TgA86 Mice overexpressing transmembrane TNF display an unexpected absence of thoracolumbar disc degeneration

TgA86 Mice overexpressing transmembrane TNF display an unexpected absence of thoracolumbar disc degeneration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report TgA86 Mice overexpressing transmembrane TNF display an unexpected absence of thoracolumbar disc degeneration Mohamed-Mehdi Raji, Liane Fontaine, Nina Bon, Claire Vinatier, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6808577/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Objective. This study evaluated the relevance of TgA86 mice, which overexpress transmembrane TNF (tmTNF), as a model for intervertebral disc degeneration (IVDD). Six middle-aged, skeletally mature, transgenic TgA86 mice were included in this study alongside three control littermates. Histopathological scoring was performed on sections of the lumbar and thoracic segments of the spine to evaluate the extent of IVDD. Results. Despite preexisting cues predicting a degenerated disc phenotype, degeneration scores were comparable between TgA86 and wild-type mice, with no evidence of any genotype-specific effects. This first report of a detailed histopathological characterization of TgA86 mice thoracolumbar intervertebral discs suggest that transmembrane TNF (tmTNF)-driven inflammation alone does not induce IVDD, limiting their utility as a model for studying disc degeneration. TgA86 mice histopathological scoring intervertebral disc degeneration Figures Figure 1 Figure 2 INTRODUCTION Low back pain (LBP) is one of the leading causes of years lived with disability, affecting all age groups across low to high-income countries( 1 ). Recent studies have estimated that the prevalence of LBP exceeds 600 million people worldwide( 2 ). The substantial socioeconomic burden associated with LBP makes it a major public health issue among noninfectious chronic diseases( 3 , 4 ). Although LBP is multifactorial and can stem from diverse etiologies, substantial evidence implicates intervertebral disc degeneration (IVDD) as a key contributor( 5 – 11 ). Current treatment strategies for LBP rely on pharmacologic pain management, lifestyle modifications or manual therapies. Some patients with advanced disease undergo invasive surgery( 12 , 13 ) due to lack of therapeutic alternatives. Thus, understanding the pathomechanisms underlying IVDD is crucial for developing innovative, evidence-based therapies, which necessitate relevant animal models that accurately mimic human pathology. Different models have been used for this purpose( 14 ). The transgenic A86 model could be a promising candidate for disease modeling and therapeutic development. TgA86 mice harbor a genetic mutation allowing the overexpression of transmembrane tumor necrosis factor (tmTNF), whose signal is transduced through the TNFR1 and TNFR2 membrane receptors( 15 ). Several studies corroborate a pathogenic role of TNF-mediated proinflammatory signaling in IVDD( 16 , 17 ). These mice are described as a model of human spondyloarthritis( 18 ), an inflammatory disease predominantly affecting the axial skeleton, notably including inflammation of the vertebrae( 19 ), i.e., anatomical structures in the vicinity of intervertebral discs (IVDs). Notably, a significant proportion of patients suffering from axial spondylarthritis also develop IVDD( 20 ). Christodoulou and colleagues studied the axial phenotype of TgA86 mice: macroscopically, these mice exhibited reduced body length and tail bending. This tail bending was shown to be accompanied by caudal IVDD( 18 , 21 ). Additionally, CT imaging revealed hyperkyphosis( 18 ). Clinical observations suggest a potential relationship between abnormal spine curvature and degenerative discopathy( 22 , 23 ) as the mechanical strains associated with aberrant spine curvature may contribute to IVDD. Conversely, the degeneration-related loss of mechanical properties of the IVD may exacerbate abnormal spine angles. Furthermore, CT imaging revealed a thinning of the intervertebral space and more compressed vertebrae at the cervical level, features reminiscent of the characteristic decrease in disc height index found in both human IVDD patients and other animal models( 24 ). They also reported roughening of vertebral surfaces, comparable to that observed in degenerating discs of ageing rats( 25 ). Histopathological analyses revealed enthesitis at intervertebral joints with immune cell infiltration, including neutrophils. This inflammatory environment could negatively impact neighboring IVDs via paracrine mechanisms, given the central role of inflammation in IVDD pathogenesis.( 16 ). Besides, increased osteoclastic activity, ectopic cartilaginous neoformation, and osteogenic marker positivity in the vertebral bodies of TgA86 mice suggest uncoordinated osteochondral remodeling. Analogous histological changes in the endplates have been reported by several studies to be associated with IVDD( 26 – 31 ), as the fate of intervertebral discs is closely linked to the health of the surrounding compartments( 32 ). Overall, TgA86 mice undergo a myriad of cellular and tissue transformations resembling features of IVDD. The present work aimed to assess the relevance of TgA86 transgenic mice as an in vivo model for IVDD. MATERIALS AND METHODS Animal model TgA86 is a transgenic model generated via site-directed mutagenesis, allowing global overexpression of transmembrane TNF, including in joints( 15 ). The mice used in this work were heterozygous for the transgene and maintained on a mixed CBA × C57BL/6J genetic background. Animal handling and surgical procedures were conducted at INSA LYON, according to the European Community Guidelines for Care and Use of Laboratory Animals (2010/63/UE) and were approved by the national ethical committee (APAFIS #37995-202207190935346) and the institutional animal welfare committee at CEEA – 102 Comité d’éthique de l’INSA de Lyon. Six transgenic mice (3 males, 3 females) and three age-matched wild-type littermates (1 male, 2 females) were euthanized at 35 weeks of age in their home cage using CO 2 inhalation to ensure minimal stress and to maintain spine integrity (6L/min, 3min dwell time). After confirmation of death by decapitation, the spines were collected for analysis. Histopathological analysis The thoracic and lumbar segments of the spines were dissected, fixed in 4% paraformaldehyde for 48 hours, decalcified in 0.5 M EDTA with 28% ammonium hydroxide for ten days, and embedded in paraffin. Four micrometer-thick mid-sagittal sections of the spine were stained with hematoxylin and eosin (H&E) and imaged using a Nanozoomer S360 digital slide scanner (Hamamatsu photonics) with a 40x objective. Three blinded raters evaluated a minimum of 3 thoracic and lumbar IVDs per mouse, using a scoring system designed specifically for grading the extent of IVDD in mice( 33 ). Graded features included the cellularity and matrix organization of Nucleus Pulposus (NP), Annulus fibrosus (AF), Endplates (EP), and the demarcation between those different compartments. A global degeneration score was determined by summing the grades obtained for each criterion. Degeneration scores were classified as follows: healthy (non-degenerated) for scores below 6, mild degeneration for scores between 7 and 13, moderate degeneration for scores between 14 and 25, and severe degeneration for scores between 26 and 33. Interscore reliability The magnitude of agreement among the three scorers was evaluated via different statistical methods included in the {irr 0.84.1} package, using {R 4.3.3} in RStudio (2023.06.0 + 421). The results are recapitulated in Supplementary Table 1 . Statistical analysis Statistical analysis was performed with GraphPad Prism {10.3.1} software. The statistical significance of differences in histopathological scores, spine curvature angles and tail length between wild-type and transgenic samples were determined using appropriate nonparametric t-test (Mann-Whitney). RESULTS Lumbar IVDs do not exhibit apparent histological signs of IVDD in TgA86 mice We first conducted a histopathological analysis of lumbar IVDs, since this level of the spine is the most commonly affected in humans( 1 ). Histopathological analysis of lumbar IVDs revealed well-organized structures in TgA86 mice, with a clear demarcation between the different compartments (Fig. 1 A, black dotted lines): a central NP surrounded by AF, and EP at the interface between IVDs and vertebrae. The central NP contained spindle-shaped cells (blue arrows) that were evenly distributed in a homogenous matrix. It was surrounded by an AF matrix consisting of concentric lamellae (red dotted lines), which is the classical architecture of a healthy disc. In between the lamellae, there were characteristic fibroblast-like cells (red arrows). Very few and small fissures could be observed in the AF of some discs, but this was the case for both the transgenic and wild-type samples. At the interface between the disc and the vertebral bodies, the cartilage EP presented as a normal hyalin cartilage comprising chondrocytes with usual morphology (green arrows). Notably, we detected porous structures (indicated by stars) in the EP of some discs. However, these porous structures were observed in both wild-type and transgenic samples, suggesting that they are more likely age-related degenerative features rather than genotype-related ones (Fig. 1 A). Histopathological scores of TgA86 mice revealed mild to moderate degeneration. There was no statistically significant difference in degeneration scores between wild-type and transgenic lumbar IVDs (Fig. 1 B). Overall, the data indicate an absence of striking signs of degeneration in TgA86 lumbar IVDs. Thoracic IVDs do not exhibit apparent histological signs of IVDD in TgA86 mice In agreement with previous findings( 18 ), the entire spine of TgA86 mice was deformed at 35 weeks of age (independently of the sex of the mice), with the occurrence of multiple and severe curvatures in the tail in particular, but also at the cervicothoracic level ( Supplementary Fig. 1 ). We then wondered whether the genotype of TgA86 mice could exacerbate a spontaneously occurring degeneration process at the thoracic level, and therefore be used as model of human thoracic degeneration( 34 ). We examined the thoracic discs following the same procedure as for lumbar IVDs. Our histopathological study highlighted the healthy structure of TgA86 thoracic IVDs (Fig. 2 ), with no genotype-associated degeneration. DISCUSSION Animal models are essential for IVDD basic and translational research ( 14 ), each offering unique advantages and limitations. TgA86 mice spines provide a set of cues suggesting tissue transformations in the IVD area that align with several features of IVDD( 18 ). However, our analysis revealed that these structural changes were not associated with a degenerative process in the lumbar and thoracic discs. The lumbar segment of the spine is the segment most frequently affected in humans( 35 ) and lumbar discs degeneration was reported in aging mice( 36 ). We therefore hypothesized that the TNF-driven inflammation in TgA86 mice exacerbates a naturally occurring degenerative process at the lumbar level. While the AF and NP compartments of the TgA86 lumbar discs displayed healthy structures, we observed porous structures in the EP. This porosity of EP has been associated with a sensory innervation and a pain-related behavior in mice( 37 ), and clinically linked with LBP in patients displaying lumbar degeneration on MRI and CT scan( 29 ). However, those porous structures were observed in both wild-type and transgenic samples, suggesting that this slight damage seemed to be solely due to the mature age of the mice, and not to their genotype. Thoracic IVDs are also prone to degeneration, which is positively correlated with the age of human patients( 34 ), and degeneration of thoracic IVDs has been described in ageing mice( 36 ). In the thoracic segment, CT imaging of TgA86 spines revealed hyperkyphosis compared with that in wild-type samples( 18 ). However, we showed that this abnormality was not accompanied by histological signs of disc degeneration. Despite evidence highlighting a pathogenic role of TNF-mediated inflammation in IVDD( 16 , 17 ), the transmembrane TNF-driven activation of the TNF axis has failed to replicate a degenerate pattern in mouse lumbar and thoracic IVDs. In another study, Gorth et al. explored the structure of lumbar IVDs in Tg197 mice, a human TNF-overexpressing model ( 38 ). As opposed to us, they noticed a decrease in NP and AF scores (modified Thompson scoring). This apparent discrepancy may stem from differences in the genetic constructs (overexpression of murine transmembrane TNF for TgA86; versus overexpression of human TNF, without distinction of the soluble and membrane forms, with several gene copies in Tg197), likely resulting in differential activation patterns of TNF signaling. Nonetheless, they observed an overall healthy tissue structure, unchanged NP aspect ratio and absence of catabolic factors expression, corroborating our concept that increased TNF transduction alone is not sufficient to satisfyingly model IVDD. The mechanistic basis of IVDD and the relative contribution of inflammation to pathogenesis may differ among species, and the pathogenic process underlying degeneration may vary according to the triggering events. A range of large and small animal models is employed in IVDD research, each relying on different IVDD induction modalities (age-related spontaneous degeneration, genetic mutation, postural, surgical, chemical, dietary or bacterial induction)( 24 , 39 , 40 ), implying mechanistic differences. Each models offers a balance between advantages and drawbacks in terms of reproducibility, costs, timeline evolution of the disease, and phenotypic outcome( 14 ). Selecting the most appropriate model requires careful consideration of these factors, depending on the research question. In conclusion, complementing Christodoulou’s work, our study provides the first detailed histopathological characterization of TgA86 IVDs. These findings suggest the limited relevance of TgA86 mice as an IVDD model but highlight the complexity of TNF role in disc disease. Future studies should explore alternative pathways and models to elucidate IVDD mechanisms and identify novel therapeutic targets. LIMITATIONS Our study has several limitations. It is possible that a pro-degenerative cascade of molecular and cellular changes was initiated in the TgA86 mice, but the selected timepoint for sacrifice may have been too early to detect substantial histological impact. In line with this observation, benefits of TNF receptors knock-out on lumbar disc structure were only detected at 21 months of age in another mouse model( 41 ). Additionally, we focused solely on structural signs of IVDD, while pain remains a central issue of this disease( 5 – 11 ). For example, in a rat model, intradiscal injection of degenerated human NP cells triggered pain-related behaviors and increased the expression of degeneration-related markers (e.g., MMP3, TNF and IL-6) by recipient NP cells, despite the absence of visible histological degeneration( 42 ). Abbreviations • LBP Low back pain • IVDD Intervertebral disc degeneration • NP Nucleus pulposus • AF Annulus fibrosus • EP Endplates • GP Growth plate Declarations ETHICS APPROVAL Animal handling and surgical procedures were conducted according to the European Community Guidelines for Care and Use of Laboratory Animals (2010/63/UE) and approved by the national ethical committee (APAFIS #37995-202207190935346) and the institutional animal welfare committee at CEEA – 102 Comité d’éthique de l’INSA de Lyon . CONSENT FOR PUBLICATION All the authors contributed to the article and approved the submitted version. DATA AVAILABILITY STATEMENT Data is provided within the manuscript or supplementary information files. Raw histological data and materials can be made available upon reasonable request to the corresponding author. AUTHORS’ CONTRIBUTIONS RG, JG and CB conceived and designed the experiments. MMR, LF, RG, NB and CB performed the experiments. MMR and RG analyzed the data. MMR and RG wrote the manuscript. JG, CV and CB reviewed the manuscript. ACKNOWLEDGMENTS The authors acknowledge the SC3M platform from the Inserm/NU/ONIRIS UMR1229 RMeS Laboratory and SFR Bonamy. They also acknowledge the MicroPICell core facility (SFR Bonamy, BioCore, Inserm UMS 016, CNRS UAR 3556, Nantes, France), member of the Scientific Interest Group (GIS) Biogenouest, IBISA, and the national infrastructure France-Bioimaging supported by the French national research agency (ANR-10-INBS-04). We thank A. Briolay and A. Debain for helping with animal care and tissue collection. FUNDING INFORMATION This study was supported by grants from the French Society of Rheumatology (Société Française de Rhumatologie). MMR was supported by the M4R Master-Doctorate program (Repair, Replace, Regenerate, Reprogram) of Nantes University, France. COMPETING INTERESTS The authors declare that they have no conflicts of interest associated with this manuscript. 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Supplementary Figure S1. Axial phenotype: tail length and spine curvature. (A) Representative pictures of a wild-type (WT, left panel) and transgenic (TgA86, right panel) tail. (B) Tail length measurements. (C) Representative pictures of H&E-stained histological sections of a Wild-Type (WT, left panel) and transgenic (TgA86, right panel) cervicothoracic spine. The angle formed by the C3-T1-T9 vertebrae is measured. (D) C3-T1-T9 angle measurements for the enrolled mice. (Mean ± SD; *: p-value ≤ 0.05). Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 07 Feb, 2026 Reviews received at journal 15 Aug, 2025 Reviewers agreed at journal 09 Aug, 2025 Reviewers invited by journal 06 Aug, 2025 Editor assigned by journal 13 Jun, 2025 Editor invited by journal 04 Jun, 2025 Submission checks completed at journal 04 Jun, 2025 First submitted to journal 04 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6808577","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":496697294,"identity":"4f36e296-a2c8-4dfa-a95a-b8c06d459a51","order_by":0,"name":"Mohamed-Mehdi Raji","email":"","orcid":"","institution":"Nantes Université, Oniris, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229","correspondingAuthor":false,"prefix":"","firstName":"Mohamed-Mehdi","middleName":"","lastName":"Raji","suffix":""},{"id":496697295,"identity":"b5807629-c436-4891-b33d-25e95f0d85ac","order_by":1,"name":"Liane Fontaine","email":"","orcid":"","institution":"Nantes Université, Oniris, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229","correspondingAuthor":false,"prefix":"","firstName":"Liane","middleName":"","lastName":"Fontaine","suffix":""},{"id":496697296,"identity":"3038b340-d170-418c-bf45-3bc7fc0afc17","order_by":2,"name":"Nina Bon","email":"","orcid":"","institution":"Nantes Université, Oniris, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229","correspondingAuthor":false,"prefix":"","firstName":"Nina","middleName":"","lastName":"Bon","suffix":""},{"id":496697297,"identity":"6cfcb129-0202-48ec-8b67-634cc807b3ec","order_by":3,"name":"Claire Vinatier","email":"","orcid":"","institution":"Nantes Université, Oniris, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229","correspondingAuthor":false,"prefix":"","firstName":"Claire","middleName":"","lastName":"Vinatier","suffix":""},{"id":496697298,"identity":"c26060ae-bcee-4519-966d-1be69e19e5d2","order_by":4,"name":"Carole Bougault","email":"","orcid":"","institution":"Université Claude Bernard Lyon 1, CNRS, UMR 5246, ICBMS","correspondingAuthor":false,"prefix":"","firstName":"Carole","middleName":"","lastName":"Bougault","suffix":""},{"id":496697299,"identity":"ad55389c-15d6-4523-a01c-d9521e24777f","order_by":5,"name":"Jérôme Guicheux","email":"","orcid":"","institution":"Nantes Université, Oniris, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229","correspondingAuthor":false,"prefix":"","firstName":"Jérôme","middleName":"","lastName":"Guicheux","suffix":""},{"id":496697300,"identity":"5ec1489b-f620-4554-895a-46919b4fa2f9","order_by":6,"name":"Romain Guiho","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYLCCBBDBzMPA8IGBgQchzHyAsBbGGXAtICG2BEJ28YAtQjIFhxbz9u7EDw93MNhtOM578LFt2x0ZgwO8Bz8X/rCxZ2DjfYBNi8yZs5slEs8wJG84zJdsnNv2jMfgAF+y9IyEtMQGNnYDbFokJHI3SCS2MSQbHOYxk85tO8wjOf+NgTRPwuEEBvk2rA4Datn8A67FEqSlgcf4N0/Cf6DD2HBp2QayxQ6shRGohZ8ByOBJOMDYgEsLz9ltFoltEgmSh3mMDXvOQbRY86QlJ7bh0sLeu/nmzzYbe77zZwwf/Cg7bM/GwGN8m8fGzp4fhxaYzsQGDDG8GoDAnoD8KBgFo2AUjGQAABsXUKPmI6YBAAAAAElFTkSuQmCC","orcid":"","institution":"Nantes Université, Oniris, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229","correspondingAuthor":true,"prefix":"","firstName":"Romain","middleName":"","lastName":"Guiho","suffix":""}],"badges":[],"createdAt":"2025-06-03 08:08:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6808577/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6808577/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88652945,"identity":"f9dde2d2-f748-49af-9f6e-68c8f0329acd","added_by":"auto","created_at":"2025-08-08 18:02:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":352100,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003eRepresentative photomicrograph of H\u0026amp;E-stained histological sections of a wild-type (WT, left side) and transgenic (TgA86, right side) lumbar IVD.\u003cem\u003e NP: nucleus pulposus; AF: annulus fibrosus; CEP: cartilage endplate\u003c/em\u003e; \u003cem\u003eGP: growth plate\u003c/em\u003e. Black dashed lines: demarcation between the \u003cem\u003eNP\u003c/em\u003e,\u003cem\u003eAF \u003c/em\u003eand \u003cem\u003eEP\u003c/em\u003e. Stars: porosity in the \u003cem\u003eEP\u003c/em\u003e. Red dashed lines: alignment of the collagen lamellae forming the \u003cem\u003eAF\u003c/em\u003e. Red arrows: \u003cem\u003eAF \u003c/em\u003efibroblast-like cells. Blue arrows: \u003cem\u003eNP \u003c/em\u003ecells. Green arrows: \u003cem\u003eCEP \u003c/em\u003echondrocytes. \u003cstrong\u003e(B) \u003c/strong\u003eDegeneration scores (mean ± SD) and scoring criteria (n.s.: not significant).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6808577/v1/ceb08543994634ee0f2ff5d6.png"},{"id":88652946,"identity":"b3029713-65c0-42a1-a222-2ea507a6b68e","added_by":"auto","created_at":"2025-08-08 18:02:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":383489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003eRepresentative photomicrograph of H\u0026amp;E-stained histological sections of a wild-type (WT, left side) and transgenic (TgA86, right side) thoracic IVD.\u003cem\u003e NP: Nucleus Pulposus; AF: annulus fibrosus; CEP: cartilage endplate\u003c/em\u003e; \u003cem\u003eGP: growth plate\u003c/em\u003e. Black dashed lines: demarcation between the \u003cem\u003eNP\u003c/em\u003e,\u003cem\u003e AF \u003c/em\u003eand \u003cem\u003eEP\u003c/em\u003e. Red dashed lines: alignment of collagen lamellae forming the \u003cem\u003eAF\u003c/em\u003e. Red arrows: \u003cem\u003eAF \u003c/em\u003efibroblast-like cells. Blue arrows: \u003cem\u003eNP \u003c/em\u003ecells. Green arrows: \u003cem\u003eCEP \u003c/em\u003echondrocytes. \u003cstrong\u003e(B) \u003c/strong\u003eDegeneration scores (mean ± SD) and scoring criteria (n.s.: not significant).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6808577/v1/a9da9cfa72932b802b00b8d8.png"},{"id":88653577,"identity":"9a3943ff-a592-47d0-afc8-7218fc6ae4f5","added_by":"auto","created_at":"2025-08-08 18:10:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1334902,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6808577/v1/22e90988-ad0a-42a7-909b-7aee9a8cf681.pdf"},{"id":88652950,"identity":"68121ad8-b545-4e42-90a2-049e7d5e8da1","added_by":"auto","created_at":"2025-08-08 18:02:54","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":332683,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1.\u003c/strong\u003e Inter-rater agreement statistics. The strength of agreement among the different raters is reported as a numerical value between 0 (no agreement) and 1 (total agreement).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure S1.\u003c/strong\u003e Axial phenotype: tail length and spine curvature. \u003cstrong\u003e(A)\u003c/strong\u003e Representative pictures of a wild-type (WT, left panel) and transgenic (TgA86, right panel) tail. \u003cstrong\u003e(B)\u003c/strong\u003e Tail length measurements. \u003cstrong\u003e(C)\u003c/strong\u003e Representative pictures of H\u0026amp;E-stained histological sections of a Wild-Type (WT, left panel) and transgenic (TgA86, right panel) cervicothoracic spine. The angle formed by the C3-T1-T9 vertebrae is measured. \u003cstrong\u003e(D)\u003c/strong\u003e C3-T1-T9 angle measurements for the enrolled mice. (Mean ± SD; *: p-value ≤ 0.05).\u003c/p\u003e","description":"","filename":"SupplementaryDataRAJITgA86ResearchNote.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6808577/v1/1c6b2c11b04e349591d0e872.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"TgA86 Mice overexpressing transmembrane TNF display an unexpected absence of thoracolumbar disc degeneration","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eLow back pain (LBP) is one of the leading causes of years lived with disability, affecting all age groups across low to high-income countries(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Recent studies have estimated that the prevalence of LBP exceeds 600\u0026nbsp;million people worldwide(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The substantial socioeconomic burden associated with LBP makes it a major public health issue among noninfectious chronic diseases(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough LBP is multifactorial and can stem from diverse etiologies, substantial evidence implicates intervertebral disc degeneration (IVDD) as a key contributor(\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9 CR10\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Current treatment strategies for LBP rely on pharmacologic pain management, lifestyle modifications or manual therapies. Some patients with advanced disease undergo invasive surgery(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) due to lack of therapeutic alternatives. Thus, understanding the pathomechanisms underlying IVDD is crucial for developing innovative, evidence-based therapies, which necessitate relevant animal models that accurately mimic human pathology. Different models have been used for this purpose(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The transgenic A86 model could be a promising candidate for disease modeling and therapeutic development.\u003c/p\u003e\u003cp\u003eTgA86 mice harbor a genetic mutation allowing the overexpression of transmembrane tumor necrosis factor (tmTNF), whose signal is transduced through the TNFR1 and TNFR2 membrane receptors(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Several studies corroborate a pathogenic role of TNF-mediated proinflammatory signaling in IVDD(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). These mice are described as a model of human spondyloarthritis(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), an inflammatory disease predominantly affecting the axial skeleton, notably including inflammation of the vertebrae(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), i.e., anatomical structures in the vicinity of intervertebral discs (IVDs). Notably, a significant proportion of patients suffering from axial spondylarthritis also develop IVDD(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Christodoulou and colleagues studied the axial phenotype of TgA86 mice: macroscopically, these mice exhibited reduced body length and tail bending. This tail bending was shown to be accompanied by caudal IVDD(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Additionally, CT imaging revealed hyperkyphosis(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Clinical observations suggest a potential relationship between abnormal spine curvature and degenerative discopathy(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) as the mechanical strains associated with aberrant spine curvature may contribute to IVDD. Conversely, the degeneration-related loss of mechanical properties of the IVD may exacerbate abnormal spine angles. Furthermore, CT imaging revealed a thinning of the intervertebral space and more compressed vertebrae at the cervical level, features reminiscent of the characteristic decrease in disc height index found in both human IVDD patients and other animal models(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). They also reported roughening of vertebral surfaces, comparable to that observed in degenerating discs of ageing rats(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Histopathological analyses revealed enthesitis at intervertebral joints with immune cell infiltration, including neutrophils. This inflammatory environment could negatively impact neighboring IVDs via paracrine mechanisms, given the central role of inflammation in IVDD pathogenesis.(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Besides, increased osteoclastic activity, ectopic cartilaginous neoformation, and osteogenic marker positivity in the vertebral bodies of TgA86 mice suggest uncoordinated osteochondral remodeling. Analogous histological changes in the endplates have been reported by several studies to be associated with IVDD(\u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), as the fate of intervertebral discs is closely linked to the health of the surrounding compartments(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Overall, TgA86 mice undergo a myriad of cellular and tissue transformations resembling features of IVDD. The present work aimed to assess the relevance of TgA86 transgenic mice as an in vivo model for IVDD.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003eAnimal model\u003c/h2\u003e\u003cp\u003eTgA86 is a transgenic model generated via site-directed mutagenesis, allowing global overexpression of transmembrane TNF, including in joints(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The mice used in this work were heterozygous for the transgene and maintained on a mixed CBA \u0026times; C57BL/6J genetic background. Animal handling and surgical procedures were conducted at INSA LYON, according to the European Community Guidelines for Care and Use of Laboratory Animals (2010/63/UE) and were approved by the national ethical committee (APAFIS #37995-202207190935346) and the institutional animal welfare committee at CEEA \u0026ndash; 102 Comit\u0026eacute; d\u0026rsquo;\u0026eacute;thique de l\u0026rsquo;INSA de Lyon.\u003c/p\u003e\u003cp\u003eSix transgenic mice (3 males, 3 females) and three age-matched wild-type littermates (1 male, 2 females) were euthanized at 35 weeks of age in their home cage using CO\u003csub\u003e2\u003c/sub\u003e inhalation to ensure minimal stress and to maintain spine integrity (6L/min, 3min dwell time). After confirmation of death by decapitation, the spines were collected for analysis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eHistopathological analysis\u003c/h3\u003e\n\u003cp\u003eThe thoracic and lumbar segments of the spines were dissected, fixed in 4% paraformaldehyde for 48 hours, decalcified in 0.5 M EDTA with 28% ammonium hydroxide for ten days, and embedded in paraffin. Four micrometer-thick mid-sagittal sections of the spine were stained with hematoxylin and eosin (H\u0026amp;E) and imaged using a Nanozoomer S360 digital slide scanner (Hamamatsu photonics) with a 40x objective. Three blinded raters evaluated a minimum of 3 thoracic and lumbar IVDs per mouse, using a scoring system designed specifically for grading the extent of IVDD in mice(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Graded features included the cellularity and matrix organization of Nucleus Pulposus (NP), Annulus fibrosus (AF), Endplates (EP), and the demarcation between those different compartments. A global degeneration score was determined by summing the grades obtained for each criterion. Degeneration scores were classified as follows: healthy (non-degenerated) for scores below 6, mild degeneration for scores between 7 and 13, moderate degeneration for scores between 14 and 25, and severe degeneration for scores between 26 and 33.\u003c/p\u003e\n\u003ch3\u003eInterscore reliability\u003c/h3\u003e\n\u003cp\u003eThe magnitude of agreement among the three scorers was evaluated via different statistical methods included in the {irr 0.84.1} package, using {R 4.3.3} in RStudio (2023.06.0\u0026thinsp;+\u0026thinsp;421). The results are recapitulated in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed with GraphPad Prism {10.3.1} software. The statistical significance of differences in histopathological scores, spine curvature angles and tail length between wild-type and transgenic samples were determined using appropriate nonparametric t-test (Mann-Whitney).\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eLumbar IVDs do not exhibit apparent histological signs of IVDD in TgA86 mice\u003c/h2\u003e\u003cp\u003eWe first conducted a histopathological analysis of lumbar IVDs, since this level of the spine is the most commonly affected in humans(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Histopathological analysis of lumbar IVDs revealed well-organized structures in TgA86 mice, with a clear demarcation between the different compartments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, black dotted lines): a central NP surrounded by AF, and EP at the interface between IVDs and vertebrae. The central NP contained spindle-shaped cells (blue arrows) that were evenly distributed in a homogenous matrix. It was surrounded by an AF matrix consisting of concentric lamellae (red dotted lines), which is the classical architecture of a healthy disc. In between the lamellae, there were characteristic fibroblast-like cells (red arrows). Very few and small fissures could be observed in the \u003cem\u003eAF\u003c/em\u003e of some discs, but this was the case for both the transgenic and wild-type samples. At the interface between the disc and the vertebral bodies, the cartilage EP presented as a normal hyalin cartilage comprising chondrocytes with usual morphology (green arrows). Notably, we detected porous structures (indicated by stars) in the EP of some discs. However, these porous structures were observed in both wild-type and transgenic samples, suggesting that they are more likely age-related degenerative features rather than genotype-related ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Histopathological scores of TgA86 mice revealed mild to moderate degeneration. There was no statistically significant difference in degeneration scores between wild-type and transgenic lumbar IVDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Overall, the data indicate an absence of striking signs of degeneration in TgA86 lumbar IVDs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eThoracic IVDs do not exhibit apparent histological signs of IVDD in TgA86 mice\u003c/h3\u003e\n\u003cp\u003eIn agreement with previous findings(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), the entire spine of TgA86 mice was deformed at 35 weeks of age (independently of the sex of the mice), with the occurrence of multiple and severe curvatures in the tail in particular, but also at the cervicothoracic level (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). We then wondered whether the genotype of TgA86 mice could exacerbate a spontaneously occurring degeneration process at the thoracic level, and therefore be used as model of human thoracic degeneration(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). We examined the thoracic discs following the same procedure as for lumbar IVDs. Our histopathological study highlighted the healthy structure of TgA86 thoracic IVDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), with no genotype-associated degeneration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAnimal models are essential for IVDD basic and translational research (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), each offering unique advantages and limitations. TgA86 mice spines provide a set of cues suggesting tissue transformations in the IVD area that align with several features of IVDD(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). However, our analysis revealed that these structural changes were not associated with a degenerative process in the lumbar and thoracic discs.\u003c/p\u003e\u003cp\u003eThe lumbar segment of the spine is the segment most frequently affected in humans(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) and lumbar discs degeneration was reported in aging mice(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). We therefore hypothesized that the TNF-driven inflammation in TgA86 mice exacerbates a naturally occurring degenerative process at the lumbar level. While the AF and NP compartments of the TgA86 lumbar discs displayed healthy structures, we observed porous structures in the EP. This porosity of EP has been associated with a sensory innervation and a pain-related behavior in mice(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), and clinically linked with LBP in patients displaying lumbar degeneration on MRI and CT scan(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). However, those porous structures were observed in both wild-type and transgenic samples, suggesting that this slight damage seemed to be solely due to the mature age of the mice, and not to their genotype.\u003c/p\u003e\u003cp\u003eThoracic IVDs are also prone to degeneration, which is positively correlated with the age of human patients(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), and degeneration of thoracic IVDs has been described in ageing mice(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In the thoracic segment, CT imaging of TgA86 spines revealed hyperkyphosis compared with that in wild-type samples(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). However, we showed that this abnormality was not accompanied by histological signs of disc degeneration.\u003c/p\u003e\u003cp\u003eDespite evidence highlighting a pathogenic role of TNF-mediated inflammation in IVDD(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), the transmembrane TNF-driven activation of the TNF axis has failed to replicate a degenerate pattern in mouse lumbar and thoracic IVDs. In another study, Gorth et al. explored the structure of lumbar IVDs in Tg197 mice, a human TNF-overexpressing model (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). As opposed to us, they noticed a decrease in NP and AF scores (modified Thompson scoring). This apparent discrepancy may stem from differences in the genetic constructs (overexpression of murine transmembrane TNF for TgA86; versus overexpression of human TNF, without distinction of the soluble and membrane forms, with several gene copies in Tg197), likely resulting in differential activation patterns of TNF signaling. Nonetheless, they observed an overall healthy tissue structure, unchanged NP aspect ratio and absence of catabolic factors expression, corroborating our concept that increased TNF transduction alone is not sufficient to satisfyingly model IVDD. The mechanistic basis of IVDD and the relative contribution of inflammation to pathogenesis may differ among species, and the pathogenic process underlying degeneration may vary according to the triggering events. A range of large and small animal models is employed in IVDD research, each relying on different IVDD induction modalities (age-related spontaneous degeneration, genetic mutation, postural, surgical, chemical, dietary or bacterial induction)(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), implying mechanistic differences. Each models offers a balance between advantages and drawbacks in terms of reproducibility, costs, timeline evolution of the disease, and phenotypic outcome(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Selecting the most appropriate model requires careful consideration of these factors, depending on the research question.\u003c/p\u003e\u003cp\u003eIn conclusion, complementing Christodoulou\u0026rsquo;s work, our study provides the first detailed histopathological characterization of TgA86 IVDs. These findings suggest the limited relevance of TgA86 mice as an IVDD model but highlight the complexity of TNF role in disc disease. Future studies should explore alternative pathways and models to elucidate IVDD mechanisms and identify novel therapeutic targets.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eLIMITATIONS\u003c/h2\u003e\u003cp\u003eOur study has several limitations. It is possible that a pro-degenerative cascade of molecular and cellular changes was initiated in the TgA86 mice, but the selected timepoint for sacrifice may have been too early to detect substantial histological impact. In line with this observation, benefits of TNF receptors knock-out on lumbar disc structure were only detected at 21 months of age in another mouse model(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Additionally, we focused solely on structural signs of IVDD, while pain remains a central issue of this disease(\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9 CR10\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). For example, in a rat model, intradiscal injection of degenerated human NP cells triggered pain-related behaviors and increased the expression of degeneration-related markers (e.g., MMP3, TNF and IL-6) by recipient NP cells, despite the absence of visible histological degeneration(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u0026bull; LBP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLow back pain\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u0026bull; IVDD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIntervertebral disc degeneration\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u0026bull; NP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNucleus pulposus\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u0026bull; AF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAnnulus fibrosus\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u0026bull; EP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEndplates\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u0026bull; GP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGrowth plate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal handling and surgical procedures were conducted according to the European Community Guidelines for Care and Use of Laboratory Animals (2010/63/UE) and approved by the national ethical committee (APAFIS #37995-202207190935346) and the institutional animal welfare committee at CEEA \u0026ndash; 102 \u003cem\u003eComit\u0026eacute; d\u0026rsquo;\u0026eacute;thique de l\u0026rsquo;INSA de Lyon\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files. Raw histological data and materials can be made available upon reasonable request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS\u0026rsquo; CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRG, JG and CB conceived and designed the experiments. MMR, LF, RG, NB and CB performed the experiments. MMR and RG analyzed the data. MMR and RG wrote the manuscript. JG, CV and CB reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the SC3M platform from the Inserm/NU/ONIRIS UMR1229 RMeS Laboratory and SFR Bonamy. \u003cstrong\u003eThey also\u0026nbsp;\u003c/strong\u003eacknowledge\u003cstrong\u003e\u0026nbsp;the MicroPICell core facility (SFR Bonamy, BioCore, Inserm UMS 016, CNRS UAR 3556, Nantes, France), member of the Scientific Interest Group (GIS) Biogenouest, IBISA, and the national infrastructure France-Bioimaging supported by the French national research agency (ANR-10-INBS-04). We thank A. Briolay and A. Debain for helping with animal care and tissue collection.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the French Society of Rheumatology (Soci\u0026eacute;t\u0026eacute; Fran\u0026ccedil;aise de Rhumatologie). MMR was supported by the M4R Master-Doctorate program (Repair, Replace, Regenerate, Reprogram) of Nantes University, France.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest associated with this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVos T, Lim SS, Abbafati C, Abbas KM, Abbasi M, Abbasifard M, et al. Global burden of 369 diseases and injuries in 204 countries and territories, 1990\u0026ndash;2019: a systematic analysis for the Global Burden of Disease Study 2019. 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Arthritis Res Ther. 2020;22(1):232.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDanve A, Deodhar A. Treatment of axial spondyloarthritis: an update. Nat Rev Rheumatol. 2022;18(4):205\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKilic G, Senol S, Baspinar S, Kilic E, Ozgocmen S. Degenerative changes of lumbar spine and their clinical implications in patients with axial spondyloarthritis. Clin Rheumatol. 2023;42(1):111\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaaij MH, Van Tok MN, Blijdorp IC, Ambarus CA, Stock M, Pots D, et al. Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis. J Exp Med. 2020;217(10):e20200288.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBae J, Lee SH, Shin SH, Seo JS, Kim KH, Jang JS. Radiological analysis of upper lumbar disc herniation and spinopelvic sagittal alignment. Eur Spine J. 2016;25(5):1382\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchneider DL, von M\u0026uuml;hlen D, Barrett-Connor E, Sartoris DJ. Kyphosis does not equal vertebral fractures: the Rancho Bernardo study. J Rheumatol. 2004;31(4):747\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang SN, Walter BA, Heimann MK, Gantt CC, Khan SN, Kokiko-Cochran ON, et al. In vivo Mouse Intervertebral Disc Degeneration Models and Their Utility as Translational Models of Clinical Discogenic Back Pain: A Comparative Review. Front Pain Res. 2022;3:894651.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGruber HE, Ashraf N, Kilburn J, Williams C, Norton HJ, Gordon BE, et al. Vertebral Endplate Architecture and Vascularization: Application of Micro-Computerized Tomography, a Vascular Tracer, and Immunocytochemistry in Analyses of Disc Degeneration in the Aging Sand Rat. 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Transgenic mice overexpressing human TNF-α experience early onset spontaneous intervertebral disc herniation in the absence of overt degeneration. Cell Death Dis. 2018;10(1):1\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDaly C, Ghosh P, Jenkin G, Oehme D, Goldschlager T. A Review of Animal Models of Intervertebral Disc Degeneration: Pathophysiology, Regeneration, and Translation to the Clinic. BioMed Res Int. 2016;2016:1\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlini M, Diwan AD, Erwin WM, Little CB, Melrose J. An update on animal models of intervertebral disc degeneration and low back pain: Exploring the potential of artificial intelligence to improve research analysis and development of prospective therapeutics. JOR SPINE. 2023;6(1):e1230.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTao C, Lin S, Shi Y, Gong W, Chen M, Li J, et al. Inactivation of Tnf-α/Tnfr signaling attenuates progression of intervertebral disc degeneration in mice. JOR Spine. 2024;7(4):e70006.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJiang W, Glaeser JD, Kaneda G, Sheyn J, Wechsler JT, Stephan S, et al. Intervertebral disc human nucleus pulposus cells associated with back pain trigger neurite outgrowth in vitro and pain behaviors in rats. Sci Transl Med. 2023;15(725):eadg7020.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"TgA86 mice, histopathological scoring, intervertebral disc degeneration","lastPublishedDoi":"10.21203/rs.3.rs-6808577/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6808577/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective.\u003c/h2\u003e\u003cp\u003eThis study evaluated the relevance of TgA86 mice, which overexpress transmembrane TNF (tmTNF), as a model for intervertebral disc degeneration (IVDD). Six middle-aged, skeletally mature, transgenic TgA86 mice were included in this study alongside three control littermates. Histopathological scoring was performed on sections of the lumbar and thoracic segments of the spine to evaluate the extent of IVDD.\u003c/p\u003e\u003ch2\u003eResults.\u003c/h2\u003e\u003cp\u003eDespite preexisting cues predicting a degenerated disc phenotype, degeneration scores were comparable between TgA86 and wild-type mice, with no evidence of any genotype-specific effects. This first report of a detailed histopathological characterization of TgA86 mice thoracolumbar intervertebral discs suggest that transmembrane TNF (tmTNF)-driven inflammation alone does not induce IVDD, limiting their utility as a model for studying disc degeneration.\u003c/p\u003e","manuscriptTitle":"TgA86 Mice overexpressing transmembrane TNF display an unexpected absence of thoracolumbar disc degeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-08 18:02:49","doi":"10.21203/rs.3.rs-6808577/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-07T16:08:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-15T11:54:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51823008159111782790491530988674925496","date":"2025-08-10T00:51:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-06T05:30:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-13T11:12:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-04T13:06:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-04T12:04:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2025-06-04T12:01:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"227b0fdf-caf9-46e7-9b78-09609448b7f2","owner":[],"postedDate":"August 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T11:53:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-08 18:02:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6808577","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6808577","identity":"rs-6808577","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}