a spin-echo sequence (TR 3,000 ms, TE 600 ms, FOV 80 mm × 80 mm, slice thickness 2 mm, no fat saturation). Disc images were evaluated by three blinded observers according to the Pfirrmann classification, and the mean gray value of the nucleus pulposus was quantified using ImageJ.

Histological evaluation

All animals were euthanized by intraperitoneal injection of an overdose of pentobarbital sodium (150 mg/kg). Disc tissues were fixed in 4% PFA, decalcified in 10% EDTA, and paraffin-embedded. Sagittal Sect.  (5 μm) were stained with H&E or safranin O–fast green (SO-FG). Degeneration was evaluated using Masuda [32] (H&E) and Tam [33] (SO-FG) scoring systems by three blinded investigators.

Mechanical pain threshold test

All rats were acclimated to the test environment for 30 min daily over two days prior to behavioral assessment. Mechanical pain thresholds at the caudal segment were measured preoperatively (Pre, baseline) and on days 3, 6, 9, 12, and 15 post-frame removal, between 8:00 AM and 12:00 PM. Von Frey filaments (Aesthesio^®) were applied perpendicularly to the ventral tail surface using an up-down paradigm: the 10 g filament was applied three times (5 s each, 15 s intervals). A positive response was defined as rapid tail withdrawal, shaking, licking, or retraction. The minimal filament force eliciting a positive response was recorded as the mechanical threshold. Mechanical pain thresholds were assessed by an experimenter who was blinded to the group assignments, ensuring objective data collection.

Immunofluorescence (IF) analysis

Human and rat disc tissues were fixed, decalcified, embedded, and sectioned (3 μm). After antigen retrieval and permeabilization, sections were incubated with primary antibodies against P2Y2 (Santa Cruz, sc-518019, 1:200), YAP1 (Novus, NB100-58358, 1:100), and TAZ (Novus, NB110-58359, 1:50), followed by fluorescent secondary antibodies (ZenBio, 1:100). Nuclei were counterstained with DAPI. Images were acquired using a BZ-9000 microscope (Keyence).

Western blot analysis

NP tissues from rats and patients were homogenized in RIPA buffer. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against YAP (CST, 1:1000), TAZ (Santa Cruz, 1:1000), P2Y2 (Santa Cruz, 1:1000), and GAPDH (Sigma, 1:5000). Band intensities were quantified using ImageJ.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.0. Continuous data are expressed as mean ± SEM, and nonparametric data as median (IQR). Normality was assessed using appropriate tests (e.g., Shapiro–Wilk or Kolmogorov–Smirnov). For two-group comparisons, normally distributed data were analyzed with the independent samples t-test (or Welch’s t-test for unequal variances), while non-normal data were compared using the Mann–Whitney U test. Multi-group comparisons were conducted using one-way ANOVA followed by Tukey’s HSD test for parametric data, or the Kruskal–Wallis test with Dunn’s post hoc test for nonparametric data. Repeated-measures ANOVA or its nonparametric equivalent was applied for longitudinal behavioral data. Pfirrmann grades and histopathological scores were analyzed using nonparametric methods. A p-value < 0.05 was considered statistically significant.

Results

The degree of caudal disc degeneration in rats worsened with increasing compression time

The severity of caudal disc degeneration in rats progressed with prolonged compression time. A disc degeneration model was established using an Ilizarov device for external compression (Fig. 1a–c). Following device removal, T2-weighted MRI was performed and evaluated via Pfirrmann grading. No significant difference in degeneration was observed between the 2WD and 2WS groups (p > 0.05, n = 8; Fig. 2a, b). In contrast, the 3WD group exhibited significantly more severe degeneration compared to both the 3WS group (p < 0.01) and the 2WD group (p < 0.05; Fig. 2a, b). Quantitative analysis of nucleus pulposus signal intensity on MRI revealed significantly lower grayscale values in the 3WD versus 3WS group (p < 0.001), and notably, unlike the Pfirrmann scores, also showed a significant reduction in the 2WD compared to the 2WS group (p < 0.01; Fig. 2a, c).

Fig. 1.

Establishment and Evaluation of IVDD Model. a Close-up views of experimental discs under unloaded conditions. b Close-up views of experimental discs under loaded conditions. c Schematic diagram of the flow of animal experiments

Fig. 2.

IVDD was assessed by Pfirrmann grading of the T2 MRI image. a Representative T2- weighted sagittal MRIs. b Changes in the Pfirrmann classification score (grades: 1–5) and in mean gray values of all pixels in the disc NP. (c) The gray value of the intervertebral disc was measured by ImageJ. n=8, **p < 0.01, ***p < 0.001, #p < 0.05

Following magnetic resonance imaging (MRI) and behavioral assessments, disc tissues from the target segments were processed for histological evaluation using hematoxylin–eosin (HE) and safranin O–fast green (SO–FG) staining (Fig. 3a, b). Histological analysis revealed degenerative alterations in both compression groups (2WD and 3WD), with more pronounced degeneration observed in the 3WD group across multiple structural features.

Fig. 3.

Histological evaluation profile in the unloaded sham, 14-day loaded 2W, and 21-day loaded 3W groups. a, b Representative HE and Safranin-O staining images of the whole experimental disc and NP, scale bar = 200 μm,100 μm or 50 μm. c, e-h Changes in the Masuda degenerative score. d, i-m Changes in the Tam grading scale. n=8, *p < 0.05, **p < 0.01, ***p < 0.001

HE-based evaluation focused on four morphological domains: the number of cells in the NP, the AF, the border between the AF and the NP and the stroma of the NP. No significant intergroup difference in NP cellularity was detected between compressed and sham-operated animals (p > 0.05, n = 8; Fig. 3e). In contrast, the 3WD group exhibited significantly exacerbated AF degeneration relative to the sham controls (p < 0.001; Fig. 3f), while the 2WD group showed no statistically significant change. Degeneration at the AF–NP junction was significantly elevated in both compressed groups (2WD: p < 0.01; 3WD: p < 0.01; Fig. 3g), as were abnormalities in NP stromal architecture (2WD: p < 0.05; 3WD: p < 0.01; Fig. 3h), with the 3WD group consistently demonstrating more severe involvement.

SO–FG staining further enabled quantitative assessment across five degenerative characteristics. Disruption of NP structure was significant in both the 2WD and 3WD groups (p < 0.05 and p < 0.01, respectively; Fig. 3i). NP cleavage was significantly increased exclusively in the 3WD group (p < 0.05; Fig. 3j). No significant differences were detected in AF architecture among the groups (Fig. 3k). AF fissuring was significantly aggravated only in the 3WD group (p < 0.05; Fig. 3l), paralleled by heightened degeneration along the AF–NP interface (p < 0.05; Fig. 3m).

These observations were corroborated by comprehensive degeneration scoring using the Masuda and Tam classification systems for HE- and SO–FG-stained sections, respectively. Total degeneration scores were significantly higher in the 2WD group compared to the 2WS controls (p < 0.05), and markedly more severe in the 3WD group relative to the 3WS group (p < 0.001; Fig. 3c, d).

Mechanical compression causes disc degeneration with associated pain

Mechanical pain thresholds were measured at the caudal target segment preoperatively and on days 3, 6, 9, 12, and 15 after removal of the Ilizarov device (Pre, D3, D6, D9, D12, D15; Fig. 4a, b). Thresholds were significantly lower on D3 compared to baseline in all groups (p < 0.05, n = 8). The 2WS group showed reduced thresholds on D3 and D6 (p < 0.05), but returned to preoperative levels thereafter. The 3WS group exhibited a significant decrease only on D3 (p < 0.05). The 2WD group exhibited sustained hypersensitivity from D3 to D15 compared to its own baseline. Furthermore, on D15, its mechanical pain threshold was significantly lower than that of the 2WS group (p < 0.05). Similarly, the 3WD group had significantly lower thresholds than the 3WS group from D6 to D15 (p < 0.05). No significant differences were observed between the 2WS and 3WS groups, or between the 2WD and 3WD groups at any time point (p > 0.05).

Fig. 4.

Mechanical pain thresholds of rats were examined by von Frey cilia needle at different time points before and after surgery. a Pain thresholds of the four groups in Pre, D3, D6, D9, D12 and D15, where "*" indicates a statistical difference with the comparison of pre-operative (Pre), and "#" indicates a statistical difference with the control group at the same time point. n=8, *p < 0.05,**p＜0.01, #p < 0.05,##p＜0.0; b Representative experimental photograph illustrating the von Frey mechanical stimulation site on the rat caudal intervertebral disc for pain threshold testing. The inset shows the von Frey filament probe used in the assay

Increased expression of P2Y2R in human degenerated disc nucleus pulposus tissue and its coexpression with YAP and TAZ

Fluorescence immunohistochemistry showed that the expression of P2Y2R was significantly increased in the group with mild IVDD (LIDD) compared to the normal controls (p < 0.05, n = 3, Fig. 5a, b). Double-labeled fluorescence immunohistochemistry showed that P2Y2R was expressed in the same cells as YAP and TAZ in the nucleus pulposus tissue of the LIDD group (Fig. 5c). Double-labeled immunofluorescence analysis of rat caudal intervertebral discs showed that the expression of P2Y2R, YAP and TAZ was significantly enhanced in the 2WD group compared with the 2WS group (Fig. 5d); compared with the 3WS group, the fluorescence expression of P2Y2R and TAZ was significantly enhanced in the 3WD group, but the expression of YAP was decreased in the 3WD group (Fig. 5e). In addition, P2Y2R, YAP and TAZ were coexpressed within nucleus pulposus tissue.

Fig. 5.

Immunofluorescence (IF) analysis of P2Y2R, YAP, and TAZ in intervertebral disc nucleus pulposus tissue. a IF analysis of P2Y2R in the nucleus pulposus tissue of normal and degenerated human intervertebral discs, scale bar = 20 μm; b Quantitative IF analysis of P2Y2R; c IF co-localization of YAP/TAZ and P2Y2R in human nucleus pulposus tissue using confocal microscopy: P2Y2Rs and YAP/TAZ are highly co-expressed in human nucleus pulposus tissue, scale bar = 50 μm; d-e IF analysis of P2Y2R and YAP/TAZ in the nucleus pulposus tissue of 4 groups of rats, scale bar = 100 μm, N=3. *p < 0.05

P2Y2R, YAP and TAZ are involved in IVDD

Western blot analysis of rat nucleus pulposus tissues revealed significantly higher protein expression of P2Y2R, YAP, and TAZ in the 2WD group compared with the 2WS group ). At the 3-week timepoint, P2Y2R and TAZ levels remained markedly increased in the 3WD group relative to the 3WS group (p, whereas YAP expression was significantly lower in the 3WD group compared with the 3WS group (p < 0.01 ;n=6; Fig. 6b–d). Western blot analysis was performed on human nucleus pulposus specimens grouped by Pfirrmann grade: LIDD (grade II), MIDD (grade III), and SIDD (grades IV–V). Western blotting was repeated to ensure uniform GAPDH band intensity across all sample lanes (Fig. 7b). Compared with the LIDD group, P2Y2 expression was significantly higher in both the MIDD and SIDD groups (p < 0.001). YAP and TAZ levels were markedly upregulated in MIDD relative to LIDD (p < 0.001) and were significantly lower in SIDD than in MIDD (p < 0.05; n = 12; Fig. 7b–e).

Fig. 6.

The expression levels of P2Y2R, YAP, and TAZ in the intervertebral disc nucleus pulpal tissues of all rat groups were analyzed by western blot. a Protein expression of P2Y2R, YAP, and TAZ in the nucleus pulposus disc in each rat model. b-d The bar graph shows the normalized quantitative analysis of P2Y2R, YAP, and TAZ gray values. n=6. *p < 0.05, **p< 0.01, ****p< 0.0001

Fig. 7.

Expression levels of P2Y2R, YAP, and TAZ in human disc pulpal nucleus by western blot analysis with different degrees of degeneration. a T2 weighted term performance of lumbar sagittal MRI with different Pfirrmann grades. b Protein expression levels of P2Y2R, YAP, and TAZ in the nucleus pulpal tissue of human intervertebral discs with different degrees of degeneration. c-e Quantitative normalization of P2Y2R, YAP, and TAZ. n=12. *p < 0.05, **p < 0.01, ***p < 0.001

Discussion

Epidemiological studies have established a strong association between disc degeneration and prolonged heavy work, bending work, and exposure to mechanical vibration [34]. Altering the mechanical environment of the spine can lead to disc changes similar to those observed in early human disc disease [35–37]. There are two types of models used to study disc degeneration: ex vivo and in vivo models. The ex vivo model involves taking a fresh animal disc and the vertebral bodies at each end and treating the disc with a compression device. The disc tissue is then collected for degeneration assessment [38–40]. In vivo experiments are performed by fixing a compression device to the vertebrae of a living animal and establishing a model of disc degeneration by applying intermittent or continuous compression loads through a spring-loaded device [5]. Depending on the compression device, this compression can be divided into axial vertical compression and axial flexion compression. Static and dynamic compression can also be used, and some studies have applied lateral bending (LB) and axial rotation (AR) to better simulate human weight-bearing exertion [41].

In this study, the Ilizarov device was used to create a rat model of IVDD, which aimed to simulate the degeneration caused by excessive stress in humans. Previous studies have used the Ilizarov device to create a rat model of disc degeneration in which a pressure of 1.3 MPa was applied, resulting in significant changes in the extracellular matrix of the disc after 7 days [5]. Another group applied mechanical pressure to the rat’s tail, resulting in a pressure load of 7.2 N, and significant disc degeneration occurred after 2 weeks [42]. Therefore, an applied force of 8 N was selected, which generated an approximate pressure of 1.13 MPa on the coccygeal intervertebral disc between Co8 and Co9. While previous studies have demonstrated that intervertebral disc degeneration can be induced by one or two weeks of abnormal loading [5, 42], the present study extended the compression duration to three weeks in an additional experimental group. This design allowed us to assess the longitudinal effects of sustained mechanical loading over two- and three-week periods on disc degeneration and the expression trends of P2Y2, YAP, and TAZ. Further studies involving longer loading durations may provide additional insights into the progression of these changes.

MRI T2-based Pfirrmann classification showed no degeneration at two weeks but detected changes at three weeks. However, T2 grayscale values, HE and SO-FG staining indicated degeneration at both time points. These findings suggest that the Pfirrmann classification may not be as sensitive as other methods in assessing degeneration in this model, possibly due to differences in the morphology, function, and amount of hyaluronic acid in the major cell types of the rodent and human nucleus pulposus [43, 44], highlighting the limitations of the Pfirrmann classification in rodents. In line with previous studies investigating disc degeneration in rat caudal models [45, 46], we utilized Pfirrmann grading as an initial and qualitative MRI-based assessment to provide a general indication of degenerative changes following mechanical compression. However, the core degenerative phenotypes in our study were definitively assessed using more objective and quantitatively reliable histological scoring systems. Discrepancies between Pfirrmann grades and histology may reflect assessment timing (MRI on day 3 vs. histology on day 15 post-removal) or species-specific grading limitations.

Histological assessment via HE and SO-FG staining revealed minimal degeneration in the 2WS and 3WS groups, with most discs remaining normal or mildly degenerated. In contrast, continuous mechanical loading for two weeks induced mild-to-moderate degeneration (Masuda grade 6–9) in most rats. Initial degenerative changes included disruption of the nucleus pulposus (NP) structure, coagulation and striation of the extracellular matrix, honeycombing of NP cells, and distortion of the NP–annulus fibrosus boundary. After three weeks, discs exhibited moderate-to-severe degeneration (Masuda grade 8–10), characterized by further NP disruption, partial cell loss, extensive matrix coagulation, and fissures extending into both the NP and annulus fibrosus. These findings indicate that continuous mechanical loading initially alters NP cell morphology, promotes extracellular matrix coagulation, and disrupts the NP–AF boundary. Prolonged loading exacerbates NP degeneration and progressively involves the annulus fibrosus.

The standard method for assessing mechanical pain in rodents involves measuring pain thresholds in the caudal region using von Frey filaments [47, 48]. While caudal root injury models are known to induce nociceptive abnormalities [49], it remains unclear whether continuous mechanical compression alone can elicit similar responses in a disc degeneration model. To minimize structural buckling often associated with multi-segment compression, we applied axial load to a single disc segment.

Mechanical pain thresholds were assessed from Day 3 after frame and pin removal to reduce confounding effects from external device weight and surgical trauma. All groups exhibited significantly reduced mechanical pain thresholds at D3 compared to preoperative levels. Control rats (2WS and 3WS) showed gradual recovery from D6 onward, indicating temporary nociceptive effects from device implantation. In contrast, rats subjected to disc-degenerating compression (2WD and 3WD) maintained significantly lowered pain thresholds through D15, demonstrating sustained hyperalgesia induced by continuous mechanical compression. These results suggest that mechanically induced disc degeneration can produce persistent nociceptive sensitization, providing a model for studying pain resulting from nonphysiological weight bearing. All animals tolerated the procedure well, exhibited normal weight gain (430–460 g after 4 weeks), and showed no infections, tissue necrosis, or neurological complications. Springs maintained consistent compression without flexion throughout the experiment.

Under static and dynamic mechanical loading, intervertebral disc cells can release significant amounts of adenosine triphosphate (ATP) to meet their energy needs. This ATP is crucial for maintaining the synthesis of extracellular matrix (ECM) by IVD cells, which in turn helps to maintain the integrity of the disc and prevent degeneration [11, 50, 51]. ATP not only provides intracellular energy but also regulates various cellular activities by binding to purinergic receptors, particularly purinergic receptor 2 (P2R), which activates intracellular signaling pathways. Pathological conditions can cause the release of large amounts of ATP (100–1000 µM) from cells or tissues, activating P2Rs such as P2X3, P2X2/3, P2X7 or P2Y2, which are involved in pathogenesis through downstream signaling pathways [23, 52, 53]. Recent studies have shown that P2X7 receptors are expressed in degenerating discs, their expression increases with the progression of degeneration, and they are involved in the formation of an inflammatory microenvironment [14]. In this study, we observed an increase in P2Y2R expression in degenerated nucleus pulposus tissues in rats and humans, with an increase in the degree of degeneration. This finding suggests that P2Y2R may be involved in the pathogenesis of disc degeneration, but its exact function is not yet clear and requires further investigation. Additionally, it is important to determine whether P2Y2R is involved in the pathogenesis of degeneration-related pain.

P2Y2R was highly co-expressed with YAP and TAZ in human degenerated NP. The Hippo pathway effectors YAP/TAZ regulate cell fate and matrix homeostasis and are mechanosensitive [24, 54]. Another study showed that YAP/TAZ regulates the degeneration-related phenotype in nucleus pulposus cells [56]. In rats, 2-week compression increased both YAP and TAZ, while 3-week compression decreased YAP but sustained TAZ elevation. Human moderately degenerated discs also showed elevated YAP/TAZ. This divergent expression suggests distinct roles: YAP may exert protective or reparative functions early in degeneration [29, 57–60], while TAZ may promote degenerative progression. However, with prolonged mechanical loading and compression, disc degeneration increased and YAP expression levels were downregulated [61], or YAP expression was decreased due to increased degeneration and further apoptosis in nucleus pulposus cells. Further studies are needed to clarify their functional mechanisms and interactions with P2Y2R.

Several limitations should be acknowledged. The compression magnitude induced slight disc flexion, and the fixation device itself restricted tail movement. K-wire insertion confounded early pain behavior, though prolonged recovery helped isolate discogenic pain. Future studies should include additional pain behavioral assays. This study demonstrated correlative changes in P2Y2R, YAP, and TAZ. Functional studies are needed to establish causality. Although the rat tail model has anatomical and biomechanical differences compared to human discs, it remains a valuable tool for studying disc degeneration pathobiology and screening therapeutic targets.

In conclusion, we established a stable rat model of compression-induced disc degeneration and associated pain. Our preliminary data implicate P2Y2R and Hippo pathway components YAP/TAZ in the degenerative process, providing a foundation for further mechanistic investigation.

Authors’ contributions

Na Li, Jiayu Chen and Fangyi Kong: were responsible for study design. Fei Chen, Junjie Pu, Yiling Deng and Junhui Qi: contributed to surgical implementation and sample processing. Fei Chen, Na Li and Junjie Pu: were responsible for data analysis. Fei Chen: drafted the manuscript. Fei Chen, Jiayu Chen and Na Li: edited and revised the manuscript. All authors have read and approved the final submitted manuscript.

Funding

This study was supported by Xingdian Talents Program (XDYC-YLWS-2024-0073), National Natural Science Foundation of China (No.81701117), The Reserve Talents for Academic and Technical Leaders of Middle-aged and Young People in Yunnan Province (202305AC160069), Yunnan Provincial “Top Young Talents Project” (No. YNWR-QNBJ-2019-184), Yunnan Provincial Key Laboratory of Digital Orthopedics (No.202005AG070004), Yunnan Clinical Medical Research Center of Orthopedics and Sports Rehabilitation (No.202102AA310068), Yunnan Provincial Spinal Cord Disease Clinical Medical Centre (No.ZX2022000101).

Data availability

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study involving human nucleus pulposus specimens and rat animal experiments was fully approved by the Medical Ethics Committee of the 920th Hospital of Joint Logistic Support Force (Approval No. 2016002). As the institution’s legally designated ethics review authority, this committee is authorized to conduct ethical audits for both human tissue research and laboratory animal experiments. All animal operations followed the ARRIVE guidelines, and written informed consent was obtained from all enrolled patients prior to tissue collection.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.