Aberrant spinal mechanical loading stress triggers intervertebral disc degeneration by inducing pyroptosis and nerve ingrowth

Aberrant mechanical factor is one of the etiologies of the intervertebral disc (IVD) degeneration (IVDD). However, the exact molecular mechanism of spinal mechanical loading stress-induced IVDD has yet to be elucidated due to a lack of an ideal and stable IVDD animal model. The present study aimed to establish a stable IVDD mouse model and evaluated the effect of aberrant spinal mechanical loading on the pathogenesis of IVDD. Eight-week-old male mice were treated with lumbar spine instability (LSI) surgery to induce IVDD. The progression of IVDD was evaluated by μCT and Safranin O/Fast green staining analysis. The metabolism of extracellular matrix, ingrowth of sensory nerves, pyroptosis in IVDs tissues were determined by immunohistological or real-time PCR analysis. The apoptosis of IVD cells was tested by TUNEL assay. IVDD modeling was successfully produced by LSI surgery, with substantial reductions in IVD height, BS/TV, Tb.N. and lower IVD score. LSI administration led to the histologic change of disc degeneration, disruption of the matrix metabolism, promotion of apoptosis of IVD cells and invasion of sensory nerves into annulus fibrosus, as well as induction of pyroptosis. Moreover, LSI surgery activated Wnt signaling in IVD tissues. Mechanical instability caused by LSI surgery accelerates the disc matrix degradation, nerve invasion, pyroptosis, and eventually lead to IVDD, which provided an alternative mouse IVDD model.


Aberrant mechanical stress disruptes the morphology of IVD tissues. To investigate whether
LSI surgery could affect the structure and composition of IVDs, we examined the histological changes of IVD between L4-L5 by Safranin O/Fast green staining. Histological stain analysis results showed that IVDs of Sham mice had well-organized tissue structures, including large vacuoles with sufficient matrix content in NP, arranged regularly AF tissues without any tearing, and no ectopic bone formation in EP. Meanwhile, LSI surgery treatment led to a significant decrease in height of discs, vacuole sizes in NP, ectopic bone formation in EP, accompanied by fissures and folds in the interlamellar of AF tissues (Fig. 2a). The degeneration of IVD was further assessed by histological score system established by Norcross et al. 28 . The score results suggested that NP and EP had significant decreases since 2-week after surgery, followed by decreases of AF scores 4-and 8-weeks after surgery (Fig. 2b). Consistent with these results, the levels of Ccn2, a factor that regulates matrix protein synthesis in IVDs and an indicator of disc degeneration 27 , were about threefold higher in LSI mice than in Sham mice (Fig. 2c,d).
Abnormal mechanical stress damages matrix composition of IVD tissues. Generally, stable matrix content maintains the physiological function of the IVD. Col2 and aggrecan are the principal components present in disc tissue, while Mmp3 and Adamts5 are known as key degradases for degrading Col2 and Aggrecan, respectively 20 . To determine the influence of mechanical instability on matrix metabolism, we first quantified the Aggrecan mRNA levels in IVD tissues 1-, 2-, 4-week post-surgery. The qRT-PCR results showed www.nature.com/scientificreports/ that LSI administration resulted in a significant decrease of Aggrecan mRNA levels since 1-week post-surgery (Fig. 3a). Then, we examined protein levels of Aggrecan, Col-II and corresponding matrix-degrading enzymes (Adamts5 and Mmp3, respectively) by immunohistochemistry (IHC) analysis. As shown in Fig. 3b-e, LSI mice exhibited a 54% down-regulation of Aggrecan and threefold upregulation of Adamts5 in the whole disc. Similarly, the expression of Col2 and Mmp3 in the EP region showed approximately the same trend as that of Aggrecan and Adamts5, respectively ( Fig. 3f-g).  www.nature.com/scientificreports/  www.nature.com/scientificreports/ Aberrant mechanical stress induces sensory nerve invasion in AF. IVDD is thought to be the cause of LBP partially due to sensory nerve ingrowth into the degenerated IVD 29 . Evidence from animal models and human studies revealed that sensory innervation of lumbar IVD and sensory nerve ingrowth into the inner layer of IVD caused painful conditions [30][31][32] . To explore the distribution of sensory nerves after IVD modeling, we detected the expression of its specific marker (Cgrp) by immunofluorescent (IF) analysis. The IF results showed that LSI surgery significantly increased the number of Cgrp-positive cells in AF region (Fig. 4a-c).
Aberrant mechanical stress aggravates apoptosis in IVD. Apoptosis, a process of cellular suicide, also plays a crucial role in IVD degeneration 33 . Then we investigated the effects of LSI surgery on apoptosis of IVD cells by TUNEL assay. We found that the rate of apoptosis in the LSI mice was significantly increased by about 2 times (Fig. 4d,e), especially in the ectopic bone formation zone of EP. These results indicated that aberrant mechanical stress strongly induced apoptosis of IVD cells.  www.nature.com/scientificreports/ Aberrant mechanical stress promotes pyroptosis of IVD. Since chronic inflammation is another key component of painful IVDs 9,10 , we subsequently determined the effect of LSI surgery on the induction of pyroptosis in IVD tissues. IF results of Nlrp3, Caspase-1 and IL-1β, the key proteins of NLRP3 inflammasome signaling, demonstrated that LSI administration induced a significantly increased expression of Nlrp3 in AF and ectopic bone formation zone in EP ( Fig. 5a-c). Similarly, LSI treatment resulted in a significant increase of Caspase-1 and IL-1β expression both in NP and AF of LSI mice ( Fig. 5d-g).

Aberrant mechanical stress activates Wnt/β-catenin signaling pathway of IVD tissues. Our
previous study showed that β-catenin protein was up-regulated in disc tissues from patients with IVDD 20 . In addition, activation of Wnt/β-catenin signaling was also involved in pyroptosis and neuropathic pain 34 . To further explored the effect of LSI treatment on Wnt/β-catenin signaling, the expression levels of β-catenin and Lef1 mRNA in IVD tissues were quantified by qRT-PCR analysis. qRT-PCR results showed that β-catenin and Lef1 mRNA levels were significantly increased in IVDs of LSI mice since 1-week post-surgery (Fig. 6a). Then we further examined protein levels of β-catenin and target proteins of Wnt signaling, including Lef1, Tcf4, and Dkk1. As expected, the IF results of β-catenin revealed that LSI surgery significantly increased the expression of β-catenin in NP and AF (Fig. 6b,d). And the IHC results of Tcf4, Lef1 and Dkk1 showed a similar pattern to β-catenin (Fig. 6c,e). These findings indicate that LSI treatment might promote pyroptosis and nerve ingrowth of IVD tissue by activating the Wnt signaling.

Discussion
Multiple clinical investigations have showed that congenital malformations of spine (including scoliosis, kyphosis, spina bifida, spondylolysis and Klippel Feil syndrome) 35,36 , accidental back injury or ligament injury [37][38][39] , occupational exposure (such as crane and car drivers, weightlifters, etc.) [40][41][42] could induce aberrant mechanical loading of lumbar spine, and finally lead to IVDD. Moreover, decompensated changes in lumbar spine structure result from abnormal mechanical environment, such as proliferation of facet joints 43 , formation of osteophytes 44 , hypertrophy of ligamentum flavum 45 or calcification of longitudinal ligament 46,47 , will fail to resist aberrant mechanical loading and eventually accelerate the process of IVDD in patients. Since there is no desirable model to mimic the clinical development process of aberrant mechanical loading induced-IVDD more accurately, the underlying molecular events of IVDD pathogenesis remains largely elusive. In this study, we constructed a reproducible mouse IVDD modeling with direct in vivo evidence of medical iconographic and morphological changes in the IVD after loading axial anomalous stress. LSI surgery could promote the progression of IVD degeneration, including notable decreases in IVD height and histological score, ectopic new bone formation in EP, folds and tears in AF, reduction of the vacuole volume in NP, acceleration of ECM degradation, innervation into AF, and induction of pyroptosis after IVDD modeling. In addition, LSI treatment resulted in the activation of Wnt/β-catenin signaling in IVDD mice, which might be responsible for the above changes (Fig. 7). Taken together, we developed a new IVDD model that could help to improve our understanding of the biological events in aberrant spinal axial mechanical loading-induced human IVDD, and presented potential targets for the clinical treatment of IVDD and LBP. The β-catenin adhesion complex is one of the central components of the cell-cell adhesion junctions, transmitting mechanical stress from cell to cell. Mechanical stress could upregulate β-catenin protein [48][49][50] , which was also significantly elevated in IVD samples obtained from patients with disc degeneration 51,52 . To develop a mouse model to mimic human IVDD, our previous study has generated an inducible β-catenin conditional activation (cAct) mice by breeding β-catenin fx(Ex3)/fx(Ex3) mice with Col2a1-CreER T2 transgenic mice, according to the upregulation of β-catenin in disc tissue of IVDD patients. Although these mice have multiple features that resemble some of the features of human IVDD, NP cells were not targeted by Col2a1-CreER T2 transgenic mice and the severely disorganized defect seen in NP tissues may result from the disruption of nutrient and solute supplies to this region and structural changes in AF tissues after the loss of the growth plate cartilage in β-catenin cAct mice 20 . Therefore, the phenotype of these mice may only partially resemble the feature of human IVDD. In the present study, we found that LSI surgery significantly increased β-catenin and its target proteins both in NP and AF tissues, which provided an alternative method for simulation of human IVDD.
Clinical evidence has reported that IVD inflammation and axonal growth of afferent fibers innervating the disc are main factors of discogenic LBP, and IL-1β is a pain-related molecule, which is significantly elevated in painful human IVD 53 . The latest research indicated that pyroptosis activation in IVD was likely responsible for the inflammatory pathology of IVDD 54 . And mechanical stress could drive inflammatory responses associated with IVDD and LBP in vitro 55 . The important finding in our study is that the pyroptosis was significantly facilitated in outer AF and ectopic bone formation zone inside EP after LSI surgery treatment. The increase of IL-1β induced by pyroptosis, a main inflammatory factor, will further inhibit ECM anabolism and promotes its catabolism in IVDs, as well as the boost of apoptosis in IVD cells.
Besides, the onset of discogenic pain is characterized by ingrowth of nerve fibers into an otherwise aneural tissues 10 . Whether mechanical stress could directly promote nerve ingrowth in AF is an under-investigated aspect. Navone et al.found that mechanical stress could affect nerve proliferation by changing the disc microenvironment 56 . Impaired AF could lead to the loss of focal proteoglycan, and leave a matrix favorable for nerve ingrowth 57 . In this study, we found that IVDD mice exhibited a significant increase in the number of Cgrppositive sensory nerves both in outer and inner AF. This finding is very similar to the phenomenon in clinical patients, that is, IVDD patients had more nociceptive sensory innervation in AF, which is an important origin of LBP in IVDD patients. In addition, a large number of cytokines released by pyroptosis cells also stimulate sensory nerves, which further leading to more severe LBP. Therefore, anti-pyroptosis and anti-innervation may serve as potential therapeutic strategies for IVDD disease and LBP. www.nature.com/scientificreports/ www.nature.com/scientificreports/ (e) Quantification of Tcf4, Lef1 and Dkk1 expression in IVD of (c). The averages of integrated optical density in interest region was calculated by Image-Pro Plus software version 6.0 (https ://www.media cy.com). Data are shown as mean ± sem. Three independent experiments were performed in triplicate. *P < 0.05, **P < 0.01 compared to Sham group. www.nature.com/scientificreports/ Previous research has reported that sustained mechanical load could activate Wnt/β-catenin signaling in the development of osteoarthritis 58 . And activation of Wnt/β-catenin signaling led to apoptosis of IVD cells and increase of ECM degradation enzymes, thereby accelerating matrix degradation and promoting IVDD progression 52,59 . However, the relationship between LSI surgery and Wnt/β-catenin signaling in IVDD mice is largely elusive. Our results found that LSI administration up-regulated Wnt/β-catenin signaling, accompanied by increased Adamts5 and Mmp3 in IVDs. Recent evidence also indicated that down-regulating Wnt/β-catenin signaling could suppress Nlrp3 inflammasome activation 60 . Consistent with the above findings, we found that the activation of Wnt/β-catenin signaling preceded the increase of Nlrp3, Caspase-1 and IL-1β in IVD tissues of IVDD mice. These findings indicated that LSI treatment might promote pyroptosis via activation of Wnt signaling.
As stated above, one limitation of this study is that not all aspects of pathological factors of IVDD can be taken into consideration in experimental animal models, and the experimental animal model can only partially resemble complex human conditions. However, this simplified model is extremely useful in investigating the reciprocal interactions between mechanical loading and IVDD progression. In this study, there was a significant induction effect on pyroptosis and innervation of IVDs in IVDD mice, which suggested that the therapy of anti-pyroptosis and anti-innervation may have therapeutic potential for clinical IVDD. However, whether the therapy was able to directly target the intervertebral disc, as well as serious side effects on the human body is still masked. In addition, Col2a1-CreER T2 transgenic mice cannot effectively target all disc cells, especially NP cells. We have constructed Aggrecan-CreER T2 transgenic mice to specifically target disc cells 61 , which enable us to www.nature.com/scientificreports/ further explore the relationships between Wnt/β-catenin signaling and mechanical stress, pyroptosis and nerve ingrowth during the pathological process of IVDD.
To sum up, we found a mouse IVDD modeling by operating the LSI surgery. Aberrant mechanical stressinduced lumbar IVDD model may expand our knowledge regarding the occurrence and development of IVDD, and also may help explain why osteophytes and spinal stenosis occur. Simultaneously, the study reminded us that restoring the normal mechanical environment of IVD, mitigating IVD pyroptosis, or inhibiting Wnt/β-catenin signaling pathway could have therapeutic potential for clinical IVDD. Surgical procedure. All mice were randomly divided into the LSI model group and the Sham group (n = 24 per group). After being anaesthetized with sodium pentobarbital, mice were placed on the surgical table in a prone position. The L5 vertebra was located on the anterior superior iliac spine. The lower dorsal skin was shaved and disinfected using iodine solution, then a longitudinal incision was created along the midline of the back to expose the lower lumbar spine. The posterior paravertebral muscles from the L3-L5 vertebrae were separated to expose the lumbar 3rd-5th spinous processes, which were further resected along with the supraspinous and interspinous ligaments using a fine scissor. Mice in the Sham group only received a detachment of the posterior paravertebral muscles from the L3-L5 vertebrae (Fig. 1b). After the surgery, the muscles and skin were successively sutured to allow the mice to recover. L3-L5 vertebrae of mice were harvested 1-, 2-, 4-, and 8-week after LSI surgery (Fig. 1a) (n = 6 per group at indicated times). μCT analysis. Before histological processing, we firstly evaluated paraformaldehyde-fixed vertebrae by μCT analysis as we previously described 63 . By using a high-resolution μCT (Skyscan 1176; Bruker μCT, Kontich, Belgium) with 90 kVp source and 300 μA current, we scanned vertebrae at a resolution of 9 μm. Three-dimension model visualization software, CTVol v2.0 (Skyscan company, San Jose, CA, USA), was employed to analyze parameters of the L4-L5 IVD with half-height of the L4 and L5 vertebrae. To quantify disc space, we measured the distance between L4 and L5 from μCT images by Image Pro For IHC staining, a horseradish peroxidase streptavidin detection system (ZSGB-BIO, Beijing, China) was subsequently used to detect the immunoactivity, followed by counterstaining with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA). For IF analysis, the slides were incubated with secondary antibodies conjugated with fluorescence at room temperature for 1 h. The morphometric study was performed using the Image Analysis System (Olympus, Japan). Triplicates of each sample were used for staining. Quantitative histomorphometric analysis was conducted in a blinded manner with Image-Pro Plus Software version 6.0 (Media Cybernetics Inc, Rockville, Maryland, USA) as we previously described 63 . Histological score was graded in a blind fashion using the definition established by Norcross et al. 28 , with some modifications (Table 1). TUNEL assay. The TUNEL assay for detecting DNA breaks was performed with TUNEL Bright Green Apoptosis Detection Kit according to the manufacturer's instruction (Vazyme Biotech; Nanjing, China) as we previously described 63 . Negative controls were incubated in a TdT free-enzyme solution. The number of positive cells was quantified in three randomly selected fields of view using three sections from each sample. DAPI staining was used to estimate the total cell number.

Methods
Real-time quantitative PCR analysis. Total RNA was extracted from L4-L5 IVD using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. The yield and purity of RNA were quantified using a Nanodrop 2000C spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA) and 2 μg of RNA were reverse-transcribed into cDNA using the Prime Script RT Reagent Kit protocol (Takara Bio, Dalian, China). Real-time PCR amplification was performed using murine gene-specific primers and TB Green Premix Ex Taq II (Takara Bio, Dalian, China). The PCR program was: initial 2 min at 95 °C denaturing; 10 s at 95 °C denaturing; and 30 s annealing/elongation for a total of 40 cycles. The primers used for the PCR are listed in www.nature.com/scientificreports/

Statistics analysis.
All the data were expressed as mean ± standard error of mean (sem), as indicated in the figure legends. Statistical significance was determined using Student's t-test. All data analysis was performed using SPSS 15.0 analysis software (SPSS Inc, Chicago, Illinois, USA) and the level of significance was defined as P < 0.05.
Received: 15 September 2020; Accepted: 28 December 2020 Table 1. Histological grading scale criteria based on Norcross et al. 28 . This scale mainly scores the disruption of nucleus pulposus central cavity, cellularity and collagen fiber orientation of annulus fibrosus and the degree of ectopia ossification of cartilage endplate. Simple radial clefting = the presence of radial gaps between AF lamellae with minimal fragmentation; complex radial clefting = the presence of radial, transverse, and/ or oblique gaps in the lamellae with significant fragmentation; junction = the triangle junction between the cartilage endplate and the annulus fibrosus.    www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.