Innovation in animal model of Pyogenic Spondylitis induced by Staphylococcus aureus

Innovation in animal model of Pyogenic Spondylitis induced by Staphylococcus aureus | 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 Article Innovation in animal model of Pyogenic Spondylitis induced by Staphylococcus aureus Qinpeng Xu, Guihe Yang, Jiaju Ma, Fei Jia, Xingzhi Jing, Xingang Cui, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7448639/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and purpose Pyogenic spondylitis (PS) is clinically challenging and induces disastrous consequences for patients. The pathogenesis of PS is difficult to explore due to a lack of ideal animal models. Thus, we aimed to reproduce the pathogenesis of PS in an innovative animal model induced by Staphylococcus aureus . Methods Rats were injected with planktonic Staphylococcus aureus (ATCC 25923) and grouped according to different concentrations. We identified the optimal bacterial inoculum concentration based on general physical signs and radiological, hematological, and histological parameters in rats. Models with the optimal bacterial concentration were used to investigate changes in physical, radiological, and inflammatory parameters at different time points. Results Our results revealed that infected rats experienced rapid weight loss, high fever, and significantly increased white blood cell count, interleukin 1β, and C-reactive protein (CRP) levels in the short term. Radiographic examination revealed bone damage in groups that received 2×10 3 /20 µl, 2×10 5 /20 µl and 2×10 7 /20 µl bacterial concentrations. The optimal concentration was identified as 2×10 5 /20 µl, based on the high survival rate, obvious bone destruction, and inflammation. Histological staining confirmed the living bacteria, inflammatory cells, bone destruction, and scarce bone formation in infected vertebrae. Conclusions This study provided an innovative PS animal model, which may improve our study of the pathological mechanism underlying PS. Health sciences/Diseases Biological sciences/Immunology Health sciences/Medical research Biological sciences/Microbiology Health sciences/Pathogenesis Pyogenic spondylitis Spinal infection Animal model Staphylococcus aureus Rat Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Spinal infections, caused by specific pathogenic microorganisms, usually involve the vertebrae, intervertebral discs, and adjacent perivertebral soft tissue. These infections were classified based on the microbial etiology into pyogenic spondylitis (PS), tuberculous spondylitis (TS), fungal spondylitis, or others [1] . PS is the most common infection, whose incidence has been increasing in recent years due to social aging and the misuse of antibiotics [2] . PS can lead to sepsis, spinal instability, and neurological deficits, and is becoming a severe and potentially life-threatening disease with a mortality rate of 4 to 29% [3, 4] . The lumbar spine is susceptible to PS involvement (58%) [5] . The most common pathogen was Staphylococcus aureus ( S. aureus ) [6] . In attempting to study its pathology, clinical manifestations, and treatment strategies, we have only partially recognized this disease. Therefore, it is pivotal to comprehensively explore the pathogenesis of PS and its influence on patients. However, the lack of a suitable animal model critically hinders further exploration of PS prevention and treatment [7] . In the pathogenesis of PS, bacteria from a distant infectious lesion may survive in the terminal microvasculature beneath the vertebral endplate [8] . The rapid growth of local bacteria leads to vessel occlusion, ischemic necrosis, osteonecrosis, and vertebral osteomyelitis. Local bacteria proliferate and damage vertebrae, cartilage endplates, and intervertebral discs owing to invasive enzymes (such as hyaluronidase secreted by S. aureus ), resulting in pyogenic discitis [9] . However, there is no suitable animal model to simulate PS originating from the primary vertebral infection. Animal studies of osteomyelitis have been reported, including the traumatic osteomyelitis model in rats [10] and the periprosthetic joint infection model in rabbits [11] . The imaging and pathological changes were narrated in these studies. Unfortunately, these models cannot appropriately simulate the characteristics of spinal infection. Bierry and Chen created models of PS in rabbits and in canines, respectively [12, 13] . In these two studies, animals were injected with a bacterial suspension into the intervertebral space, which differs from the pathogenesis of human PS. Sweet created a rabbit infectious model by laminectomy, which can be used to mimic surgical site infection after metal implantation [14] . These models have obvious limitations, including high technical requirements and poor imitation. PS imposes a great burden on families and society, thus innovative animal models are necessary to further explore the pathogenesis of PS and potential treatment strategies. Rats are commonly used due to their low cost, minimal individual differences, antibiotic tolerance, and manageable body size [15] . It is vital to establish an animal model to reproduce the pathogenesis of PS in order to investigate the mechanisms underlying inflammation and bone metabolism [16] . The objective of this study was to establish a new, reliable animal model to precisely mimic the pathogenesis of PS, and further explore novel anti-infective strategies. 2. Materials and methods 2.1 Animal selection and grouping This study was approved by the Animal Ethics Committee and Institutional Review Board at our hospital (NO. SDNSFC 2023 − 0198). We confirmed that all experiments were performed in accordance with relevant guidelines and regulations. Seventy-five male Wistar rats aged 8 weeks old were used in this study. The mean weight of the animals was 250 g. The animals were housed in individual cages at least six days before surgery and had free access to food and water. To determine the optimal S. aureus concentration, rats were randomly divided into five groups. The experimental groups were administered a 20 µl S. aureus suspension of varying concentrations: 1×10 3 (G1 group, n = 7), 1×10 5 (G2 group, n = 7), 1×10 7 (G3 group, n = 7), 1×10 9 colony forming units (CFU)/ml (G4 group, n = 7). The control group received 20 µl phosphate buffer saline (PBS). Postoperatively, body weight and anal temperature was measured daily. Rats reached the endpoint of humanitarian execution when weight loss was > 20% or anal temperature was < 36 ℃. 2.2 Preparation of bacterial inoculum S. aureus (ATCC 25923) was incubated in trypticase soy agar (TSA) medium at 37°C for 18 h. After 18 h, S. aureus was harvested and resuspended in sterile PBS at a concentration of 1×10 9 CFU /ml according to optical density measurements. The suspension was diluted into various bacterial concentrations (1×10 3 , 1×10 5 ,1×10 7 CFU /ml). A preliminary experiment was first performed to identify the appropriate concentration of bacterial inoculum. 2.3 Surgical procedure The anal temperature and weight of each rat were measured before surgery. To prepare a 100% Avertin stock solution, 10 g of tribromoethanol was dissolved in 10 mL of tertiary amyl alcohol in a centrifuge tube by gently shaking by hand until fully dissolved. The solution was then filtered through a 0.22 µm membrane. The 100% Avertin stock was diluted with 0.9% NaCl physiological saline to a final concentration of 2.5% (1:40 dilution). Avertin (2.5%) was administered via intraperitoneal injection at a dose of 250 mg/kg for each rat. After shaving and sterilization with povidone-iodine, the lumbar vertebra was exposed posteriorly. The skin and fascia were incised sequentially with tissue scissors and a scalpel. Thereafter, the right lamina, facets, and transverse processes were exposed. The puncture point was located at the transition point between the superior articular process and the transverse process. First, the outer layer of the puncture point was opened with a needle (0.45 mm × 16 mm) from a 1 ml syringe, then the puncture channel was made by a needle (0.33 × 12 mm) through the vertebral pedicle. The channel with a depth of 3 mm ended at the anterior section under the endplate. Subsequently, a 20 µl bacterial solution was injected into the vertebral body through the established channel. After injection, the puncture site was sealed using bone wax. Finally, the muscle, fascia, and skin were closed layer by layer after washing (Fig. 1 ). All procedures were conducted aseptically. No antibiotic was administered preoperatively, intraoperatively, or postoperatively. 2.4 Blood assays Blood was collected by cardiac sampling at the endpoints of 7, 14, 21, and 28 days postoperatively. WBC number and morphological differences were determined from blood smears using CellaVision® DM9600. Serum was obtained from blood by centrifugation at 2000 × g for 15 min. According to the manufacturer's instructions, the CRP and interleukin 1β (IL-1β) levels were determined using an enzyme-linked immunosorbent assay (ELISA) (Abcam, Cambridge, Massachusetts, USA). 2.5 Radiographic evaluation Rats were executed at their endpoints post operation. The lumbar spine was harvested and fixed in 4% paraformaldehyde, then scanned using microcomputed tomography (µCT) (Scanco Viva-CT80, Scanco Medical AG, Basserdorf, Switzerland). The data processing and 3D reconstruction were performed using Built-in software. Vertebral destruction was calculated using embedded 3D measurement techniques. Spinal destruction was scored using a modified An and Friedman scoring system (Table 1 ) by three individuals blinded to the experimental protocol. 2.6 Histological analysis After µCT measurement and decalcification, the infected vertebrae and adjacent discs were separated and embedded in paraffin. Next, samples were sectioned axially at 5 µm thickness to obtain anatomical sections, which were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) according to routine protocols. Goldner trichrome staining was used to assess bone mineralization. In addition, Gram staining was performed to determine bacterial infection in the tissue. Immunohistochemical staining was used to evaluate the expression of inflammatory factors. Sections were blocked with 5% BSA at 37°C for 30 minutes. 2.7 Statistical analysis The data is presented as mean ± standard deviation (SD). The Student's t-test was used to evaluate differences between the two groups. One-way analysis of variance (ANOVA) was used when there were more than two groups (SPSS V19.0, SPSS Inc., Chicago, IL, USA). P -values < 0.05 were deemed significant. 2.8 Euthanasia method Five to eight rats were placed in the euthanasia box. The CO 2 cylinder switch was turned on, and CO 2 was introduced into the box at a rate of 10 − 30% of the box volume per minute, until the animals no longer moved, breathes, or exhibited pupil constriction. The CO 2 cylinder switch was then turned off, and the animals were observed for an additional 2–3 minutes to confirm death. Table 1 Modified An and Friedman scoring system. Score Osteolysis Osteogenesis Vertebral body wall destruction Paravertebral abscess 0 not present not present not present not present 1 Unifocal lesion Focal osteosclerosis within the vertebra Single focal lesion Focal abscess involved in a single vertebra 2 Multifocal lesions Generalized osteosclerosis within the vertebra Multiple focal lesions Generalized abscess involved in a single vertebra and adjacent disc 3 Lesions involved in two vertebrae Osteogenesis evident outside the vertebral body Lesions exceeding half of the vertebra Abscess involved in two vertebrae 4 Lesions involved in multiple vertebrae (> 2 vertebrae) Significant osteogenesis involved in at least two vertebrae Extensive lesions involved in at least two vertebrae Abscess involved in multiple vertebrae (> 2 vertebrae) 3. Results 3.1 Vital signs and mortality All rats were observed for 14 days after surgery. There was no statistically significant difference in body weight between the control group and the G1 ~ G3 groups after surgery. However, rats in the G4 group, receiving the highest dose of bacteria (10 9 CFU/ml), experienced the greatest weight loss after bacterial inoculation. All groups showed high body temperature on the first day post-surgery, but the mean temperature of the G1-G4 group peaked 3–5-day post operation and remained significantly higher than that of the control group. However, three rats in the G4 group showed cachexia, rapid weight loss, abnormally decreased temperature, and eventually died. The mortality rate of the G4 group was 42.86%. In contrast, the survival rates of the control group and the G1-G3 groups were greater than 80% (Fig. 2 a-c). 3.2 Blood assays WBC and CRP are well-established biomarkers for detecting infections. Blood smear analysis revealed a significantly increased WBC in the infected group compared with the control group, while there were no significant differences between infected groups. The CRP level in the G3 and G4 groups was higher than that in the control group. Due to significant individual variations, the CRP level in the G1 and G2 groups showed no statistical significance compared with that of the control group. Additionally, the G3 and G4 groups displayed higher IL-1β levels compared with the control group and G1 and G2 groups, which was similar to observed CRP expression levels (Figure S1 a-c). 3.3 Radiographs µCT scans showed that G3 and G4 groups exhibited significant bone destruction, hyperostosis, and sclerosis, while G1 and G2 groups had lower levels of bone destruction compared to G3 and G4 groups, and the control group had narrow channels surrounded by obvious sclerotic bone (Fig. 3 ). Based on the modified An and Friedman rating system (Table 1 ), the scores of G3 and G4 groups were significantly higher than that observed in G1 and G2 groups. The control group displayed the minimum score (Figure S2 ). 3.4 Identifying the optimal bacterial concentration Based on physical signs, bone destruction on CT images, and levels of inflammatory markers, we aimed to identify the optimal bacterial concentration to induce PS. Although obvious bone destruction was observed in both G3 and G4 groups, the maximal concentration in the G4 group was not optimal due to the excessive weight loss and low survival rate. Despite improved survival rates, the G1 and G2 groups showed lower imaging scores and inconsistent levels of inflammatory markers. However, some rats in G1 and G2 groups were not successfully infected by the any bacterial suspension. Therefore, we ultimately identified the 10 7 CFU/ml (20 µl) in the G3 group as the optimal inoculation concentration for subsequent experiments. 3.5 Histology and bacteriologic analyses We performed H&E staining for spinal tissues after rat execution. The control group showed normal trabecular morphology, puncture channels, and surrounding sclerosis (Fig. 4 a). Rats infected by bacterial suspension showed obvious bone destruction filled with fibrous tissue, massive inflammatory cell infiltration, and periosteal reaction (Fig. 4 b). According to the histological scoring criteria of bone infection proposed by Smeltzer et al [17] , the scores in the G3 and G4 groups were significantly higher than that observed in other groups (Figure S3 ). In addition, we performed Goldner staining to evaluate osteogenesis in spinal infection. Mild mineralization was observed in G3 and G4 groups using Goldner staining. In contrast, the active bone-forming response was observed in the control group and in certain rats in G1 and G2 groups. These results were consistent with the µCT imaging findings (Fig. 4 b). Gram staining performed at execution points revealed the presence of gram-positive bacteria around the surgical puncture site (Fig. 4 b). The infected vertebrae were collected and inoculated. Numerous colonies of bacteria were observed and identified as S. aureus (ATCC25923) using mass spectrometry analysis. The bacterial culture showed no bacteria in the liver and blood from the PS group, which indicated that the animal model displayed local infection without systemic infection. 3.6 Dynamic changes to physical characteristics After confirming the inoculating concentration, we monitored consecutive changes to physical characteristics and inflammatory parameters for four weeks. The PS group presented with higher body temperatures than the control group from 3 to 15 days post operation, but there was no statistical difference 20 days after injection. The body weight of the infected rats was lower than that of the control group. In addition, the survival rates at the endpoint were 100% and 80% in the control and PS groups, respectively (Fig. 5 a-c). Inflammatory markers showed dynamic changes in both groups after surgery. Moreover, CRP levels in the PS group were higher than those observed in the control group (Fig. 5 e). We observed similar trends in IL-1β levels, WBC count, and CRP levels (Fig. 5 d and f). 4. Discussion In recent years, the incidence of PS has increased due to the increased life expectancy of patients with chronic diseases and the misuse of antimicrobial drugs [18] . PS generally involves bone and intervertebral discs, thereby inducing several complications that may lead to spinal instability, vertebral collapse and kyphosis, and neurological dysfunction. These complications are common and unique in PS. Moreover, discitis and epidural abscess is a specific presentation of PS. However, the vertebra has no medullary cavity but adjacent discs, which are distinct to the tubular bone. PS is characterized by inflammatory infiltration, bone destruction, and intervertebral or intravertebral abscess, leading to inflammatory reactions, spinal instability, and neurological dysfunction (Figure S4 ). Most animal models of osteomyelitis focus on the bones of the limbs, such as the tibia and femur [10, 19] . Most of these models were used for implant infection or for the evaluation of antibiotic therapy in the tubular bone. Unfortunately, these findings were not suitable for PS due to the distinct anatomy and pathogenesis. There are several animal models for spinal infection. Chen et al. reported a canine model of intervertebral disc infection and evaluated it through microbiology, imaging, and histopathology [13] . Owing to its high cost and the difficult surgical procedure, this animal model was not widely used for anti-infection strategies. Rabbits are commonly used as animal models for spinal infections. Guiboux et al [20] . and Bierry et al [12] . produced a PS animal model by injecting S. aureus into the intervertebral disc and Bostian et al. established a similar model in rats [21] . Small animal models are often preferred due to their lower costs and ease of handling. Ofluoglu et al. established a PS infection model in rats by inserting a pedicle screw with S. aureus [22] . However, these models were inconsistent with the pathogenesis of human PS. In adults, the disc is avascular and resistant to some bacteria, but S. aureus and other enzyme-producing bacteria can spread directly to the intervertebral disc by disrupting the endplate. PS is typically initialized by bacterial colonization in slow-flowing capillaries beneath the vertebral endplate and is characterized by localized bone erosion in this region. Thus, infection emanating from the vertebra is similar to the pathogenesis of PS. In this study, the pathogenic bacterium was injected into the vertebra under the endplate, which agrees with the pathogenesis of PS [8] . Other studies have found that injury can significantly increase the likelihood of bone infection [23] . In this animal model, we first induced vertebral bone injury by puncture and then inoculated bacteria into the lumbar vertebral body of rats, which is consistent with the pathogenesis of PS. Imaging and histological analyses showed that infected rats exhibited early-stage vertebral destruction, which subsequently progressed and spread to the intervertebral disc through the compromised endplate, closely mimicking the pathological progression of human PS. We identified the optimal inoculum concentration (10 7 CFU/ml) of S. aureus based on basic vital signs, imaging, hematology, histology, and bacteriology. Infected rats exhibited high fever and weight loss in the first week, then returned to normal temperature and weight around the second week after inoculation. The levels of WBC and CRP also increased first and then decreased after infection. In addition, µCT images showed significant bone destruction and slight osteogenesis surrounding the lesion. H&E staining showed bone destruction without normal bone trabeculae in the vertebrae, which were replaced by fibrous tissue and inflammatory cells. Meanwhile, Gram staining and bacterial culture results showed the activity of S. aureus in the PS model group. In summary, the above results demonstrate that this animal model can reliably mimic the pathogenesis of PS in humans. This study has several limitations. Firstly, we conducted CT scanning and inflammatory tests on the 14th day after inoculation when the rats displayed obvious bone destruction. Earlier evaluation may help to understand the pathogenesis during PS onset. Secondly, the PS model in this study was tested using a very common bacterium. It may be useful to evaluate the animal model in response to other pathogens. 5. Conclusions In conclusion, we successfully established a PS animal model by injecting S. aureus into the vertebral body below the endplate. Although this model has certain flaws, it is highly mimetic to human PS and provides a significant step toward exploring the pathogenesis of PS and identifying effective treatment strategies. Declarations This study is reported in accordance with ARRIVE guidelines. Conflicts of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding The present study was supported by the Natural Science Foundation of Shandong Province (grant no. ZR2023MH159). Author Contribution Qinpeng Xu: Conceptualization, Writing-original draft; Guihe Yang: Writing-original draft, Validation; Jiaju Ma: Data curation, Software; Xingzhi Jing: Investigation; Fei Jia: Data curation, Software; Xingang Cui: Supervision; Jianlong Li: Methodology; Xiaoyang Liu: Conceptualization, Writing-review & editing. Hongdong Tan: Conceptualization, Writing-review & editing. Data Availability The data used to support the findings of this study are included within the article. References Yokota H, Tali ET. Spinal Infections. 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Spine (Phila Pa 1976) 1998;23:653-6. https://doi.org/10.1097/00007632-199803150-00002. Bostian PA, Karnes JM, Cui S, Robinson LJ, Daffner SD, Witt MR, et al. Novel rat tail discitis model using bioluminescent Staphylococcus aureus. J Orthop Res 2017;35:2075-81. https://doi.org/10.1002/jor.23497. Ofluoglu EA, Zileli M, Aydin D, Baris YS, Kuçukbasmaci O, Gonullu N, et al. Implant-related infection model in rat spine. Arch Orthop Trauma Surg 2007;127:391-6. https://doi.org/10.1007/s00402-007-0365-0. Heng BC, Bai Y, Li X, Lim LW, Li W, Ge Z, et al. Electroactive Biomaterials for Facilitating Bone Defect Repair under Pathological Conditions. Adv Sci (Weinh) 2023;10:e2204502. https://doi.org/10.1002/advs.202204502. Additional Declarations No competing interests reported. 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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-7448639","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":512725061,"identity":"4562c03d-d0d8-4bb6-b297-6586b0541c83","order_by":0,"name":"Qinpeng Xu","email":"","orcid":"","institution":"Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qinpeng","middleName":"","lastName":"Xu","suffix":""},{"id":512725062,"identity":"ace69848-7423-4790-bc76-4e8021201c52","order_by":1,"name":"Guihe Yang","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guihe","middleName":"","lastName":"Yang","suffix":""},{"id":512725066,"identity":"ee38b601-9a6f-4b51-8050-4798e621b7e4","order_by":2,"name":"Jiaju Ma","email":"","orcid":"","institution":"Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiaju","middleName":"","lastName":"Ma","suffix":""},{"id":512725067,"identity":"84967ae1-0ec0-4bbb-bdc5-7dc471142d70","order_by":3,"name":"Fei Jia","email":"","orcid":"","institution":"Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Jia","suffix":""},{"id":512725068,"identity":"9e7148fe-c5e3-4e33-aaac-f37405f8f172","order_by":4,"name":"Xingzhi Jing","email":"","orcid":"","institution":"Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xingzhi","middleName":"","lastName":"Jing","suffix":""},{"id":512725069,"identity":"26c4d0c5-54ba-4e9c-b348-8622c9ec4e88","order_by":5,"name":"Xingang Cui","email":"","orcid":"","institution":"Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xingang","middleName":"","lastName":"Cui","suffix":""},{"id":512725070,"identity":"2ca3b200-f45e-4df0-8d09-a21c1e17cd80","order_by":6,"name":"Jianlong Li","email":"","orcid":"","institution":"Shandong Public Health Clinical Center Affiliated to Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Jianlong","middleName":"","lastName":"Li","suffix":""},{"id":512725071,"identity":"2181c8c8-aca0-4e03-90a5-9bce657fc419","order_by":7,"name":"Xiaoyang Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYBACPmYgkQBhMz5gYJMDsyTwaWFD0sJswMBmTIQWZLYEcVrYecwkHtTYJPbPbr9W+aPMINrgAPPB2zwMdnm4HcZjbJBwLC1xxp0zZbd5zhnkbjjAlmzNw5BcjEeL4YMEtsOJDTdy0m4ztv0BauExk+ZhOJDYgFuLwYGEf4cT5wO1FP5sA9nC/42QFsMHiW2HEzfcSD/GwAvWwsNGQAtbsUFiX5rxxhs5zNIgv8w8zGZsOccgGacWfv7D2yR/fLORnXcj/eFHYIjl9h1vfnjjTYUdTi0w4NjAwGMAYYIil8GAgHogsGdgYH9AWNkoGAWjYBSMSAAAGHhVyoyziIoAAAAASUVORK5CYII=","orcid":"","institution":"Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xiaoyang","middleName":"","lastName":"Liu","suffix":""},{"id":512725072,"identity":"75c239f5-8723-4db1-ab3c-13879b091350","order_by":8,"name":"Hongdong Tan","email":"","orcid":"","institution":"Shandong Public Health Clinical Center Affiliated to Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Hongdong","middleName":"","lastName":"Tan","suffix":""}],"badges":[],"createdAt":"2025-08-25 00:53:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7448639/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7448639/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91194464,"identity":"30cb37d4-0b9b-48c2-acb4-a4a4ec035bb4","added_by":"auto","created_at":"2025-09-12 14:52:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":600356,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSurgical procedure diagram.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003e(a)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e The diagram indicates the surgical procedures conducted during the study. The lumbar spine of the rat was exposed. The puncture point was the junction of the transverse and articular processes and the channel reached the anterior part of the vertebra. The bacterial solution was injected into the vertebral body (below the endplate) using a 25 μl syringe.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e (b)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Operative feature. Yellow arrows show the insertion position.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7448639/v1/1f8fafd52aed8935843f78d1.png"},{"id":91193403,"identity":"60bfc1c6-54fc-4601-a579-8afb48547123","added_by":"auto","created_at":"2025-09-12 14:44:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":62232,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysical markers of pyogenic infections.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a, b)\u003c/strong\u003e Body weight and temperature of rats among different groups after surgery.\u003cstrong\u003e (c) \u003c/strong\u003eSurvival rate curves of rats after surgery in different groups. Data are presented as mean ± standard deviation (SD). ns-no significance, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7448639/v1/d0bd5f1e127057776fb8e360.png"},{"id":91195930,"identity":"07367275-ded1-4b1d-ac91-e5237ecc7a4d","added_by":"auto","created_at":"2025-09-12 15:00:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":511324,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImaging findings of pyogenic infections.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicro-CT imaging shows bone destruction in different groups two weeks after surgery. Bone infections are indicated by green arrows.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7448639/v1/f61a7c261389d8b4ba5b572d.png"},{"id":91195931,"identity":"c2a145b4-7da8-4b86-b9a7-a502a9b5df81","added_by":"auto","created_at":"2025-09-12 15:00:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1092169,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological evidence of pyogenic spondylitis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) Hematoxylin and eosin (H\u0026amp;E) \u003c/strong\u003estaining of vertebrae in Control and G3 groups revealed obvious bone destruction and massive inflammatory cell infiltration. Scale bar = 500 μm and 200 μm. \u003cstrong\u003e(b) \u003c/strong\u003eGoldner trichrome staining indicated milder bone-forming in PS rats compared with that in the control group. Gram staining indicated the colonies of injected \u003cem\u003eS. aureus\u003c/em\u003e in the PS model group. The green arrowhead indicates gram-positive bacterial colonies. Scale bar = 500μm.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7448639/v1/c8a324f0937a8ee0f6b08d8c.png"},{"id":91193408,"identity":"e1d323df-5cd1-46a3-9e3b-11a48fcf9fae","added_by":"auto","created_at":"2025-09-12 14:44:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":46159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVital signs of rats within 28 days.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Body weight and \u003cstrong\u003e(b)\u003c/strong\u003e temperature of rats among different groups after surgery. \u003cstrong\u003e(c) \u003c/strong\u003eSurvival rate curves of rats after surgery in different groups. ns-no significance. \u003cstrong\u003e(d) \u003c/strong\u003eSerum interleukin-1β (IL-1β), \u003cstrong\u003e(e) \u003c/strong\u003eC-reactive protein (CRP), and \u003cstrong\u003e(f) \u003c/strong\u003ewhite blood cell (WBC) values in different groups of rats 28 days after surgery.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7448639/v1/666ec0c45f0db7f5ecbbd5a8.png"},{"id":107027017,"identity":"240d78b4-84fa-4c01-96d2-fc3f8b30c2f8","added_by":"auto","created_at":"2026-04-16 01:40:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3013611,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7448639/v1/00f31cbf-e020-4127-a50d-01a22334f441.pdf"},{"id":91194468,"identity":"ac7426a8-54e8-4c61-bc7c-0df9a94c972b","added_by":"auto","created_at":"2025-09-12 14:52:54","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2282759,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7448639/v1/d0ea4c2d25103e89af0cc70f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Innovation in animal model of Pyogenic Spondylitis induced by Staphylococcus aureus","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSpinal infections, caused by specific pathogenic microorganisms, usually involve the vertebrae, intervertebral discs, and adjacent perivertebral soft tissue. These infections were classified based on the microbial etiology into pyogenic spondylitis (PS), tuberculous spondylitis (TS), fungal spondylitis, or others\u003csup\u003e[1]\u003c/sup\u003e. PS is the most common infection, whose incidence has been increasing in recent years due to social aging and the misuse of antibiotics\u003csup\u003e[2]\u003c/sup\u003e. PS can lead to sepsis, spinal instability, and neurological deficits, and is becoming a severe and potentially life-threatening disease with a mortality rate of 4 to 29%\u003csup\u003e[3, 4]\u003c/sup\u003e. The lumbar spine is susceptible to PS involvement (58%)\u003csup\u003e[5]\u003c/sup\u003e. The most common pathogen was \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e)\u003csup\u003e[6]\u003c/sup\u003e. In attempting to study its pathology, clinical manifestations, and treatment strategies, we have only partially recognized this disease. Therefore, it is pivotal to comprehensively explore the pathogenesis of PS and its influence on patients. However, the lack of a suitable animal model critically hinders further exploration of PS prevention and treatment\u003csup\u003e[7]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn the pathogenesis of PS, bacteria from a distant infectious lesion may survive in the terminal microvasculature beneath the vertebral endplate\u003csup\u003e[8]\u003c/sup\u003e. The rapid growth of local bacteria leads to vessel occlusion, ischemic necrosis, osteonecrosis, and vertebral osteomyelitis. Local bacteria proliferate and damage vertebrae, cartilage endplates, and intervertebral discs owing to invasive enzymes (such as hyaluronidase secreted by \u003cem\u003eS. aureus\u003c/em\u003e), resulting in pyogenic discitis\u003csup\u003e[9]\u003c/sup\u003e. However, there is no suitable animal model to simulate PS originating from the primary vertebral infection.\u003c/p\u003e\u003cp\u003eAnimal studies of osteomyelitis have been reported, including the traumatic osteomyelitis model in rats\u003csup\u003e[10]\u003c/sup\u003e and the periprosthetic joint infection model in rabbits\u003csup\u003e[11]\u003c/sup\u003e. The imaging and pathological changes were narrated in these studies. Unfortunately, these models cannot appropriately simulate the characteristics of spinal infection. Bierry and Chen created models of PS in rabbits and in canines, respectively\u003csup\u003e[12, 13]\u003c/sup\u003e. In these two studies, animals were injected with a bacterial suspension into the intervertebral space, which differs from the pathogenesis of human PS. Sweet created a rabbit infectious model by laminectomy, which can be used to mimic surgical site infection after metal implantation\u003csup\u003e[14]\u003c/sup\u003e. These models have obvious limitations, including high technical requirements and poor imitation. PS imposes a great burden on families and society, thus innovative animal models are necessary to further explore the pathogenesis of PS and potential treatment strategies.\u003c/p\u003e\u003cp\u003eRats are commonly used due to their low cost, minimal individual differences, antibiotic tolerance, and manageable body size\u003csup\u003e[15]\u003c/sup\u003e. It is vital to establish an animal model to reproduce the pathogenesis of PS in order to investigate the mechanisms underlying inflammation and bone metabolism\u003csup\u003e[16]\u003c/sup\u003e. The objective of this study was to establish a new, reliable animal model to precisely mimic the pathogenesis of PS, and further explore novel anti-infective strategies.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animal selection and grouping\u003c/h2\u003e\u003cp\u003eThis study was approved by the Animal Ethics Committee and Institutional Review Board at our hospital (NO. SDNSFC 2023\u0026thinsp;\u0026minus;\u0026thinsp;0198). We confirmed that all experiments were performed in accordance with relevant guidelines and regulations. Seventy-five male Wistar rats aged 8 weeks old were used in this study. The mean weight of the animals was 250 g. The animals were housed in individual cages at least six days before surgery and had free access to food and water. To determine the optimal \u003cem\u003eS. aureus\u003c/em\u003e concentration, rats were randomly divided into five groups. The experimental groups were administered a 20 \u0026micro;l \u003cem\u003eS. aureus\u003c/em\u003e suspension of varying concentrations: 1\u0026times;10\u003csup\u003e3\u003c/sup\u003e (G1 group, n\u0026thinsp;=\u0026thinsp;7), 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e (G2 group, n\u0026thinsp;=\u0026thinsp;7), 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e (G3 group, n\u0026thinsp;=\u0026thinsp;7), 1\u0026times;10\u003csup\u003e9\u003c/sup\u003e colony forming units (CFU)/ml (G4 group, n\u0026thinsp;=\u0026thinsp;7). The control group received 20 \u0026micro;l phosphate buffer saline (PBS). Postoperatively, body weight and anal temperature was measured daily. Rats reached the endpoint of humanitarian execution when weight loss was \u0026gt;\u0026thinsp;20% or anal temperature was \u0026lt;\u0026thinsp;36 ℃.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Preparation of bacterial inoculum\u003c/h2\u003e\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e (ATCC 25923) was incubated in trypticase soy agar (TSA) medium at 37\u0026deg;C for 18 h. After 18 h, \u003cem\u003eS. aureus\u003c/em\u003e was harvested and resuspended in sterile PBS at a concentration of 1\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU /ml according to optical density measurements. The suspension was diluted into various bacterial concentrations (1\u0026times;10\u003csup\u003e3\u003c/sup\u003e, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e,1\u0026times;10\u003csup\u003e7\u003c/sup\u003e CFU /ml). A preliminary experiment was first performed to identify the appropriate concentration of bacterial inoculum.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Surgical procedure\u003c/h2\u003e\u003cp\u003eThe anal temperature and weight of each rat were measured before surgery. To prepare a 100% Avertin stock solution, 10 g of tribromoethanol was dissolved in 10 mL of tertiary amyl alcohol in a centrifuge tube by gently shaking by hand until fully dissolved. The solution was then filtered through a 0.22 \u0026micro;m membrane. The 100% Avertin stock was diluted with 0.9% NaCl physiological saline to a final concentration of 2.5% (1:40 dilution). Avertin (2.5%) was administered via intraperitoneal injection at a dose of 250 mg/kg for each rat. After shaving and sterilization with povidone-iodine, the lumbar vertebra was exposed posteriorly. The skin and fascia were incised sequentially with tissue scissors and a scalpel. Thereafter, the right lamina, facets, and transverse processes were exposed. The puncture point was located at the transition point between the superior articular process and the transverse process. First, the outer layer of the puncture point was opened with a needle (0.45 mm \u0026times; 16 mm) from a 1 ml syringe, then the puncture channel was made by a needle (0.33 \u0026times; 12 mm) through the vertebral pedicle. The channel with a depth of 3 mm ended at the anterior section under the endplate. Subsequently, a 20 \u0026micro;l bacterial solution was injected into the vertebral body through the established channel. After injection, the puncture site was sealed using bone wax. Finally, the muscle, fascia, and skin were closed layer by layer after washing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All procedures were conducted aseptically. No antibiotic was administered preoperatively, intraoperatively, or postoperatively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Blood assays\u003c/h2\u003e\u003cp\u003eBlood was collected by cardiac sampling at the endpoints of 7, 14, 21, and 28 days postoperatively. WBC number and morphological differences were determined from blood smears using CellaVision\u0026reg; DM9600. Serum was obtained from blood by centrifugation at 2000 \u0026times; g for 15 min. According to the manufacturer's instructions, the CRP and interleukin 1β (IL-1β) levels were determined using an enzyme-linked immunosorbent assay (ELISA) (Abcam, Cambridge, Massachusetts, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Radiographic evaluation\u003c/h2\u003e\u003cp\u003eRats were executed at their endpoints post operation. The lumbar spine was harvested and fixed in 4% paraformaldehyde, then scanned using microcomputed tomography (\u0026micro;CT) (Scanco Viva-CT80, Scanco Medical AG, Basserdorf, Switzerland). The data processing and 3D reconstruction were performed using Built-in software. Vertebral destruction was calculated using embedded 3D measurement techniques. Spinal destruction was scored using a modified An and Friedman scoring system (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) by three individuals blinded to the experimental protocol.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Histological analysis\u003c/h2\u003e\u003cp\u003eAfter \u0026micro;CT measurement and decalcification, the infected vertebrae and adjacent discs were separated and embedded in paraffin. Next, samples were sectioned axially at 5 \u0026micro;m thickness to obtain anatomical sections, which were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H\u0026amp;E) according to routine protocols. Goldner trichrome staining was used to assess bone mineralization. In addition, Gram staining was performed to determine bacterial infection in the tissue. Immunohistochemical staining was used to evaluate the expression of inflammatory factors. Sections were blocked with 5% BSA at 37\u0026deg;C for 30 minutes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e\u003cp\u003eThe data is presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The Student's t-test was used to evaluate differences between the two groups. One-way analysis of variance (ANOVA) was used when there were more than two groups (SPSS V19.0, SPSS Inc., Chicago, IL, USA). \u003cem\u003eP\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were deemed significant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Euthanasia method\u003c/h2\u003e\u003cp\u003eFive to eight rats were placed in the euthanasia box. The CO\u003csub\u003e2\u003c/sub\u003e cylinder switch was turned on, and CO\u003csub\u003e2\u003c/sub\u003e was introduced into the box at a rate of 10\u0026thinsp;\u0026minus;\u0026thinsp;30% of the box volume per minute, until the animals no longer moved, breathes, or exhibited pupil constriction. The CO\u003csub\u003e2\u003c/sub\u003e cylinder switch was then turned off, and the animals were observed for an additional 2\u0026ndash;3 minutes to confirm death.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eModified An and Friedman scoring system.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eScore\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsteolysis\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOsteogenesis\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVertebral body wall destruction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eParavertebral\u003c/p\u003e\u003cp\u003eabscess\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003enot present\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003enot present\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003enot present\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003enot present\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnifocal lesion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFocal osteosclerosis within the vertebra\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSingle focal lesion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFocal abscess involved in a single vertebra\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMultifocal lesions\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGeneralized osteosclerosis within the vertebra\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMultiple focal lesions\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGeneralized abscess involved in a single vertebra and adjacent disc\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLesions involved in two vertebrae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOsteogenesis evident outside the vertebral body\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLesions exceeding half of the vertebra\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAbscess involved in two vertebrae\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLesions involved in multiple vertebrae (\u0026gt;\u0026thinsp;2 vertebrae)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSignificant osteogenesis involved in at least two vertebrae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eExtensive lesions involved in at least two vertebrae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAbscess involved in multiple vertebrae (\u0026gt;\u0026thinsp;2 vertebrae)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Vital signs and mortality\u003c/h2\u003e\u003cp\u003eAll rats were observed for 14 days after surgery. There was no statistically significant difference in body weight between the control group and the G1\u0026thinsp;~\u0026thinsp;G3 groups after surgery. However, rats in the G4 group, receiving the highest dose of bacteria (10\u003csup\u003e9\u003c/sup\u003e CFU/ml), experienced the greatest weight loss after bacterial inoculation. All groups showed high body temperature on the first day post-surgery, but the mean temperature of the G1-G4 group peaked 3\u0026ndash;5-day post operation and remained significantly higher than that of the control group. However, three rats in the G4 group showed cachexia, rapid weight loss, abnormally decreased temperature, and eventually died. The mortality rate of the G4 group was 42.86%. In contrast, the survival rates of the control group and the G1-G3 groups were greater than 80% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Blood assays\u003c/h2\u003e\u003cp\u003eWBC and CRP are well-established biomarkers for detecting infections. Blood smear analysis revealed a significantly increased WBC in the infected group compared with the control group, while there were no significant differences between infected groups. The CRP level in the G3 and G4 groups was higher than that in the control group. Due to significant individual variations, the CRP level in the G1 and G2 groups showed no statistical significance compared with that of the control group. Additionally, the G3 and G4 groups displayed higher IL-1β levels compared with the control group and G1 and G2 groups, which was similar to observed CRP expression levels (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea-c).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Radiographs\u003c/h2\u003e\u003cp\u003e\u0026micro;CT scans showed that G3 and G4 groups exhibited significant bone destruction, hyperostosis, and sclerosis, while G1 and G2 groups had lower levels of bone destruction compared to G3 and G4 groups, and the control group had narrow channels surrounded by obvious sclerotic bone (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Based on the modified An and Friedman rating system (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the scores of G3 and G4 groups were significantly higher than that observed in G1 and G2 groups. The control group displayed the minimum score (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Identifying the optimal bacterial concentration\u003c/h2\u003e\u003cp\u003eBased on physical signs, bone destruction on CT images, and levels of inflammatory markers, we aimed to identify the optimal bacterial concentration to induce PS. Although obvious bone destruction was observed in both G3 and G4 groups, the maximal concentration in the G4 group was not optimal due to the excessive weight loss and low survival rate. Despite improved survival rates, the G1 and G2 groups showed lower imaging scores and inconsistent levels of inflammatory markers. However, some rats in G1 and G2 groups were not successfully infected by the any bacterial suspension. Therefore, we ultimately identified the 10\u003csup\u003e7\u003c/sup\u003e CFU/ml (20 \u0026micro;l) in the G3 group as the optimal inoculation concentration for subsequent experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Histology and bacteriologic analyses\u003c/h2\u003e\u003cp\u003eWe performed H\u0026amp;E staining for spinal tissues after rat execution. The control group showed normal trabecular morphology, puncture channels, and surrounding sclerosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Rats infected by bacterial suspension showed obvious bone destruction filled with fibrous tissue, massive inflammatory cell infiltration, and periosteal reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). According to the histological scoring criteria of bone infection proposed by Smeltzer et al\u003csup\u003e[17]\u003c/sup\u003e, the scores in the G3 and G4 groups were significantly higher than that observed in other groups (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition, we performed Goldner staining to evaluate osteogenesis in spinal infection. Mild mineralization was observed in G3 and G4 groups using Goldner staining. In contrast, the active bone-forming response was observed in the control group and in certain rats in G1 and G2 groups. These results were consistent with the \u0026micro;CT imaging findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Gram staining performed at execution points revealed the presence of gram-positive bacteria around the surgical puncture site (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The infected vertebrae were collected and inoculated. Numerous colonies of bacteria were observed and identified as \u003cem\u003eS. aureus\u003c/em\u003e (ATCC25923) using mass spectrometry analysis. The bacterial culture showed no bacteria in the liver and blood from the PS group, which indicated that the animal model displayed local infection without systemic infection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Dynamic changes to physical characteristics\u003c/h2\u003e\u003cp\u003eAfter confirming the inoculating concentration, we monitored consecutive changes to physical characteristics and inflammatory parameters for four weeks. The PS group presented with higher body temperatures than the control group from 3 to 15 days post operation, but there was no statistical difference 20 days after injection. The body weight of the infected rats was lower than that of the control group. In addition, the survival rates at the endpoint were 100% and 80% in the control and PS groups, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c). Inflammatory markers showed dynamic changes in both groups after surgery. Moreover, CRP levels in the PS group were higher than those observed in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). We observed similar trends in IL-1β levels, WBC count, and CRP levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ed and f).\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn recent years, the incidence of PS has increased due to the increased life expectancy of patients with chronic diseases and the misuse of antimicrobial drugs\u003csup\u003e[18]\u003c/sup\u003e. PS generally involves bone and intervertebral discs, thereby inducing several complications that may lead to spinal instability, vertebral collapse and kyphosis, and neurological dysfunction. These complications are common and unique in PS. Moreover, discitis and epidural abscess is a specific presentation of PS. However, the vertebra has no medullary cavity but adjacent discs, which are distinct to the tubular bone. PS is characterized by inflammatory infiltration, bone destruction, and intervertebral or intravertebral abscess, leading to inflammatory reactions, spinal instability, and neurological dysfunction (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Most animal models of osteomyelitis focus on the bones of the limbs, such as the tibia and femur\u003csup\u003e[10, 19]\u003c/sup\u003e. Most of these models were used for implant infection or for the evaluation of antibiotic therapy in the tubular bone. Unfortunately, these findings were not suitable for PS due to the distinct anatomy and pathogenesis.\u003c/p\u003e\u003cp\u003eThere are several animal models for spinal infection. Chen et al. reported a canine model of intervertebral disc infection and evaluated it through microbiology, imaging, and histopathology\u003csup\u003e[13]\u003c/sup\u003e. Owing to its high cost and the difficult surgical procedure, this animal model was not widely used for anti-infection strategies. Rabbits are commonly used as animal models for spinal infections. Guiboux et al\u003csup\u003e[20]\u003c/sup\u003e. and Bierry et al\u003csup\u003e[12]\u003c/sup\u003e. produced a PS animal model by injecting \u003cem\u003eS. aureus\u003c/em\u003e into the intervertebral disc and Bostian et al. established a similar model in rats\u003csup\u003e[21]\u003c/sup\u003e. Small animal models are often preferred due to their lower costs and ease of handling. Ofluoglu et al. established a PS infection model in rats by inserting a pedicle screw with \u003cem\u003eS. aureus\u003c/em\u003e\u003csup\u003e[22]\u003c/sup\u003e. However, these models were inconsistent with the pathogenesis of human PS.\u003c/p\u003e\u003cp\u003eIn adults, the disc is avascular and resistant to some bacteria, but \u003cem\u003eS. aureus\u003c/em\u003e and other enzyme-producing bacteria can spread directly to the intervertebral disc by disrupting the endplate. PS is typically initialized by bacterial colonization in slow-flowing capillaries beneath the vertebral endplate and is characterized by localized bone erosion in this region. Thus, infection emanating from the vertebra is similar to the pathogenesis of PS. In this study, the pathogenic bacterium was injected into the vertebra under the endplate, which agrees with the pathogenesis of PS\u003csup\u003e[8]\u003c/sup\u003e. Other studies have found that injury can significantly increase the likelihood of bone infection\u003csup\u003e[23]\u003c/sup\u003e. In this animal model, we first induced vertebral bone injury by puncture and then inoculated bacteria into the lumbar vertebral body of rats, which is consistent with the pathogenesis of PS. Imaging and histological analyses showed that infected rats exhibited early-stage vertebral destruction, which subsequently progressed and spread to the intervertebral disc through the compromised endplate, closely mimicking the pathological progression of human PS.\u003c/p\u003e\u003cp\u003eWe identified the optimal inoculum concentration (10\u003csup\u003e7\u003c/sup\u003e CFU/ml) of \u003cem\u003eS. aureus\u003c/em\u003e based on basic vital signs, imaging, hematology, histology, and bacteriology. Infected rats exhibited high fever and weight loss in the first week, then returned to normal temperature and weight around the second week after inoculation. The levels of WBC and CRP also increased first and then decreased after infection. In addition, \u0026micro;CT images showed significant bone destruction and slight osteogenesis surrounding the lesion. H\u0026amp;E staining showed bone destruction without normal bone trabeculae in the vertebrae, which were replaced by fibrous tissue and inflammatory cells. Meanwhile, Gram staining and bacterial culture results showed the activity of \u003cem\u003eS. aureus\u003c/em\u003e in the PS model group. In summary, the above results demonstrate that this animal model can reliably mimic the pathogenesis of PS in humans.\u003c/p\u003e\u003cp\u003eThis study has several limitations. Firstly, we conducted CT scanning and inflammatory tests on the 14th day after inoculation when the rats displayed obvious bone destruction. Earlier evaluation may help to understand the pathogenesis during PS onset. Secondly, the PS model in this study was tested using a very common bacterium. It may be useful to evaluate the animal model in response to other pathogens.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn conclusion, we successfully established a PS animal model by injecting \u003cem\u003eS. aureus\u003c/em\u003e into the vertebral body below the endplate. Although this model has certain flaws, it is highly mimetic to human PS and provides a significant step toward exploring the pathogenesis of PS and identifying effective treatment strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThis study is reported in accordance with ARRIVE guidelines.\u003c/p\u003e\u003ch2\u003eConflicts of Interest\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe present study was supported by the Natural Science Foundation of Shandong Province (grant no. ZR2023MH159).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eQinpeng Xu: Conceptualization, Writing-original draft; Guihe Yang: Writing-original draft, Validation; Jiaju Ma: Data curation, Software; Xingzhi Jing: Investigation; Fei Jia: Data curation, Software; Xingang Cui: Supervision; Jianlong Li: Methodology; Xiaoyang Liu: Conceptualization, Writing-review \u0026amp; editing. Hongdong Tan: Conceptualization, Writing-review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data used to support the findings of this study are included within the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYokota H, Tali ET. Spinal Infections. Neuroimaging Clin N Am 2023;33:167\u0026thinsp;\u0026minus;\u0026thinsp;83. https://doi.org/10.1016/j.nic.2022.07.015.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlmansour H, Pepke W, Akbar M. 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J Orthop Res 2017;35:2075-81. https://doi.org/10.1002/jor.23497.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOfluoglu EA, Zileli M, Aydin D, Baris YS, Ku\u0026ccedil;ukbasmaci O, Gonullu N, et al. Implant-related infection model in rat spine. Arch Orthop Trauma Surg 2007;127:391-6. https://doi.org/10.1007/s00402-007-0365-0.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHeng BC, Bai Y, Li X, Lim LW, Li W, Ge Z, et al. Electroactive Biomaterials for Facilitating Bone Defect Repair under Pathological Conditions. Adv Sci (Weinh) 2023;10:e2204502. https://doi.org/10.1002/advs.202204502.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pyogenic spondylitis, Spinal infection, Animal model, Staphylococcus aureus, Rat","lastPublishedDoi":"10.21203/rs.3.rs-7448639/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7448639/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and purpose\u003c/h2\u003e\u003cp\u003ePyogenic spondylitis (PS) is clinically challenging and induces disastrous consequences for patients. The pathogenesis of PS is difficult to explore due to a lack of ideal animal models. Thus, we aimed to reproduce the pathogenesis of PS in an innovative animal model induced by \u003cem\u003eStaphylococcus aureus\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eRats were injected with planktonic \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (ATCC 25923) and grouped according to different concentrations. We identified the optimal bacterial inoculum concentration based on general physical signs and radiological, hematological, and histological parameters in rats. Models with the optimal bacterial concentration were used to investigate changes in physical, radiological, and inflammatory parameters at different time points.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eOur results revealed that infected rats experienced rapid weight loss, high fever, and significantly increased white blood cell count, interleukin 1β, and C-reactive protein (CRP) levels in the short term. Radiographic examination revealed bone damage in groups that received 2\u0026times;10\u003csup\u003e3\u003c/sup\u003e/20 \u0026micro;l, 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e/20 \u0026micro;l and 2\u0026times;10\u003csup\u003e7\u003c/sup\u003e/20 \u0026micro;l bacterial concentrations. The optimal concentration was identified as 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e/20 \u0026micro;l, based on the high survival rate, obvious bone destruction, and inflammation. Histological staining confirmed the living bacteria, inflammatory cells, bone destruction, and scarce bone formation in infected vertebrae.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study provided an innovative PS animal model, which may improve our study of the pathological mechanism underlying PS.\u003c/p\u003e","manuscriptTitle":"Innovation in animal model of Pyogenic Spondylitis induced by Staphylococcus aureus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 14:44:49","doi":"10.21203/rs.3.rs-7448639/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9b18d10b-a1a0-4a78-bce2-0e578495f998","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54465452,"name":"Health sciences/Diseases"},{"id":54465453,"name":"Biological sciences/Immunology"},{"id":54465454,"name":"Health sciences/Medical research"},{"id":54465455,"name":"Biological sciences/Microbiology"},{"id":54465456,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2026-04-16T01:39:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-12 14:44:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7448639","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7448639","identity":"rs-7448639","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}