Additional benefits of combined ceftriaxone and adipose-derived mesenchymal stem cells on revamping the outcomes in rodent after acute spinal infection

Additional benefits of combined ceftriaxone and adipose-derived mesenchymal stem cells on revamping the outcomes in rodent after acute spinal infection | 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 Research Article Additional benefits of combined ceftriaxone and adipose-derived mesenchymal stem cells on revamping the outcomes in rodent after acute spinal infection Tsung-Cheng Yin, Hung-Sheng Lin, Pei-Hsun Sung, John Y. Chiang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5310715/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jan, 2025 Read the published version in Journal of Molecular Histology → Version 1 posted 9 You are reading this latest preprint version Abstract This study tested whether combined ceftriaxone and adipose-derived mesenchymal stem cells (ADMSCs) would defend the spinal cord against acute spinal infection (ASI) in rodent . Adult-Male- SD rats were grouped into groups 1 (SC)/2 (ASI)/3 (ASI + ceftriaxone from days 2 to 28 after ASI induction)/4 (ASI + allogenic ADMSCs from day 2 for a total of 3 doses/3 consecutive intervals by intravenous injection)/5 (ASS + combined ceftriaxone and ADMSC) and spinal cord tissues were harvested by day 28. Circulatory levels of TNF-α/IL-6 at days 7 and 28, and these two parameters in spinal fluid at day 28 were lowest in group 1, highest in group 2, significantly lower in group 5 than in groups 3/4, and significantly lower in group 3 than in group 4 (all p < 0.0001). The day-28 bacterial colony formation unit ( CFU ) in vertebral bone and circulatory WBC counts at the time points of days 7/14/28, and the protein expressions of upstream (TRL-2/TLR-4/MYD88/TRAF6/IKKα/IKKβ /IKBβ/p-NF-κB) and downstream (IL-1β/IL-6/TNF-α/IFN-γ/iNOS) inflammatory signalings displayed a similar pattern of inflammatory biomarkers in spinal fluid among the groups (all p < 0.0001). By day 28, the bone injury score/bone marrow density/ratio of bone volume (BV) to the bone tissue volume (TV)/ratio of bone surface (BS) to BV/ratio of BS to bone TV/trabecular number exhibited an opposite, whereas the trabecular space exhibited an alike pattern of inflammatory biomarkers among the groups (all p < 0.0001). Combined ceftriaxone and ADMSCs therapy offered an additional benefit on protecting the vertebral bone/spinal cord against ASI damage. acute spinal cord infection antibiotics mesenchymal stem cells inflammatory reaction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Spinal infection (SI) is an infectious disease involving the intervertebral disc, vertebral body, and/or adjacent paraspinal tissue (Lener et al. 2018 ). The SI contributes to 2–7% of all musculoskeletal infections in the clinical observation, usually resulting in seriously destructive spinal cord tissues if not treated promptly and properly (Lener et al. 2018 ). The histopathological findings have identified that the sources of infection can be spread to the spine by hematogenous dissemination from distant site, by diffusion from contiguous tissues, or/and by external inoculation (trauma, surgery) (Lener et al. 2018 ; Gregori et al. 2019 ). The mortality rate of SI has been reported to be 2% and 20%, respectively (Lener et al. 2018 ; Aagaard et al. 2016 ; Artenstein et al. 2016 ; Fantoni et al. 2012 ). Patients who suffer from SI generally express non-specific symptoms, including back pain (85%), fever (48%), and paresis (32%) (Fantoni et al. 2012 ; Gregori et al. 2019 ; Lener et al. 2018 ). Although treatment strategies of SI remain controversial and sometimes ineffective, use of concomitant antibiotics treatment under non-surgical (i.e., immobilization with rigid orthosis) or surgical managements (i.e., debridement, decompression, etc.) is still the common choice of treatment for SI, depending on different clinical manifestation and disease course as well as the general conditions (Gregori et al. 2019 ; Lener et al. 2018 ). Conclusively, the management of SI is still an unmet need that raises the urgency to conduct a comprehensive investigation to establish an effective and innocuous treatment for SI patients. In most countries, Staphylococcus aureus is still one of the most common pathogens that causes SI. According to the previous reports, the incidence of methicillin-resistant S. aureus (MRSA) accounts from 6.8–30% of pathogens for SI (Gregori et al. 2019 ; Lener et al. 2018 ). Such pathogens might further generate complicated process of localized or systemic inflammatory reactions, such as overwhelming immune response, induction of oxidative stress and reactive oxygen species (ROS) as well as over-compensatory inflammatory reaction (Gregori et al. 2019 ; Lener et al. 2018 ; Sung et al. 2016 ). Although use of antimicrobial agents is the gold standard treatment of bacterial-induced infection, the morbidity, mortality, hospital length of stay (LOS), and health costs are still relatively high (Camino Willhuber et al. 2019 ). This could be mainly due to the reason that antimicrobial agents cannot effectively control the exaggerated immune response and overwhelming inflammatory reaction effectively (Camino Willhuber et al. 2019 ; Sung et al. 2016 ). Therefore, in addition to the advisable choice of antimicrobial agents, control of the exaggerated immune response and overwhelming inflammatory reaction may be critical to reduce SI-induced morbidity and mortality (Artenstein et al. 2016 ; Camino Willhuber et al. 2019 ). It is well known that ceftriaxone (Sileshi et al. 2016 ; Hedlund 2011 ; Raja et al. 2020 ) is an antibiotic that pertains to a category of medicine recounted to as cephalosporin antibiotics, and it effectively treats a variety of bacterial infections by ceasing the growth of bacteria in variety of bacterial infections in different organs, including bone and spinal cord bacterial infection. However, growing data have shown that ceftriaxone-resistant Gram-negative bacilli , especially those caused by extended-spectrum β-lactamases (Paterson et al. 2020 ; Harris et al. 2018 ) becomes more and more popular in our daily clinical practice. Not only has abundant evidence showed that stem cell therapy attenuates inflammation and innate/adaptive immune responses (Sung et al. 2016 ; Le Blanc et al. 2003 ; Sun et al. 2011 ), but adipose-derived mesenchymal stem cells (ADMSCs) have also been shown to possess the intrinsic capacity of immunomodulation and tissue regeneration (Sun et al. 2011 ; Camino Willhuber et al. 2019 ; Sung et al. 2016 ). Interestingly, limited investigations suggest that, in the setting of ischemia-reperfusion injury and sepsis, ADMSCs treatment could attenuate morbidity and mortality through regulation of ischemia-reperfusion injury and sepsis-induced exaggerated immune response and overwhelming inflammatory reaction as well as control of ROS and oxidative stress generation (Sung et al. 2016 ; Krasnodembskaya et al. 2012 ; Mei et al. 2010 ). Additionally, our previously experimental studies have demonstrated that ADMSCs therapy effectively controlled sepsis syndrome (SS) (Sung et al. 2016 ; Chen et al. 2014 ), whereas combined therapy with antibiotics and ADMSCs even more effectively controlled the SS and reduced the rodent mortality (Sung et al. 2016 ). On the other hand, there has been no experimental study that focuses on the therapeutic effect of stem cell therapy compared with that of ceftriaxone antimicrobial agents as well as that when the two strategies are combined in the setting of experimental model of SI. Therefore, we conducted this study to test the hypothesis that ceftriaxone-ADMSC combined therapy was superior to ceftriaxone monotherapy in animal model of microbial-induced SI to attenuate inflammation/oxidative stress as well as morbidity and mortality. Materials and methods Ethical statement All animal procedures were approved by the Institute of Animal Care and Use Committee at Kaohsiung Chang Gung Memorial Hospital (Affidavit of Approval of Animal Use Protocol No. 2020121605) and performed in accordance with the Guide for the Care and Use of Laboratory Animals. Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC; Frederick, MD, USA)-accredited animal facility in Kaohsiung Chang Gung Memorial Hospital under temperature (22 ± 1 ◦C), humidity (55 ± 5%) and light (12: 12/light: dark photo-cycle, light on at 08:00) control. Standard laboratory rat chow and tap water were available ad libitum. All animals were allowed to acclimatize to the animal facility for 14 days prior to experimental manipulations. To create an animal model of acute bacterial spinal vertebral bone infection (ASI) in SD rat The procedure and protocol were based on the previous study with some modification (Ofluoglu et al. 2007; Dworsky et al. 2017) with some modification. Pathogen-free, adult male Sprague-Dawley (SD) rats , weighing 320-350 g (Charles River Technology, BioLASCO Taiwan Co. Ltd., Taiwan), were used in the present study. In detail, the animals were anesthetized by inhalation of isoflurane, i.e., isoflurane was generally used at concentrations of 5% for induction, and 2 % for maintenance of general anesthesia and 1.0% for recovery stage and placed prone on a warming pad. The hair on the skin on the lower back around the spine was shaved. The skin was incised at the midline of the spine at the T11 - L2 level, and the paraspinal muscles were carefully dissected subperiosteally to expose the lamina. A sterile self-tapping screw (diameter 1 mm, length 4 mm) was screwed into the vertebral bone between T13 - L1, following by unscrewing the above screw, the Staphylococcus aureus subsp. aureus ( S. aureus , ATCC® 19636 ™) suspension (5×105 cells in 50μL) was inject into the hole that was drilled by the screw, and then reset the screw. Finally, the incision was sutured, and the animals were left awake on a warm pad at 37°C. Animal grouping and animals were euthanized Adult male SD rats (n = 42) were categorized into group 1 [sham-operated control (SC), i.e., merely opening the skin and muscle layer of spinal cord, followed by closure of these two layers over the spinal cord, n = 8], group 2 [acute spine infection (ASI), n = 10], group 3 [ASI + ceftriaxone (5 mg/kg/day by subcutaneous administration over the spinal area from days 2 to 28 after ASI induction, n = 8), group 4 [ASI + allogenic ADMSCs (1.0 x 10 6 cells/rat from day 2 for a total of 3 doses/for 3 consecutive intervals with 9 days after ASS by intravenous injection), n = 8] and group 5 (ASS + combined ceftriaxone and ADMSC, n = 8), respectively. The dosage of antibiotics to be utilized in the present study was based on the previous report with some modification (Sung et al. 2016). On the other hand, the dosage of ADMSCs to be utilized in the present study was based on our previous reports with some modification (Chang et al. 2018; Chang et al. 2019). Euthanasia of animals was performed under anesthesia with overdose of isoflurane inhalation (i.e., more than 5.0%) and collected the blood by more than 10 ml by day 28 after ASI induction. The vertebral bone, cartilage and spinal cord tissues were harvested after the respiratory arrest for individual study. Methodology for preparing and culturing allogenic ADMSCs Additional 20 rats were utilized for allogenic ADMSCs isolation. The procedure and protocol for allogenic ADMSC isolation and culture have been described in our previous reports (Fantoni et al. 2012; Sung et al. 2016). Briefly, adipose tissue surrounding the epididymis was dissected, excised and prepared by day 14 prior to acute kidney IR induction. For purification, the harvested cells were cultured in Dulbecco’s modified Eagle's medium (DMEM)-low glucose medium containing 10% FBS for 14 days. By this time, plentiful ADMSCs (i.e., approximately 2.5 - 3.0 x 10 6 cells) were obtained in the culture plate and were collected to treat ASI animals. ELISA examination Collect the circulatory blood samples at days 14 and 28 after ASI induction in each group of the animals for determining the circulating levels of IL-6 and TNF-α levels. Additionally, these parameters of spinal fluid were also collected and measured at the end of study period, i.e., by day 28 after ASI induction. These biomarkers were measured by ELISA standard method was according to the manufacturer’s instruction. Histopathological examination By day 28 after ASI induction, the histopathological examination was performed after routine fixation, decalcification, and paraffin embedding for assessment of vertebral bone/cartilage destruction, the bacterial counts in the bone and adjacent region and inflammatory cell infiltration (i.e., polymorphonuclear cells). Western blot analysis from isolated spinal-cord tissues The protocol and procedure were described by our previous reports (Chang et al. 2019; Chang et al. 2018; Sung et al. 2016; An et al. 2006). Briefly, equal amounts (50 µg) of protein extracts were loaded and separated by SDS-PAGE using 8-12% acrylamide gradients. After electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences). Nonspecific sites were blocked by incubation of the membrane in blocking buffer (1X TBS, 0.1% Tween-20 with 5% w/v nonfat dry milk) overnight. The membrane was incubated with indicated primary antibodies Toll-like receptor 2 (TLR2) (1:1000, Abcam), TLR4 (1:1000, Novusbio), tumor necrosis factor receptor associated factor 6 (TRAF6)1:1000, Abcam), phosphorylated nuclear factor (p-NF)-κB (1:1000, Abcam), interleukin (IL)-1ß (1:1000, Cell Signaling), IL-6 (1:1000, Abcam), IKKα(1:5000, Abcam), IKKβ (1:1000, Cell Signaling), IKBβ (1:1000, Abcam), interferon (IFN)-γ (1:4000, Abcam), tumor necrosis (TNF)-α (1:1000, Cell Signaling), inducible nitric oxide synthase (iNOS) (1:1000, Abcam), MYD88 (1:1000, Abcam) and Actin (1:6000, Millipore)] for 1 hour at room temperature. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin IgG (1:5000, Sigma) was used as a secondary antibody for one-hour incubation at room temperature. The washing procedure was repeated eight times within one hour. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences, Amersham, UK) and exposed to Biomax L film (Kodak, Rochester, NY, US). For the purpose of quantification, ECL signals were digitized using Labwork software (UVP, Waltham, MA, US). Micro-CT examination of decalcified vertebral/spinal-cord bone By day 28, i.e., at the end of study period, the animals were euthanized, and the bone-articular joints were harvested from each animal. After well preparation, these specimens were assessed by the Bruker Micro-CT (SkyScan 1176, Bruker BioSpin, Germany, with Bruker CTAn Micro-CT Software for analysis) for verifying the destructive features of the vertebral/spinal-cord bone, respectively. Definition for BMD, ratios of BV/TV, BS/BV and BS/TV, TN and TS Bone mineral density (BMD) measures the density of minerals in the bone. It can be used to detect osteoporosis, determine the risk of future fractures, and assess the outcome of osteoporosis treatment. Ratio of bone volume (BV) to bone tissue volume (TV) (i.e., percentage of bone volume) refers to the proportion of deducted dense bone to bone TV in all tissues. This index is a common index used for evaluating the bone mass of compact bone and spongy bone. An increase in this value indicates that bone metabolism is greater than catabolism and bone mass increases correspondingly, and vice versa, which indirectly reflects the state of bone metabolism. Ratio of bone surface to bone volume (BS/BV) indirectly reflects the amount of bone mass. Ratio of bone surface to bone tissue volume (BS/TV) also indirectly reflects the amount of bone mass. Trabecular number (TN) refers to the number of intersection points produced by the intersection of the plate-shaped structure and the round rod-shaped structure, and the structure woven by these intersection points is the trabecular bone. When osteoporosis occurs, the TN value will decrease relatively. Trabecular separation (TS) refers to the average width of the medullary cavity between the trabecular bone. The increase of TS corresponds the increase of bone loss leading to the increase of cavity space between bone trabeculae, so osteoporosis may occur. Definitions of Classification of bone destructive core and infectious bone deposition The definitions were based on the previous report (Ofluoglu et al. 2007) with minimal modifications: (1) Classification of vertebral bone destruction: on a scoring of 1 to 5, indicating that the bone was damaged (i.e., by infection) and could not be repaired by itself, which included the bone marrow destructive space. (2) Classification of infectious bone deposition: including the severity of scale from 1 to 5, indicating abnormal bone hyperplasia/deposition. Thus, bone injury score was the comprehensive score of cartilage destruction (1) and infectious bone deposition (2) in the current study. Statistical analysis Quantitative data were expressed as mean ± SD. Statistical analysis was adequately performed by ANOVA, followed by Bonferroni multiple comparison post hoc test. SAS statistical software for Windows version 8.2 (SAS Institute, Cary, NC, USA) was utilized. A probability value <0.05 was considered statistically significant. Results Body weight, mortality rate, and time courses of circulatory level of white blood cell counts (WBC) (Fig. 1) The ratio of the initial (i.e., by day 0) to final (i.e., by day 28) body weight significantly lower in group 4 (ASI + ADMSC) than in groups 1 (SC), 2 (ASI), 3 (ASI + ceftriaxone), and 5 (ASS + combined ceftriaxone and ADMSC)] and significantly lower in groups 2 and 5 than in groups 1 and 3, but it showed no difference between groups 2 and 5 or between 1 and 3 (n = 8 for each group). The total mortality rate within 48h prior to grouping was 9.1% (5/55). Animals were groups at day 2 prior to the treatment and the mortality rate after grouping prior to being euthanized by day 28 were 0% (0/8) in group 1, 30.0% (3/10) in group 2 and 0% (0/8) in groups 3, 4 and 5, respectively. Regarding the statistical analysis of the mortality rate, the variables were compared with one-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) for comparison of the difference between two groups. Difference of alphabetic characters ( a or b ) in superscript indicates statistical significance of p-value < 0.05 after comparison, p = 0.012 (n = 10 for each group). The circulating level of WBCs at baseline did not differ among the five groups (n = 8 for each group). However, by day 3 after the ASI induction, the WBC count was significantly lower in group 1 than in other groups, significantly lower in group 5 than in groups 2, 3 and 4. However, this parameter did not differ among the latter three groups. Additionally, by days 7 and 14 after ASI, this parameter was significantly lower in 1 than in other groups, and significantly higher in group 2 than in groups 3, 4, and 5, but it was similar among the groups 3 to 5. However, by days 28 after ASI induction, the WBC count was lowest in group 1, highest in group 2, significantly lower in group 5 than in groups 3 and 4, but it showed no difference between the latter two groups (n = 8 for each group). Circulatory and spinal cord fluid levels of proinflammatory cytokines and bacterial CFU in vertebral bone and circulation (Fig. 2 and Table 1) To elucidate whether the ceftriaxone or ADMSCs treatment would suppress the circulating and spinal cord fluid levels of IL-6 and TNF-α, the ELISA methodology was utilized in the present study. The result showed that by day 7 after ASI induction, the circulating levels of IL-6 and TNF-α, were lowest in group 1, highest in group 2, significantly lower in group 5 than in groups 3 and 4 and significantly lower in group 3 than in group 4 (n = 8 for each group). Additionally, by day 28 after ASI induction, the circulating and spinal cord fluid levels of these two parameters displayed an alike pattern as those of day 7 (n = 6–8 for each group). Additionally, Table 1 illustrated that the bacterial CFU in vertebral bone (n = 6 for each group) was significantly higher in group 2 than in groups 3, 4, and 5, and significantly higher in groups 3 and 4 than in group 5, but it showed no difference between groups 3 and 4. Furthermore, bacterial CFU in circulation (n = 8 for each group) was significantly higher in group 2 than in groups 3 to 5, but this parameter did not differ within these latter three groups. Protein levels of upstream and downstream inflammatory signalings (Fig. 3) To verify the impact of ceftriaxone and ADMSCs treatments on alleviating the inflammatory reaction, the Western blot analysis (n = 6 for each group) for the protein levels of parameters were conducted in the present study. The result demonstrated that protein expressions of TRL-2, TLR-4, MYD88, TRAF6, IKK-α, IKK-β, IKB-β and p-NFκ-B, eight upstream inflammatory signalings, were lowest in group 1, highest in group 2, significantly lower in group 5 than in groups 3 and 4, and significantly lower in group 3 than in group 4. Additionally, the protein expressions of IL-1β, IL-6, TNF-α, IFN-γ and iNOS, five downstream inflammatory signalings, displayed an identical pattern among the five groups, implicating that combined Cef and ADMSCs offered additional benefit than merely one on suppressing ASI induced inflammatory reaction. To clarify the impact of ceftriaxone and ADMSCs treatments on attenuating the bone injury score (Fig. 4) To verify whether ceftriaxone-ADMSCs would offer benefit on protecting the bone and cartilage against ASI damage, the H&E staining (n = 5–10 for each group) was conducted in the present study. The result showed that vertebral bone destructive score and infectious bone deposition score, i.e., defined as the bone injury score, were significantly higher in group 2 than in other groups, significantly higher in groups 3 and 4 than in groups 1 and 5 and significantly higher in group 5 than in group 1, but it showed no difference between groups 3 and 4. Micro-CT examination of vertebral/spinal-cord bone for identification of bone/bone marrow damage (Fig. 5 and Table 2) To verify the ceftriaxone and ADMSCs on protecting the vertebral bone against ASI induced damage, the micro-CT specific for small animals was utilized in the present study (n = 6 for each group). The result showed that the bone marrow density (BMD) was significantly higher in group 5 than in other groups, significantly higher in group 1 than in groups 2 to 4 and significantly higher in group 3 and 4 than in group 2, but it showed no difference between the groups 3 and 4. Our finding implied that combined ceftriaxone and ADMSCs treatment increasing the BMD could be mainly due to the tissue regeneration effect of ADMSCS. Additionally, the BV/TV bone volume/trabecular volume (i.e., BV/TV), a ratio of bone volume to the trabecular volume, was significantly higher in groups 1 and 5 than in the groups 2 to 4 and significantly higher in groups 3 and 4 than in group 2, but it showed no difference between groups 1 and 5 or between 3 and 4 (n = 6 for each group), implicating that the combined ceftriaxone and ADMSCs was superior to merely one treatment on protecting the bone materials again infection-induced loss. Furthermore, the bone surface/trabecular volume (i.e., BS/TV), i.e., a ratio of bone surface to trabecular volume reflecting the bone density, and the numbers of trabeculae, an indicator of trabecular bone density, were significantly higher in groups 1 and 3 to 5 than in those of the group 2, but these parameters were similar among the groups 1 and 3 to 5 (n = 6 for each group), implicating that ceftriaxone-ADMSCs therapy significantly protected bone materials against the bacterial infection damage. Schematically illustrated the underlying mechanism of bacterial spinal cord infection in rodent and the therapeutic impact of ceftriaxone-ADMSCs on ASI (Fig. 6) Finally, through the results from Figs. 1 to 6, we could graphically depict the underlying mechanistic basis of overwhelming inflammatory reactions in circulatory levels and bone tissue, resulting in vertebral bone and cartilage destruction that were remarkably reversed by ceftriaxone-ADMSCs therapy. Discussion Our present study which investigated the therapeutic effect of ceftriaxone-DMSCs on ASI delivered some striking connotations. First, we successfully created a reproducible animal model of ASI for testing the aftermath of ceftriaxone-ADMSCs on this disease entity and could be extended to the other strategic management of ASI. Second, the result of the current study demonstrated that ADMSCs were comparable with ceftriaxone for securing the spinal cord against the ASI damage. Third, combined ceftriaxone and ADMSCs therapy was superior to either one on sheltering the spinal cord against the ASI injury. Finally, we found that upstream and downstream inflammatory signaling acted as a foremost role on spinal cord damage in setting of ASI. Despite various modalities have been reported for the treatment (Artenstein et al. 2016 ; Camino Willhuber et al. 2019 ; Gregori et al. 2019 ; Lener et al. 2018 ), there is still lacking an unanimously acknowledged effective management that could rationally explained why the morbidity, mortality, hospital length of stay and health costs are still unacceptably high in ASI and chronic SI setting (Camino Willhuber et al. 2019 ). One important finding in this preclinical study demonstrated that one kind of antibiotic (i.e., ceftriaxone) was identified to be comparable with ADMSCs for the efficacy on safeguarding the spinal cord damage and bettering the outcomes in ASI rodent. The most important finding in this preclinical study proclaimed that combined antibiotic and ADMSCs therapy was superior to just one therapy for boosting the outcomes in ASI rodent. In this way, our findings, in addition to extending the findings from previous studies (Artenstein et al. 2016 ; Camino Willhuber et al. 2019 ; Gregori et al. 2019 ; Lener et al. 2018 ), highlight that this combined regimen may be a therapeutic novelty for ASI patients, especially for those patients who are refractory to the conventional therapy (Camino Willhuber et al. 2019 ). Two essential findings from this current study were that the bacteria in the circulation and derived from vertebral bone were remarkably higher in ASI animals than those of ASI animals after receiving ceftriaxone-ADMSCs treatment. Interestingly, when looked at the findings from H&E staining, we identified that the bone injury score was remarkably increased in ASI animals without than with the ceftriaxone-ADMSCs treatment. It is well known that the bacteria in chronic osteomyelitis patients are always too difficult to be eradicated through antibiotics (i.e., by bactericidal effect) mainly due to the antibiotics are rather difficult to penetrate in the bone parenchyma and could be also due to the bone distinctively complicated structure always serving as a good harbor for bacteria concealed there. Perhaps, our findings (i.e., by H.E. staining) of destructive vertebral bone (i.e., including bone marrow, trabeculae, and cartilage) would serve as a good sanctuary for bacterial occultation. We were very concerned the underlying mechanism for how ceftriaxone-ADMSCs treatment ensured the spinal cord/vertebral bone in setting of ASI. It is well recognized that MYD88 played the role as an essential innate immune signal transduction adaptor (Deguine et al. 2014). Additionally, plentiful studies have established that this inflammatory-immune signaling takes part in damage of the tissues/organs in organ ischemic settings (Chen et al. 2020 ; Yang et al. 2021 ). Intriguingly, when looked at MYD88 innate immune signal, we found that the protein expressions of upstream and downstream inflammation/innate immune signalings were much increased in ASI animals than in SC animals. Our finding was consistent with the findings of previous studies (Chen et al. 2020 ; Deguine et al. 2014; Yang et al. 2021 ). Of paramount important finding in the present study was that these molecular-cellular perturbations were noteworthily repressed by ceftriaxone or ADMSCs therapy and further noteworthily repressed by combined ceftriaxone + ADMSCs treatment, highlighting that our finding in addition to extending the findings of previous studies (Chen et al. 2020 ; Deguine et al. 2014; Yang et al. 2021 ), encourages the use of these two regimens for those acute or chronic SI or even extrapolates to the treatment of osteomyelitis patients, especially when they are refractory to conventional therapy. We suggest this combination therapy offering great benefits to those of ASI animals and improving their outcomes could be due to the synergic effect of anti-inflammation (i.e., the essential effect of ceftriaxone) and anti-inflammation/immunomodulation (i.e., the principal therapeutic effect of ADMSCs) along with possible tissue regeneration (another therapeutic impact of MSCs). An especially interesting finding in the present study was that as compared with the ASI animals without treatment, the mortality rate was significantly lower in those of ASI animals treated by ADMSCs. Undoubtedly, MSCs treatment could not directly killed the bacterial. However, when we looked at the circulatory derived CFU , we found that the CUF was markedly reduced in ASI animals with than in without ADMSCs treatment. Additionally, it is well known that systemic inflammatory response syndrome (SIRS) is an exaggerated defense response of the body to a noxious stressor, i.e., such as bacterial infection, trauma, surgery, or acute inflammation…etc. Based on the aforementioned issues, we suggested that perhaps, the mortality rate was notably lower in ASI animals with than in without ADMSCs treatment could be, at least in part, due to the fact that HUCDMSCs has the property of anti-inflammation and immunomodulation (Le Blanc et al. 2003 ; Sun et al. 2011 ; Sung et al. 2016 ). Scientists would be interesting in understanding the underlying mechanism for how the combined ceftriaxone and ADMSCs could offer synergic effect on protecting the vertebral bone and cartilage against ASI damage. In fact, abundant data have shown that ceftriaxone kills many types of bacteria by suppressing the mucopeptide synthesis of bacterial cell wall. The β-lactam core of ceftriaxone irreversibly binds to carboxypeptidases, endopeptidases, and transpeptidases in the bacterial cytoplasmic membrane (Lamb et al. 2002 ). Thus, ceftriaxone is characterized as one kind of bactericidal antibiotics. During bacterial infection, especially those of gram-negative bacteria always release endotoxin that would elicit overwhelming inflammatory reaction, enhance innate immune reaction and inflammatory cell infiltration in infectious organs, resulting in activate the inflammatory signalings and generation of proinflammatory cytokines, subsequently destructs the tissues, organs and bone as well as the cartilage. On the other hand, the ADMSCs have been shown to have intrinsic ability of anti-inflammation, immunomodulation and tissue regeneration (Sun et al. 2011 ; Camino Willhuber et al. 2019 ; Sung et al. 2016 ). Accordingly, the above-mentioned issues (Sun et al. 2011 ; Camino Willhuber et al. 2019 ; Sung et al. 2016 ; Lamb et al. 2002 ) and the findings of the present study could, at least in part, explain why combined ceftriaxone and ADMSCs therapy was superior to merely one on protecting the vertebral bone and cartilage against ASI damage (referred to Fig. 7). Study limitations Our study has some constraints. Frist, the study period was only 28 days, implicating that it was relatively short, i.e., just reflected the ASI outcomes. Accordingly, the chronic outcome of SI after receiving these therapeutic management is currently unclear. Second, the underlying mechanism of bacterial spinal cord injury in rodent and therapeutic impact of ceftriaxone-ADMSCs on ASI was merely dependent on the results of our study that may not really delineate the complicated mechanistic basis of vertebral bone/spinal-cord damage in the setting of ASI. Conclusions The complications of spinal cord infection are commonly observed in gravely destructive spinal cord tissues and uncontrolled infection, consequently, leave grievous sequalae and unacceptable high morbidity and mortality. On the other hand, how to effective treatment for ASI, especially in those patients who already develop destructive vertebral bone and cartilage damage, remains a formidable challenge. Our study demonstrated that combined ceftriaxone-ADMSCs treatment provided synergic effect on safeguarding the vertebral bone/spinal cord in opposition to ASI damage. Declarations Acknowledgments This study was supported by a program grant from Chang Gung Memorial Hospital, Chang Gung University (CMRPG8M0581). Availability of data and materials The datasets of present study can be available from the corresponding author upon request. Authors’ contributions T. -C. Y., P.-H. S., H.-K. Y. designed the study. T. -C. Y., H. -S. L., P.-H. S., C.-H. Y., investigated experiments. T. -C. Y., P.-H. S., C.-H. Y., H.-K. Y curated data. H. -S. L., P.-H. S., K.-H. C., C.-H. Y. and H.-K. Y. did formal analysis. K.-H. C. was responsible for funding acquisition. H.-K. Y. and K.-H. C. administered and supervised the project. K.-H. C., J.-Y. C., H.-K. Y. wrote the first draft of the manuscript and all named authors contributed in revising the manuscript. Ethics approval and consent to participate All animal procedures were approved by the Institute of Animal Care and Use Committee at Kaohsiung Chang Gung Memorial Hospital (Affidavit of Approval of Animal Use Protocol No. 2020121605) and performed in accordance with the Guide for the Care and Use of Laboratory Animals. Patient consent for publication Not applicable. Conflict of interest The authors declare that they have no competing interests. Funding This study was supported by a program grant from Chang Gung Memorial Hospital, Chang Gung University (CMRPG8M0581). References Aagaard T, Roed C, Dahl B, Obel N (2016) Long-term prognosis and causes of death after spondylodiscitis: A Danish nationwide cohort study. Infect Dis (Lond) 48:201–208. https://doi.org/10.3109/23744235.2015.1103897 An YH, Kang QK, Arciola CR (2006) Animal models of osteomyelitis. Int J Artif Organs 29:407–420. https://doi.org/10.1177/039139880602900411 Artenstein AW, Friderici J, Holers A, Lewis D, Fitzgerald J, Visintainer P (2016) Spinal Epidural Abscess in Adults: A 10-Year Clinical Experience at a Tertiary Care Academic Medical Center. Open Forum Infect Dis 3:ofw191. https://doi.org/10.1093/ofid/ofw191 Camino Willhuber G, Elizondo C, Slullitel P (2019) Analysis of Postoperative Complications in Spinal Surgery, Hospital Length of Stay, and Unplanned Readmission: Application of Dindo-Clavien Classification to Spine Surgery. Global Spine J 9:279–286. https://doi.org/10.1177/2192568218792053 Chang CL, Chen HH, Chen KH, Chiang JY, Li YC, Lin HS, Sung PH, Yip HK (2019) Adipose-derived mesenchymal stem cell-derived exosomes markedly protected the brain against sepsis syndrome induced injury in rat. Am J Transl Res 11:3955-71. https://doi.org/ Chang CL, Sung PH, Chen KH, Shao PL, Yang CC, Cheng BC, Lin KC, Chen CH, Chai HT, Chang HW, Yip HK, Chen HH (2018) Adipose-derived mesenchymal stem cell-derived exosomes alleviate overwhelming systemic inflammatory reaction and organ damage and improve outcome in rat sepsis syndrome. Am J Transl Res 10:1053-70. https://doi.org/ Chen HH, Lin KC, Wallace CG, Chen YT, Yang CC, Leu S, Chen YC, Sun CK, Tsai TH, Chen YL, Chung SY, Chang CL, Yip HK (2014) Additional benefit of combined therapy with melatonin and apoptotic adipose-derived mesenchymal stem cell against sepsis-induced kidney injury. J Pineal Res 57:16–32. https://doi.org/10.1111/jpi.12140 Chen KH, Lin KC, Ko SF, Chiang JY, Guo J, Yip HK (2020) Melatonin against acute ischaemic stroke dependently via suppressing both inflammatory and oxidative stress downstream signallings. J Cell Mol Med 24:10402–10419. https://doi.org/10.1111/jcmm.15654 Deguine J, Barton GM (2014) MyD88: a central player in innate immune signaling. F1000Prime Rep. 6:97. https://doi.org/10.12703/P6-97 Dworsky EM, Hegde V, Loftin AH, Richman S, Hu Y, Lord E, Francis KP, Miller LS, Wang JC, Scaduto A, Bernthal NM (2017) Novel in vivo mouse model of implant related spine infection. J Orthop Res 35:193–199. https://doi.org/10.1002/jor.23273 Fantoni M, Trecarichi EM, Rossi B, Mazzotta V, Di Giacomo G, Nasto LA, Di Meco E, Pola E (2012) Epidemiological and clinical features of pyogenic spondylodiscitis. Eur Rev Med Pharmacol Sci 16 Suppl 2:2–7. https://doi.org/ Gregori F, Grasso G, Iaiani G, Marotta N, Torregrossa F, Landi A (2019) Treatment algorithm for spontaneous spinal infections: A review of the literature. J Craniovertebr Junction Spine 10:3–9. https://doi.org/10.4103/jcvjs.JCVJS_115_18 Harris PNA, Tambyah PA, Lye DC, Mo Y, Lee TH, Yilmaz M, Alenazi TH, Arabi Y, Falcone M, Bassetti M, Righi E, Rogers BA, Kanj S, Bhally H, Iredell J, Mendelson M, Boyles TH, Looke D, Miyakis S, Walls G, Al Khamis M, Zikri A, Crowe A, Ingram P, Daneman N, Griffin P, Athan E, Lorenc P, Baker P, Roberts L, Beatson SA, Peleg AY, Harris-Brown T, Paterson DL (2018) Investigators MT, and the Australasian Society for Infectious Disease Clinical Research N Effect of Piperacillin-Tazobactam vs Meropenem on 30-Day Mortality for Patients With E coli or Klebsiella pneumoniae Bloodstream Infection and Ceftriaxone Resistance: A Randomized Clinical Trial. JAMA 320:984 – 94. https://doi.org/10.1001/jama.2018.12163 Hedlund E (2011) The protective effects of beta-lactam antibiotics in motor neuron disorders. Exp Neurol 231:14–18. https://doi.org/10.1016/j.expneurol.2011.06.002 Krasnodembskaya A, Samarani G, Song Y, Zhuo H, Su X, Lee JW, Gupta N, Petrini M, Matthay MA (2012) Human mesenchymal stem cells reduce mortality and bacteremia in gram-negative sepsis in mice in part by enhancing the phagocytic activity of blood monocytes. Am J Physiol Lung Cell Mol Physiol 302:L1003–L1013. https://doi.org/10.1152/ajplung.00180.2011 Lamb HM, Ormrod D, Scott LJ, Figgitt DP (2002) Ceftriaxone: an update of its use in the management of community-acquired and nosocomial infections. Drugs 62:1041–1089. https://doi.org/10.2165/00003495-200262070-00005 Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden O (2003) Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 57:11–20. https://doi.org/10.1046/j.1365-3083.2003.01176.x Lener S, Hartmann S, Barbagallo GMV, Certo F, Thome C, Tschugg A (2018) Management of spinal infection: a review of the literature. Acta Neurochir (Wien) 160:487–496. https://doi.org/10.1007/s00701-018-3467-2 Mei SH, Haitsma JJ, Dos Santos CC, Deng Y, Lai PF, Slutsky AS, Liles WC, Stewart DJ (2010) Mesenchymal stem cells reduce inflammation while enhancing bacterial clearance and improving survival in sepsis. Am J Respir Crit Care Med 182:1047–1057. https://doi.org/10.1164/rccm.201001-0010OC Ofluoglu EA, Zileli M, Aydin D, Baris YS, Kucukbasmaci O, Gonullu N, Ofluoglu O, Toplamaoglu H (2007) Implant-related infection model in rat spine. Arch Orthop Trauma Surg 127:391–396. https://doi.org/10.1007/s00402-007-0365-0 Paterson DL, Henderson A, Harris PNA (2020) Current evidence for therapy of ceftriaxone-resistant Gram-negative bacteremia. Curr Opin Infect Dis 33:78–85. https://doi.org/10.1097/QCO.0000000000000623 Raja DA, Musharraf SG, Shah MR, Jabbar A, Bhanger MI, Malik MI (2020) Poly (propylene glycol) stabilized gold nanoparticles: an efficient colorimetric assay for ceftriaxone. Journal of Industrial and Engineering Chemistry 87:180-6. https://doi.org/ Sileshi A, Tenna A, Feyissa M, Shibeshi W (2016) Evaluation of ceftriaxone utilization in medical and emergency wards of Tikur Anbessa specialized hospital: a prospective cross-sectional study. BMC Pharmacol Toxicol 17:7. https://doi.org/10.1186/s40360-016-0057-x Sun CK, Yen CH, Lin YC, Tsai TH, Chang LT, Kao YH, Chua S, Fu M, Ko SF, Leu S, Yip HK (2011) Autologous transplantation of adipose-derived mesenchymal stem cells markedly reduced acute ischemia-reperfusion lung injury in a rodent model. J Transl Med 9:118. https://doi.org/10.1186/1479-5876-9-118 Sung PH, Chiang HJ, Chen CH, Chen YL, Huang TH, Zhen YY, Chang MW, Liu CF, Chung SY, Chen YL, Chai HT, Sun CK, Yip HK (2016) Combined Therapy With Adipose-Derived Mesenchymal Stem Cells and Ciprofloxacin Against Acute Urogenital Organ Damage in Rat Sepsis Syndrome Induced by Intrapelvic Injection of Cecal Bacteria. Stem Cells Transl Med 5:782–792. https://doi.org/10.5966/sctm.2015-0116 Yang CC, Sung PH, Chiang JY, Chai HT, Chen CH, Chu YC, Li YC, Yip HK (2021) Combined tacrolimus and melatonin effectively protected kidney against acute ischemia-reperfusion injury. FASEB J 35:e21661. https://doi.org/10.1096/fj.202100174R Tables Table 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Table2.docx Cite Share Download PDF Status: Published Journal Publication published 06 Jan, 2025 Read the published version in Journal of Molecular Histology → Version 1 posted Editorial decision: Revision requested 18 Nov, 2024 Reviews received at journal 18 Nov, 2024 Reviews received at journal 14 Nov, 2024 Reviewers agreed at journal 08 Nov, 2024 Reviewers agreed at journal 06 Nov, 2024 Reviewers invited by journal 23 Oct, 2024 Editor assigned by journal 23 Oct, 2024 Submission checks completed at journal 22 Oct, 2024 First submitted to journal 22 Oct, 2024 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-5310715","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":369724491,"identity":"1beb8059-f8cc-49f4-a7a3-41a3702016af","order_by":0,"name":"Tsung-Cheng Yin","email":"","orcid":"","institution":"Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tsung-Cheng","middleName":"","lastName":"Yin","suffix":""},{"id":369724492,"identity":"a546c4f2-d06c-45b0-8c0f-c75b5da29cd4","order_by":1,"name":"Hung-Sheng Lin","email":"","orcid":"","institution":"Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hung-Sheng","middleName":"","lastName":"Lin","suffix":""},{"id":369724493,"identity":"fdedfb20-e3f9-4a28-b209-cde51ba2e13e","order_by":2,"name":"Pei-Hsun Sung","email":"","orcid":"","institution":"Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Pei-Hsun","middleName":"","lastName":"Sung","suffix":""},{"id":369724495,"identity":"f0cd8f06-699a-482c-ab0e-e05ef92dc0ed","order_by":3,"name":"John Y. Chiang","email":"","orcid":"","institution":"National Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"Y.","lastName":"Chiang","suffix":""},{"id":369724497,"identity":"7fd00d23-35ab-41a3-8b8e-f9b792decd61","order_by":4,"name":"Chien-Hui Yang","email":"","orcid":"","institution":"Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chien-Hui","middleName":"","lastName":"Yang","suffix":""},{"id":369724500,"identity":"23a33a6d-ef71-4ea6-901a-34b7422cf6ed","order_by":5,"name":"Hon-Kan Yip","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBACAxiDH0QwNkiQoEWyAaaFDcRNIEKLwQGwFgbCWszZzx5g/Np2x27zteMPPzDusMjjl29ge/DxB24tlj15Ccyybc+St93OMZZgPCNRLNnGwG44A5/DDuQYMEu2HU42u53DIMHYJpG44RgDmzQPPi3n30C0GM9Of/wDpGU/SMsffFpu5Bgwfmw7bGcgnWAGsYUNqAVviN14Y3CY4dzhBInbOWYWiUAtM44ltkn2pOFzWI7hwx9lh+35gQ678bGtLrG/+fAxiR82uLWAwGEeBobEBhAL4hxw7OAHjMBIsCeoahSMglEwCkYuAACPMlI5rdi1MwAAAABJRU5ErkJggg==","orcid":"","institution":"Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Hon-Kan","middleName":"","lastName":"Yip","suffix":""},{"id":369724502,"identity":"858f061f-ff25-4742-af7f-7fd96ade4f89","order_by":6,"name":"Kuan-Hung Chen","email":"","orcid":"","institution":"Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kuan-Hung","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-10-22 10:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5310715/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5310715/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10735-024-10344-9","type":"published","date":"2025-01-06T15:56:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":67403428,"identity":"22f85770-3656-44f4-98d4-80eebb54cf57","added_by":"auto","created_at":"2024-10-24 13:38:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":233638,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBody weight, mortality rate, and time courses of circulatory level of white blood cell counts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e Ratio of final (i.e., at day 28) to initial (i.e., at baseline) body weight (BW), * vs. other group with different symbols (†, ‡), all p \u0026lt;0.001. \u003cstrong\u003eB)\u003c/strong\u003e Accumulated mortality rate after animals grouping, * vs. †, p =0.012. \u003cstrong\u003eC)\u003c/strong\u003e Circulatory level of white blood cell (WBC) count at baseline, p\u0026gt;0.5. \u003cstrong\u003eD)\u003c/strong\u003eCirculatory level of WBC count at day 3, * vs. other group with different symbols (†, ‡), all p\u0026lt;0.001. \u003cstrong\u003eE)\u003c/strong\u003e Circulatory level of WBC count at day 7, * vs. other group with different symbols (†, ‡), all p\u0026lt;0.0001. \u003cstrong\u003eF)\u003c/strong\u003e Circulatory level of WBC count at day 14, *vs. other group with different symbols (†, ‡), all p\u0026lt;0.0001. \u003cstrong\u003eF)\u003c/strong\u003e Circulatory level of WBC count at day 28, *vs. other group with different symbols (†, ‡, §), all p\u0026lt;0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=6 to 8 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level). \u0026nbsp;SC = sham-operated control; ASI = acute spinal infection; Cef = ceftriaxone; adipose-derived mesenchymal stem cell (ADMSC).\u003c/p\u003e","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5310715/v1/7bc9c20244c9f8ce2807a71c.png"},{"id":67401855,"identity":"587a79b8-eac8-4a36-832c-b98d29cfef4e","added_by":"auto","created_at":"2024-10-24 13:22:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":276731,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCirculatory and spinal cord fluid levels of proinflammatory cytokines \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003eA)\u003c/strong\u003e Plasma level of interleukin (IL-6) at day 7, * vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001. \u0026nbsp;\u003cstrong\u003eB)\u003c/strong\u003e Plasma level of tumor necrosis factor (TNF)-α at day 7, * vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001. \u003cstrong\u003eC)\u003c/strong\u003e Plasma level of IL-6 at day 28, *vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001. \u0026nbsp;\u003cstrong\u003eD)\u003c/strong\u003e Plasma level of TNF-α at day 28, * vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001. \u003cstrong\u003eE)\u003c/strong\u003e Spinal fluid level of IL-6 at day 28, *vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001. \u003cstrong\u003eF)\u003c/strong\u003e Spinal fluid level of TNF-α at day 28, *vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=6 to 8 for each group). \u0026nbsp;Symbols (*, †, ‡) indicate significance for each other (at 0.05 level). SC = sham-operated control; ASI = acute spinal infection; Cef = ceftriaxone; adipose-derived mesenchymal stem cell (ADMSC).\u003c/p\u003e","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5310715/v1/821af40bc50c2f3c830a52ef.png"},{"id":67401501,"identity":"7141d385-937e-49a9-96a3-ef126f8b4cbd","added_by":"auto","created_at":"2024-10-24 13:14:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1013496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein levels of upstream and downstream inflammatory signaling biomarkers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Illustrating the densitometry analysis by Western blot analysis.\u003cstrong\u003e (B)\u003c/strong\u003e Illustrating the analytical result of protein expression among the different groups of each parameter.\u003c/p\u003e\n\u003cp\u003eProtein quantity of toll-like receptor (TRL-2), * vs. other group with different symbols (†, ‡, §), p\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003eProtein quantity of TLR-4, * vs. other group with different symbols (†, ‡, §), p\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003eProtein quantity of Kinase α (IKK-α), * vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003eProtein quantity of IKK-β, * vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003eProtein quantity of phosphorylated nuclear factor (p-NF)-κB, * vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003eProtein quantity of myeloid differentiation primary response 88 (MYD88), * vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003eProtein quantity interferon (IFN)-γ, * vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003eProtein quantity of inducible nitric oxide synthase (iNOS), * vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003eProtein quantity of TNF receptor associated factor 6 (TRAF6), * vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003eProtein quantity of interleukin (IL)-1β, * vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003eProtein quantity of IL-6, * vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003eProtein quantity TNF-α, * vs. other group with different symbols (†, ‡, §, ¶), p\u0026lt;0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=6 for each group). \u0026nbsp;Symbols (*, †, ‡, §, ¶) indicate significance for each other (at 0.05 level). SC = sham-operated control; ASI = acute spinal infection; Cef = ceftriaxone; adipose-derived mesenchymal stem cell (ADMSC).\u003c/p\u003e","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5310715/v1/fb0619b9007c938d7c5c9a69.png"},{"id":67402966,"identity":"0d9ec2f8-514b-4151-87da-3627e5c6f3cf","added_by":"auto","created_at":"2024-10-24 13:30:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2146362,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLight Microscopic findings for clarifying the impact of ceftriaxone and ADMSCs on attenuating the bone injury score\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA to E)\u003c/strong\u003e Illustrating the light microscopic finding (12.5x, 40x, 100x) of H\u0026amp;E stain for identification bone injury score, i.e., black arrow heads indicated the vertebral bone destruction whereas the red arrow heads indicated the infectious bone deposition. Additionally, the integrity of the vertebral bone and the number of trabeculae were observed to be markedly destroyed in ASI group (Ba, Bb, Bc) than in other groups (Aa-Ac, Ca-Cc, Da-Dc, Ea-Ec). Scale bar in right lower corner represents 600µm for 12.5x, 200µm for 40x and 100µm for100x, respectively. \u003cstrong\u003eF) \u003c/strong\u003eAnalytical result of vertebral bone destructive score, * vs. other group with different symbols (†, ‡, §), p\u0026lt;0.0001. \u003cstrong\u003eG) \u003c/strong\u003eAnalytical result of infectious bone deposition score, * vs. other group with different symbols (†, ‡, §), p\u0026lt;0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=5-10 for each group). \u0026nbsp;Symbols (†, ‡, §) indicate significance for each other (at 0.05 level). SC = sham-operated control; ASI = acute spinal infection; Cef = ceftriaxone; adipose-derived mesenchymal stem cell (ADMSC).\u003c/p\u003e","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5310715/v1/263828cdd1e90739900bad93.png"},{"id":67402967,"identity":"69354f85-cf45-4134-b346-c787353a92b8","added_by":"auto","created_at":"2024-10-24 13:30:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":443759,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUtilization of micro-CT for identification of the destructive vertebral bone (also refer to Table 2)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo verify the ceftriaxone and ADMSCs on protecting the vertebral bone against ASI induced damage, the micro-CT was utilized in the present study. \u003cstrong\u003eA to E)\u003c/strong\u003e Illustrating the transverse plan of micro-CT finding. The vertebral bone was destroyed (red arrows) more severely in ASI group (B) than in other groups. \u0026nbsp;\u003cstrong\u003eF to J)\u003c/strong\u003eShowing the 3-D of micro-CT finding for anatomical structure of vertebral bone of spinal cord. The result showed that the surface of vertebral bone was substantially destroyed (red arrows indicated plentiful punch-point lesions) in ASI group (G) than in other groups.\u003c/p\u003e","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5310715/v1/faa2478a786a5d831dc0320e.png"},{"id":67401503,"identity":"abf266ef-84f7-45e4-b78e-50b46e9d371d","added_by":"auto","created_at":"2024-10-24 13:14:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":216988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIllustrating the underlying mechanism of bacterial infection-caused spinal cord injury and the effective treatment of the combined antibiotics and ADMSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eADMSCs = adipose-derived mesenchymal stem cells; WBC = white blood cell; CFU = Colony formation unit.\u003c/p\u003e","description":"","filename":"OnlineFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5310715/v1/3200698d9f0c1bc94ce7a5fa.png"},{"id":73693860,"identity":"59486489-6a74-4800-8355-1b5b782de127","added_by":"auto","created_at":"2025-01-13 16:08:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7039953,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5310715/v1/7b9a84f2-b802-4045-8bf7-713d2e43a053.pdf"},{"id":67401499,"identity":"f944da91-6347-40f9-9c6a-ee298a30bf96","added_by":"auto","created_at":"2024-10-24 13:14:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":64783,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5310715/v1/27b89c0b3b51f75dc329816a.docx"},{"id":67401500,"identity":"85e9f027-0dd2-4240-8de7-23672d99a962","added_by":"auto","created_at":"2024-10-24 13:14:12","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":108305,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5310715/v1/8171158478469d1672075243.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Additional benefits of combined ceftriaxone and adipose-derived mesenchymal stem cells on revamping the outcomes in rodent after acute spinal infection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpinal infection (SI) is an infectious disease involving the intervertebral disc, vertebral body, and/or adjacent paraspinal tissue (Lener et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The SI contributes to 2\u0026ndash;7% of all musculoskeletal infections in the clinical observation, usually resulting in seriously destructive spinal cord tissues if not treated promptly and properly (Lener et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The histopathological findings have identified that the sources of infection can be spread to the spine by hematogenous dissemination from distant site, by diffusion from contiguous tissues, or/and by external inoculation (trauma, surgery) (Lener et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Gregori et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe mortality rate of SI has been reported to be 2% and 20%, respectively (Lener et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Aagaard et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Artenstein et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Fantoni et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Patients who suffer from SI generally express non-specific symptoms, including back pain (85%), fever (48%), and paresis (32%) (Fantoni et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Gregori et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lener et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Although treatment strategies of SI remain controversial and sometimes ineffective, use of concomitant antibiotics treatment under non-surgical (i.e., immobilization with rigid orthosis) or surgical managements (i.e., debridement, decompression, etc.) is still the common choice of treatment for SI, depending on different clinical manifestation and disease course as well as the general conditions (Gregori et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lener et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Conclusively, the management of SI is still an unmet need that raises the urgency to conduct a comprehensive investigation to establish an effective and innocuous treatment for SI patients.\u003c/p\u003e \u003cp\u003eIn most countries, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e is still one of the most common pathogens that causes SI. According to the previous reports, the incidence of \u003cem\u003emethicillin-resistant S. aureus (MRSA)\u003c/em\u003e accounts from 6.8\u0026ndash;30% of pathogens for SI (Gregori et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lener et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Such pathogens might further generate complicated process of localized or systemic inflammatory reactions, such as overwhelming immune response, induction of oxidative stress and reactive oxygen species (ROS) as well as over-compensatory inflammatory reaction (Gregori et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lener et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sung et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Although use of antimicrobial agents is the gold standard treatment of bacterial-induced infection, the morbidity, mortality, hospital length of stay (LOS), and health costs are still relatively high (Camino Willhuber et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This could be mainly due to the reason that antimicrobial agents cannot effectively control the exaggerated immune response and overwhelming inflammatory reaction effectively (Camino Willhuber et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sung et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, in addition to the advisable choice of antimicrobial agents, control of the exaggerated immune response and overwhelming inflammatory reaction may be critical to reduce SI-induced morbidity and mortality (Artenstein et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Camino Willhuber et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is well known that ceftriaxone (Sileshi et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hedlund \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Raja et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) is an antibiotic that pertains to a category of medicine recounted to as cephalosporin antibiotics, and it effectively treats a variety of bacterial infections by ceasing the growth of \u003cem\u003ebacteria\u003c/em\u003e in variety of bacterial infections in different organs, including bone and spinal cord bacterial infection. However, growing data have shown that ceftriaxone-resistant \u003cem\u003eGram-negative bacilli\u003c/em\u003e, especially those caused by extended-spectrum \u003cem\u003eβ-lactamases\u003c/em\u003e (Paterson et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Harris et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) becomes more and more popular in our daily clinical practice.\u003c/p\u003e \u003cp\u003eNot only has abundant evidence showed that stem cell therapy attenuates inflammation and innate/adaptive immune responses (Sung et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Le Blanc et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), but adipose-derived mesenchymal stem cells (ADMSCs) have also been shown to possess the intrinsic capacity of immunomodulation and tissue regeneration (Sun et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Camino Willhuber et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sung et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Interestingly, limited investigations suggest that, in the setting of ischemia-reperfusion injury and sepsis, ADMSCs treatment could attenuate morbidity and mortality through regulation of ischemia-reperfusion injury and sepsis-induced exaggerated immune response and overwhelming inflammatory reaction as well as control of ROS and oxidative stress generation (Sung et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Krasnodembskaya et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mei et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Additionally, our previously experimental studies have demonstrated that ADMSCs therapy effectively controlled sepsis syndrome (SS) (Sung et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), whereas combined therapy with antibiotics and ADMSCs even more effectively controlled the SS and reduced the rodent mortality (Sung et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the other hand, there has been no experimental study that focuses on the therapeutic effect of stem cell therapy compared with that of ceftriaxone antimicrobial agents as well as that when the two strategies are combined in the setting of experimental model of SI. Therefore, we conducted this study to test the hypothesis that ceftriaxone-ADMSC combined therapy was superior to ceftriaxone monotherapy in animal model of microbial-induced SI to attenuate inflammation/oxidative stress as well as morbidity and mortality.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003eEthical statement \u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were approved by the Institute of Animal Care and Use Committee at Kaohsiung Chang Gung Memorial Hospital (Affidavit of Approval of Animal Use Protocol No. 2020121605) and performed in accordance with the Guide for the Care and Use of Laboratory Animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnimals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC; Frederick, MD, USA)-accredited animal facility in Kaohsiung Chang Gung Memorial Hospital under temperature (22 \u0026plusmn; 1 ◦C), humidity (55 \u0026plusmn; 5%) and light (12: 12/light: dark photo-cycle, light on at 08:00) control. Standard laboratory rat chow and tap water were available ad libitum. All animals were allowed to acclimatize to the animal facility for 14 days prior to experimental manipulations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTo create an animal model of acute bacterial spinal vertebral bone infection (ASI)\u0026nbsp;\u003c/em\u003e\u003cem\u003ein SD rat\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe procedure and protocol were based on the previous study with some modification (Ofluoglu et al. 2007; Dworsky et al. 2017) with some modification. Pathogen-free, adult male \u003cem\u003eSprague-Dawley (SD) rats\u003c/em\u003e, weighing 320-350 g (Charles River Technology, BioLASCO Taiwan Co. Ltd., Taiwan), were used in the present study. In detail, the animals were anesthetized by inhalation of isoflurane, i.e., isoflurane was generally used at concentrations of 5% for induction, and 2 % for maintenance of general anesthesia and 1.0% for recovery stage and placed prone on a warming pad. The hair on the skin on the lower back around the spine was shaved. The skin was incised at the midline of the spine at the T11 - L2 level, and the paraspinal muscles were carefully dissected subperiosteally to expose the lamina. A sterile self-tapping screw (diameter 1 mm, length 4 mm) was screwed into the vertebral bone between T13 - L1, following by unscrewing the above screw, the \u003cem\u003eStaphylococcus aureus subsp. aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e, ATCC\u0026reg; 19636 \u0026trade;) suspension (5\u0026times;105 cells in 50\u0026mu;L) was inject into the hole that was drilled by the screw, and then reset the screw. Finally, the incision was sutured, and the animals were left awake on a warm pad at 37\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnimal grouping and animals were euthanized\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAdult male\u003cem\u003e\u0026nbsp;SD rats\u003c/em\u003e (n = 42) were categorized into group 1 [sham-operated control (SC), i.e., merely opening the skin and muscle layer of spinal cord, followed by closure of these two layers over the spinal cord, n = 8], group 2 [acute spine infection (ASI), n = 10], group 3 [ASI + ceftriaxone (5 mg/kg/day by subcutaneous administration over the spinal area from days 2 to 28 after ASI induction, n = 8), group 4 [ASI + allogenic ADMSCs (1.0 x 10\u003csup\u003e6\u003c/sup\u003e cells/rat from day 2 for a total of 3 doses/for 3 consecutive intervals with 9 days after ASS by intravenous injection), n = 8] and group 5 (ASS + combined ceftriaxone and ADMSC, n = 8), respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe dosage of antibiotics to be utilized in the present study was based on the previous report with some modification (Sung et al. 2016). On the other hand, the dosage of ADMSCs to be utilized in the present study was based on our previous reports with some modification (Chang et al. 2018; Chang et al. 2019).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEuthanasia of animals was performed under anesthesia with overdose of isoflurane inhalation (i.e., more than 5.0%) and collected the blood by more than 10 ml by day 28 after ASI induction. \u0026nbsp;The vertebral bone, cartilage and spinal cord tissues were harvested after the respiratory arrest for individual study.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMethodology for preparing and culturing allogenic ADMSCs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAdditional 20 \u003cem\u003erats\u003c/em\u003e were utilized for allogenic ADMSCs isolation. The procedure and protocol for allogenic ADMSC isolation and culture have been described in our previous reports (Fantoni et al. 2012; Sung et al. 2016). Briefly, adipose tissue surrounding the epididymis was dissected, excised and prepared by day 14 prior to acute kidney IR induction. For purification, the harvested cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026apos;s medium (DMEM)-low glucose medium containing 10% FBS for 14 days. By this time, plentiful ADMSCs (i.e., approximately 2.5 - 3.0 x 10\u003csup\u003e6\u003c/sup\u003ecells) were obtained in the culture plate and were collected to treat ASI animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eELISA examination\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCollect the circulatory blood samples at days 14 and 28 after ASI induction in each group of the animals for determining the circulating levels of IL-6 and TNF-\u0026alpha; levels. Additionally, these parameters of spinal fluid were also collected and measured at the end of study period, i.e., by day 28 after ASI induction. These biomarkers were measured by ELISA standard method was according to the manufacturer\u0026rsquo;s instruction. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHistopathological examination\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBy day 28 after ASI induction, the histopathological examination was performed after routine fixation, decalcification, and paraffin embedding for assessment of vertebral bone/cartilage destruction, the bacterial counts in the bone and adjacent region and inflammatory cell infiltration (i.e., polymorphonuclear cells).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWestern blot analysis from isolated spinal-cord tissues\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe protocol and procedure were described by our previous reports (Chang et al. 2019; Chang et al. 2018; Sung et al. 2016; An et al. 2006). \u0026nbsp;Briefly, equal amounts (50 \u0026micro;g) of protein extracts were loaded and separated by SDS-PAGE using 8-12% acrylamide gradients. After electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences). Nonspecific sites were blocked by incubation of the membrane in blocking buffer (1X TBS, 0.1% Tween-20 with 5% w/v nonfat dry milk) overnight. The membrane was incubated with indicated primary antibodies Toll-like receptor 2 (TLR2) (1:1000, Abcam), TLR4 (1:1000, Novusbio), tumor necrosis factor receptor associated factor 6 (TRAF6)1:1000, Abcam), phosphorylated nuclear factor (p-NF)-\u0026kappa;B (1:1000, Abcam), interleukin (IL)-1\u0026szlig; (1:1000, Cell Signaling), IL-6 (1:1000, Abcam), IKK\u0026alpha;(1:5000, Abcam), IKK\u0026beta; (1:1000, Cell Signaling), IKB\u0026beta; (1:1000, Abcam), interferon (IFN)-\u0026gamma; (1:4000, Abcam), tumor necrosis (TNF)-\u0026alpha; (1:1000, Cell Signaling), inducible nitric oxide synthase (iNOS) (1:1000, Abcam), MYD88 (1:1000, Abcam) and Actin (1:6000, Millipore)] for 1 hour at room temperature. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin IgG (1:5000, Sigma) was used as a secondary antibody for one-hour incubation at room temperature. The washing procedure was repeated eight times within one hour. \u0026nbsp;Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences, Amersham, UK) and exposed to Biomax L film (Kodak, Rochester, NY, US). For the purpose of quantification, ECL signals were digitized using Labwork software (UVP, Waltham, MA, US).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicro-CT examination of decalcified vertebral/spinal-cord bone\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBy day 28, i.e., at the end of study period, the animals were euthanized, and the bone-articular joints were harvested from each animal. After well preparation, these specimens were assessed by the Bruker Micro-CT (SkyScan 1176, Bruker BioSpin, Germany, with Bruker CTAn Micro-CT Software for analysis) for verifying the destructive features of the vertebral/spinal-cord bone, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDefinition for BMD, ratios of BV/TV,\u0026nbsp;\u003c/em\u003e\u003cem\u003eBS/BV and BS/TV, TN\u003c/em\u003e\u003cem\u003e\u0026nbsp;and TS\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBone mineral density (BMD) measures the density of minerals in the bone. It can be used to detect osteoporosis, determine the risk of future fractures, and assess the outcome of osteoporosis treatment.\u003c/p\u003e\n\u003cp\u003eRatio of bone volume (BV) to bone tissue volume (TV) (i.e., percentage of bone volume) refers to the proportion of deducted dense bone to bone TV in all tissues. This index is a common index used for evaluating the bone mass of compact bone and spongy bone. An increase in this value indicates that bone metabolism is greater than catabolism and bone mass increases correspondingly, and vice versa, which indirectly reflects the state of bone metabolism. Ratio of bone surface to bone volume (BS/BV) indirectly reflects the amount of bone mass. Ratio of bone surface to bone tissue volume (BS/TV)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ealso indirectly reflects the amount of bone mass.\u003c/p\u003e\n\u003cp\u003eTrabecular number (TN)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003erefers to the number of intersection points produced by the intersection of the plate-shaped structure and the round rod-shaped structure, and the structure woven by these intersection points is the trabecular bone. When osteoporosis occurs, the TN value will decrease relatively.\u003c/p\u003e\n\u003cp\u003eTrabecular separation (TS)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003erefers to the average width of the medullary cavity between the trabecular bone. The increase of TS corresponds the increase of bone loss leading to the increase of cavity space between bone trabeculae, so osteoporosis may occur.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDefinitions of Classification of bone destructive core and infectious bone deposition\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe definitions were based on the previous report (Ofluoglu et al. 2007) with minimal modifications:\u003c/p\u003e\n\u003cp\u003e(1) Classification of vertebral bone destruction: on a scoring of 1 to 5, indicating that the bone was damaged (i.e., by infection) and could not be repaired by itself, which included the bone marrow destructive space.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(2) Classification of infectious bone deposition: including the severity of scale from 1 to 5, indicating abnormal bone hyperplasia/deposition. Thus, bone injury score was the comprehensive score of cartilage destruction (1) and infectious bone deposition (2) in the current study. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative data were expressed as mean \u0026plusmn; SD. Statistical analysis was adequately performed by ANOVA, followed by Bonferroni multiple comparison post hoc test. SAS statistical software for Windows version 8.2 (SAS Institute, Cary, NC, USA) was utilized. A probability value \u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":" \u003cp\u003e \u003cem\u003eBody weight, mortality rate, and time courses of circulatory level of white blood cell counts (WBC) (Fig.\u0026nbsp;1)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe ratio of the initial (i.e., by day 0) to final (i.e., by day 28) body weight significantly lower in group 4 (ASI + ADMSC) than in groups 1 (SC), 2 (ASI), 3 (ASI + ceftriaxone), and 5 (ASS + combined ceftriaxone and ADMSC)] and significantly lower in groups 2 and 5 than in groups 1 and 3, but it showed no difference between groups 2 and 5 or between 1 and 3 (n = 8 for each group).\u003c/p\u003e \u003cp\u003eThe total mortality rate within 48h prior to grouping was 9.1% (5/55). Animals were groups at day 2 prior to the treatment and the mortality rate after grouping prior to being euthanized by day 28 were 0% (0/8) in group 1, 30.0% (3/10) in group 2 and 0% (0/8) in groups 3, 4 and 5, respectively. Regarding the statistical analysis of the mortality rate, the variables were compared with one-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) for comparison of the difference between two groups. Difference of alphabetic characters (\u003csup\u003ea or b\u003c/sup\u003e) in superscript indicates statistical significance of p-value \u0026lt; 0.05 after comparison, p = 0.012 (n = 10 for each group).\u003c/p\u003e \u003cp\u003eThe circulating level of WBCs at baseline did not differ among the five groups (n = 8 for each group). However, by day 3 after the ASI induction, the WBC count was significantly lower in group 1 than in other groups, significantly lower in group 5 than in groups 2, 3 and 4. However, this parameter did not differ among the latter three groups. Additionally, by days 7 and 14 after ASI, this parameter was significantly lower in 1 than in other groups, and significantly higher in group 2 than in groups 3, 4, and 5, but it was similar among the groups 3 to 5. However, by days 28 after ASI induction, the WBC count was lowest in group 1, highest in group 2, significantly lower in group 5 than in groups 3 and 4, but it showed no difference between the latter two groups (n = 8 for each group).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCirculatory and spinal cord fluid levels of proinflammatory cytokines and bacterial CFU in vertebral bone and circulation (Fig.\u0026nbsp;2 and Table\u0026nbsp;1)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo elucidate whether the ceftriaxone or ADMSCs treatment would suppress the circulating and spinal cord fluid levels of IL-6 and TNF-α, the ELISA methodology was utilized in the present study. The result showed that by day 7 after ASI induction, the circulating levels of IL-6 and TNF-α, were lowest in group 1, highest in group 2, significantly lower in group 5 than in groups 3 and 4 and significantly lower in group 3 than in group 4 (n = 8 for each group). Additionally, by day 28 after ASI induction, the circulating and spinal cord fluid levels of these two parameters displayed an alike pattern as those of day 7 (n = 6–8 for each group).\u003c/p\u003e \u003cp\u003eAdditionally, Table\u0026nbsp;1 illustrated that the bacterial \u003cem\u003eCFU\u003c/em\u003e in vertebral bone (n = 6 for each group) was significantly higher in group 2 than in groups 3, 4, and 5, and significantly higher in groups 3 and 4 than in group 5, but it showed no difference between groups 3 and 4. Furthermore, bacterial \u003cem\u003eCFU\u003c/em\u003e in circulation (n = 8 for each group) was significantly higher in group 2 than in groups 3 to 5, but this parameter did not differ within these latter three groups.\u003c/p\u003e \u003cp\u003e \u003cem\u003eProtein levels of upstream and downstream inflammatory signalings (Fig.\u0026nbsp;3)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo verify the impact of ceftriaxone and ADMSCs treatments on alleviating the inflammatory reaction, the Western blot analysis (n = 6 for each group) for the protein levels of parameters were conducted in the present study. The result demonstrated that protein expressions of TRL-2, TLR-4, MYD88, TRAF6, IKK-α, IKK-β, IKB-β and p-NFκ-B, eight upstream inflammatory signalings, were lowest in group 1, highest in group 2, significantly lower in group 5 than in groups 3 and 4, and significantly lower in group 3 than in group 4. Additionally, the protein expressions of IL-1β, IL-6, TNF-α, IFN-γ and iNOS, five downstream inflammatory signalings, displayed an identical pattern among the five groups, implicating that combined Cef and ADMSCs offered additional benefit than merely one on suppressing ASI induced inflammatory reaction.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTo clarify the impact of ceftriaxone and ADMSCs treatments on attenuating the bone injury score (Fig.\u0026nbsp;4)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo verify whether ceftriaxone-ADMSCs would offer benefit on protecting the bone and cartilage against ASI damage, the H\u0026amp;E staining (n = 5–10 for each group) was conducted in the present study. The result showed that vertebral bone destructive score and infectious bone deposition score, i.e., defined as the bone injury score, were significantly higher in group 2 than in other groups, significantly higher in groups 3 and 4 than in groups 1 and 5 and significantly higher in group 5 than in group 1, but it showed no difference between groups 3 and 4.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMicro-CT examination of vertebral/spinal-cord bone for identification of bone/bone marrow damage (Fig.\u0026nbsp;5 and Table\u0026nbsp;2)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo verify the ceftriaxone and ADMSCs on protecting the vertebral bone against ASI induced damage, the micro-CT specific for small animals was utilized in the present study (n = 6 for each group). The result showed that the bone marrow density (BMD) was significantly higher in group 5 than in other groups, significantly higher in group 1 than in groups 2 to 4 and significantly higher in group 3 and 4 than in group 2, but it showed no difference between the groups 3 and 4. Our finding implied that combined ceftriaxone and ADMSCs treatment increasing the BMD could be mainly due to the tissue regeneration effect of ADMSCS.\u003c/p\u003e \u003cp\u003eAdditionally, the BV/TV bone volume/trabecular volume (i.e., BV/TV), a ratio of bone volume to the trabecular volume, was significantly higher in groups 1 and 5 than in the groups 2 to 4 and significantly higher in groups 3 and 4 than in group 2, but it showed no difference between groups 1 and 5 or between 3 and 4 (n = 6 for each group), implicating that the combined ceftriaxone and ADMSCs was superior to merely one treatment on protecting the bone materials again infection-induced loss.\u003c/p\u003e \u003cp\u003eFurthermore, the bone surface/trabecular volume (i.e., BS/TV), i.e., a ratio of bone surface to trabecular volume reflecting the bone density, and the numbers of trabeculae, an indicator of trabecular bone density, were significantly higher in groups 1 and 3 to 5 than in those of the group 2, but these parameters were similar among the groups 1 and 3 to 5 (n = 6 for each group), implicating that ceftriaxone-ADMSCs therapy significantly protected bone materials against the bacterial infection damage.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSchematically illustrated the underlying mechanism of bacterial spinal cord infection in rodent and the therapeutic impact of ceftriaxone-ADMSCs on ASI (Fig.\u0026nbsp;6)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eFinally, through the results from Figs.\u0026nbsp;1 to 6, we could graphically depict the underlying mechanistic basis of overwhelming inflammatory reactions in circulatory levels and bone tissue, resulting in vertebral bone and cartilage destruction that were remarkably reversed by ceftriaxone-ADMSCs therapy.\u003c/p\u003e "},{"header":"Discussion","content":"\u003cp\u003eOur present study which investigated the therapeutic effect of ceftriaxone-DMSCs on ASI delivered some striking connotations. First, we successfully created a reproducible animal model of ASI for testing the aftermath of ceftriaxone-ADMSCs on this disease entity and could be extended to the other strategic management of ASI. Second, the result of the current study demonstrated that ADMSCs were comparable with ceftriaxone for securing the spinal cord against the ASI damage. Third, combined ceftriaxone and ADMSCs therapy was superior to either one on sheltering the spinal cord against the ASI injury. Finally, we found that upstream and downstream inflammatory signaling acted as a foremost role on spinal cord damage in setting of ASI.\u003c/p\u003e\u003cp\u003eDespite various modalities have been reported for the treatment (Artenstein et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Camino Willhuber et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gregori et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lener et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), there is still lacking an unanimously acknowledged effective management that could rationally explained why the morbidity, mortality, hospital length of stay and health costs are still unacceptably high in ASI and chronic SI setting (Camino Willhuber et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). One important finding in this preclinical study demonstrated that one kind of antibiotic (i.e., ceftriaxone) was identified to be comparable with ADMSCs for the efficacy on safeguarding the spinal cord damage and bettering the outcomes in ASI rodent. The most important finding in this preclinical study proclaimed that combined antibiotic and ADMSCs therapy was superior to just one therapy for boosting the outcomes in ASI rodent. In this way, our findings, in addition to extending the findings from previous studies (Artenstein et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Camino Willhuber et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gregori et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lener et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), highlight that this combined regimen may be a therapeutic novelty for ASI patients, especially for those patients who are refractory to the conventional therapy (Camino Willhuber et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTwo essential findings from this current study were that the bacteria in the circulation and derived from vertebral bone were remarkably higher in ASI animals than those of ASI animals after receiving ceftriaxone-ADMSCs treatment. Interestingly, when looked at the findings from H\u0026amp;E staining, we identified that the bone injury score was remarkably increased in ASI animals without than with the ceftriaxone-ADMSCs treatment. It is well known that the bacteria in chronic osteomyelitis patients are always too difficult to be eradicated through antibiotics (i.e., by bactericidal effect) mainly due to the antibiotics are rather difficult to penetrate in the bone parenchyma and could be also due to the bone distinctively complicated structure always serving as a good harbor for bacteria concealed there. Perhaps, our findings (i.e., by H.E. staining) of destructive vertebral bone (i.e., including bone marrow, trabeculae, and cartilage) would serve as a good sanctuary for bacterial occultation.\u003c/p\u003e\u003cp\u003eWe were very concerned the underlying mechanism for how ceftriaxone-ADMSCs treatment ensured the spinal cord/vertebral bone in setting of ASI. It is well recognized that MYD88 played the role as an essential innate immune signal transduction adaptor (Deguine et al. 2014). Additionally, plentiful studies have established that this inflammatory-immune signaling takes part in damage of the tissues/organs in organ ischemic settings (Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Intriguingly, when looked at MYD88 innate immune signal, we found that the protein expressions of upstream and downstream inflammation/innate immune signalings were much increased in ASI animals than in SC animals. Our finding was consistent with the findings of previous studies (Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Deguine et al. 2014; Yang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Of paramount important finding in the present study was that these molecular-cellular perturbations were noteworthily repressed by ceftriaxone or ADMSCs therapy and further noteworthily repressed by combined ceftriaxone + ADMSCs treatment, highlighting that our finding in addition to extending the findings of previous studies (Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Deguine et al. 2014; Yang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), encourages the use of these two regimens for those acute or chronic SI or even extrapolates to the treatment of osteomyelitis patients, especially when they are refractory to conventional therapy. We suggest this combination therapy offering great benefits to those of ASI animals and improving their outcomes could be due to the synergic effect of anti-inflammation (i.e., the essential effect of ceftriaxone) and anti-inflammation/immunomodulation (i.e., the principal therapeutic effect of ADMSCs) along with possible tissue regeneration (another therapeutic impact of MSCs).\u003c/p\u003e\u003cp\u003eAn especially interesting finding in the present study was that as compared with the ASI animals without treatment, the mortality rate was significantly lower in those of ASI animals treated by ADMSCs. Undoubtedly, MSCs treatment could not directly killed the bacterial. However, when we looked at the circulatory derived \u003cem\u003eCFU\u003c/em\u003e, we found that the \u003cem\u003eCUF\u003c/em\u003e was markedly reduced in ASI animals with than in without ADMSCs treatment. Additionally, it is well known that systemic inflammatory response syndrome (SIRS) is an exaggerated defense response of the body to a noxious stressor, i.e., such as bacterial infection, trauma, surgery, or acute inflammation…etc. Based on the aforementioned issues, we suggested that perhaps, the mortality rate was notably lower in ASI animals with than in without ADMSCs treatment could be, at least in part, due to the fact that HUCDMSCs has the property of anti-inflammation and immunomodulation (Le Blanc et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sung et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eScientists would be interesting in understanding the underlying mechanism for how the combined ceftriaxone and ADMSCs could offer synergic effect on protecting the vertebral bone and cartilage against ASI damage. In fact, abundant data have shown that ceftriaxone kills many types of bacteria by suppressing the mucopeptide synthesis of bacterial cell wall. The \u003cem\u003eβ-lactam\u003c/em\u003e core of ceftriaxone irreversibly binds to carboxypeptidases, endopeptidases, and transpeptidases in the bacterial cytoplasmic membrane (Lamb et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Thus, ceftriaxone is characterized as one kind of bactericidal antibiotics. During bacterial infection, especially those of \u003cem\u003egram-negative bacteria\u003c/em\u003e always release endotoxin that would elicit overwhelming inflammatory reaction, enhance innate immune reaction and inflammatory cell infiltration in infectious organs, resulting in activate the inflammatory signalings and generation of proinflammatory cytokines, subsequently destructs the tissues, organs and bone as well as the cartilage. On the other hand, the ADMSCs have been shown to have intrinsic ability of anti-inflammation, immunomodulation and tissue regeneration (Sun et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Camino Willhuber et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sung et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Accordingly, the above-mentioned issues (Sun et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Camino Willhuber et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sung et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lamb et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) and the findings of the present study could, at least in part, explain why combined ceftriaxone and ADMSCs therapy was superior to merely one on protecting the vertebral bone and cartilage against ASI damage (referred to Fig.\u0026nbsp;7).\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eStudy limitations\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eOur study has some constraints. Frist, the study period was only 28 days, implicating that it was relatively short, i.e., just reflected the ASI outcomes. Accordingly, the chronic outcome of SI after receiving these therapeutic management is currently unclear. Second, the underlying mechanism of bacterial spinal cord injury in rodent and therapeutic impact of ceftriaxone-ADMSCs on ASI was merely dependent on the results of our study that may not really delineate the complicated mechanistic basis of vertebral bone/spinal-cord damage in the setting of ASI.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe complications of spinal cord infection are commonly observed in gravely destructive spinal cord tissues and uncontrolled infection, consequently, leave grievous sequalae and unacceptable high morbidity and mortality. On the other hand, how to effective treatment for ASI, especially in those patients who already develop destructive vertebral bone and cartilage damage, remains a formidable challenge. Our study demonstrated that combined ceftriaxone-ADMSCs treatment provided synergic effect on safeguarding the vertebral bone/spinal cord in opposition to ASI damage.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a program grant from Chang Gung Memorial Hospital, Chang Gung University (CMRPG8M0581).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets of present study can be available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT. -C. Y., P.-H. S., H.-K. Y. designed the study. T. -C. Y., H. -S. L., P.-H. S., C.-H. Y., investigated experiments. T. -C. Y., P.-H. S., C.-H. Y., H.-K. Y curated data. H. -S. L., P.-H. S., K.-H. C., C.-H. Y. and H.-K. Y. did formal analysis. K.-H. C. was responsible for funding acquisition. H.-K. Y. and K.-H. C. administered and supervised the project. K.-H. C., J.-Y. C., H.-K. Y. wrote the first draft of the manuscript and all named authors contributed in revising the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were approved by the Institute of Animal Care and Use Committee at Kaohsiung Chang Gung Memorial Hospital (Affidavit of Approval of Animal Use Protocol No. 2020121605) and performed in accordance with the Guide for the Care and Use of Laboratory Animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a program grant from Chang Gung Memorial Hospital, Chang Gung University (CMRPG8M0581).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAagaard T, Roed C, Dahl B, Obel N (2016) Long-term prognosis and causes of death after spondylodiscitis: A Danish nationwide cohort study. Infect Dis (Lond) 48:201\u0026ndash;208. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3109/23744235.2015.1103897\u003c/span\u003e\u003cspan address=\"10.3109/23744235.2015.1103897\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAn YH, Kang QK, Arciola CR (2006) Animal models of osteomyelitis. Int J Artif Organs 29:407\u0026ndash;420. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/039139880602900411\u003c/span\u003e\u003cspan address=\"10.1177/039139880602900411\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArtenstein AW, Friderici J, Holers A, Lewis D, Fitzgerald J, Visintainer P (2016) Spinal Epidural Abscess in Adults: A 10-Year Clinical Experience at a Tertiary Care Academic Medical Center. Open Forum Infect Dis 3:ofw191. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/ofid/ofw191\u003c/span\u003e\u003cspan address=\"10.1093/ofid/ofw191\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCamino Willhuber G, Elizondo C, Slullitel P (2019) Analysis of Postoperative Complications in Spinal Surgery, Hospital Length of Stay, and Unplanned Readmission: Application of Dindo-Clavien Classification to Spine Surgery. Global Spine J 9:279\u0026ndash;286. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/2192568218792053\u003c/span\u003e\u003cspan address=\"10.1177/2192568218792053\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang CL, Chen HH, Chen KH, Chiang JY, Li YC, Lin HS, Sung PH, Yip HK (2019) Adipose-derived mesenchymal stem cell-derived exosomes markedly protected the brain against sepsis syndrome induced injury in rat. Am J Transl Res 11:3955-71. https://doi.org/\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang CL, Sung PH, Chen KH, Shao PL, Yang CC, Cheng BC, Lin KC, Chen CH, Chai HT, Chang HW, Yip HK, Chen HH (2018) Adipose-derived mesenchymal stem cell-derived exosomes alleviate overwhelming systemic inflammatory reaction and organ damage and improve outcome in rat sepsis syndrome. Am J Transl Res 10:1053-70. https://doi.org/\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen HH, Lin KC, Wallace CG, Chen YT, Yang CC, Leu S, Chen YC, Sun CK, Tsai TH, Chen YL, Chung SY, Chang CL, Yip HK (2014) Additional benefit of combined therapy with melatonin and apoptotic adipose-derived mesenchymal stem cell against sepsis-induced kidney injury. J Pineal Res 57:16\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jpi.12140\u003c/span\u003e\u003cspan address=\"10.1111/jpi.12140\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen KH, Lin KC, Ko SF, Chiang JY, Guo J, Yip HK (2020) Melatonin against acute ischaemic stroke dependently via suppressing both inflammatory and oxidative stress downstream signallings. J Cell Mol Med 24:10402\u0026ndash;10419. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jcmm.15654\u003c/span\u003e\u003cspan address=\"10.1111/jcmm.15654\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeguine J, Barton GM (2014) MyD88: a central player in innate immune signaling. F1000Prime Rep. 6:97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.12703/P6-97\u003c/span\u003e\u003cspan address=\"10.12703/P6-97\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDworsky EM, Hegde V, Loftin AH, Richman S, Hu Y, Lord E, Francis KP, Miller LS, Wang JC, Scaduto A, Bernthal NM (2017) Novel in vivo mouse model of implant related spine infection. J Orthop Res 35:193\u0026ndash;199. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jor.23273\u003c/span\u003e\u003cspan address=\"10.1002/jor.23273\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFantoni M, Trecarichi EM, Rossi B, Mazzotta V, Di Giacomo G, Nasto LA, Di Meco E, Pola E (2012) Epidemiological and clinical features of pyogenic spondylodiscitis. Eur Rev Med Pharmacol Sci 16 Suppl 2:2\u0026ndash;7. https://doi.org/\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGregori F, Grasso G, Iaiani G, Marotta N, Torregrossa F, Landi A (2019) Treatment algorithm for spontaneous spinal infections: A review of the literature. J Craniovertebr Junction Spine 10:3\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4103/jcvjs.JCVJS_115_18\u003c/span\u003e\u003cspan address=\"10.4103/jcvjs.JCVJS_115_18\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarris PNA, Tambyah PA, Lye DC, Mo Y, Lee TH, Yilmaz M, Alenazi TH, Arabi Y, Falcone M, Bassetti M, Righi E, Rogers BA, Kanj S, Bhally H, Iredell J, Mendelson M, Boyles TH, Looke D, Miyakis S, Walls G, Al Khamis M, Zikri A, Crowe A, Ingram P, Daneman N, Griffin P, Athan E, Lorenc P, Baker P, Roberts L, Beatson SA, Peleg AY, Harris-Brown T, Paterson DL (2018) Investigators MT, and the Australasian Society for Infectious Disease Clinical Research N Effect of Piperacillin-Tazobactam vs Meropenem on 30-Day Mortality for Patients With E coli or Klebsiella pneumoniae Bloodstream Infection and Ceftriaxone Resistance: A Randomized Clinical Trial. JAMA 320:984\u0026thinsp;\u0026ndash;\u0026thinsp;94. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1001/jama.2018.12163\u003c/span\u003e\u003cspan address=\"10.1001/jama.2018.12163\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHedlund E (2011) The protective effects of beta-lactam antibiotics in motor neuron disorders. Exp Neurol 231:14\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.expneurol.2011.06.002\u003c/span\u003e\u003cspan address=\"10.1016/j.expneurol.2011.06.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrasnodembskaya A, Samarani G, Song Y, Zhuo H, Su X, Lee JW, Gupta N, Petrini M, Matthay MA (2012) Human mesenchymal stem cells reduce mortality and bacteremia in gram-negative sepsis in mice in part by enhancing the phagocytic activity of blood monocytes. Am J Physiol Lung Cell Mol Physiol 302:L1003\u0026ndash;L1013. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1152/ajplung.00180.2011\u003c/span\u003e\u003cspan address=\"10.1152/ajplung.00180.2011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLamb HM, Ormrod D, Scott LJ, Figgitt DP (2002) Ceftriaxone: an update of its use in the management of community-acquired and nosocomial infections. Drugs 62:1041\u0026ndash;1089. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2165/00003495-200262070-00005\u003c/span\u003e\u003cspan address=\"10.2165/00003495-200262070-00005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLe Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden O (2003) Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 57:11\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1365-3083.2003.01176.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-3083.2003.01176.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLener S, Hartmann S, Barbagallo GMV, Certo F, Thome C, Tschugg A (2018) Management of spinal infection: a review of the literature. Acta Neurochir (Wien) 160:487\u0026ndash;496. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00701-018-3467-2\u003c/span\u003e\u003cspan address=\"10.1007/s00701-018-3467-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMei SH, Haitsma JJ, Dos Santos CC, Deng Y, Lai PF, Slutsky AS, Liles WC, Stewart DJ (2010) Mesenchymal stem cells reduce inflammation while enhancing bacterial clearance and improving survival in sepsis. Am J Respir Crit Care Med 182:1047\u0026ndash;1057. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1164/rccm.201001-0010OC\u003c/span\u003e\u003cspan address=\"10.1164/rccm.201001-0010OC\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOfluoglu EA, Zileli M, Aydin D, Baris YS, Kucukbasmaci O, Gonullu N, Ofluoglu O, Toplamaoglu H (2007) Implant-related infection model in rat spine. Arch Orthop Trauma Surg 127:391\u0026ndash;396. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00402-007-0365-0\u003c/span\u003e\u003cspan address=\"10.1007/s00402-007-0365-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaterson DL, Henderson A, Harris PNA (2020) Current evidence for therapy of ceftriaxone-resistant Gram-negative bacteremia. Curr Opin Infect Dis 33:78\u0026ndash;85. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/QCO.0000000000000623\u003c/span\u003e\u003cspan address=\"10.1097/QCO.0000000000000623\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaja DA, Musharraf SG, Shah MR, Jabbar A, Bhanger MI, Malik MI (2020) Poly (propylene glycol) stabilized gold nanoparticles: an efficient colorimetric assay for ceftriaxone. Journal of Industrial and Engineering Chemistry 87:180-6. https://doi.org/\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSileshi A, Tenna A, Feyissa M, Shibeshi W (2016) Evaluation of ceftriaxone utilization in medical and emergency wards of Tikur Anbessa specialized hospital: a prospective cross-sectional study. BMC Pharmacol Toxicol 17:7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s40360-016-0057-x\u003c/span\u003e\u003cspan address=\"10.1186/s40360-016-0057-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun CK, Yen CH, Lin YC, Tsai TH, Chang LT, Kao YH, Chua S, Fu M, Ko SF, Leu S, Yip HK (2011) Autologous transplantation of adipose-derived mesenchymal stem cells markedly reduced acute ischemia-reperfusion lung injury in a rodent model. J Transl Med 9:118. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1479-5876-9-118\u003c/span\u003e\u003cspan address=\"10.1186/1479-5876-9-118\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSung PH, Chiang HJ, Chen CH, Chen YL, Huang TH, Zhen YY, Chang MW, Liu CF, Chung SY, Chen YL, Chai HT, Sun CK, Yip HK (2016) Combined Therapy With Adipose-Derived Mesenchymal Stem Cells and Ciprofloxacin Against Acute Urogenital Organ Damage in Rat Sepsis Syndrome Induced by Intrapelvic Injection of Cecal Bacteria. Stem Cells Transl Med 5:782\u0026ndash;792. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5966/sctm.2015-0116\u003c/span\u003e\u003cspan address=\"10.5966/sctm.2015-0116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang CC, Sung PH, Chiang JY, Chai HT, Chen CH, Chu YC, Li YC, Yip HK (2021) Combined tacrolimus and melatonin effectively protected kidney against acute ischemia-reperfusion injury. FASEB J 35:e21661. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1096/fj.202100174R\u003c/span\u003e\u003cspan address=\"10.1096/fj.202100174R\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-histology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hijo","sideBox":"Learn more about [Journal of Molecular Histology](https://www.springer.com/journal/10735)","snPcode":"10735","submissionUrl":"https://submission.springernature.com/new-submission/10735/3","title":"Journal of Molecular Histology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"acute spinal cord infection, antibiotics, mesenchymal stem cells, inflammatory reaction","lastPublishedDoi":"10.21203/rs.3.rs-5310715/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5310715/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study tested whether combined ceftriaxone and adipose-derived mesenchymal stem cells (ADMSCs) would defend the spinal cord against acute spinal infection (ASI) in \u003cem\u003erodent\u003c/em\u003e. Adult-Male-\u003cem\u003eSD rats\u003c/em\u003e were grouped into groups 1 (SC)/2 (ASI)/3 (ASI\u0026thinsp;+\u0026thinsp;ceftriaxone from days 2 to 28 after ASI induction)/4 (ASI\u0026thinsp;+\u0026thinsp;allogenic ADMSCs from day 2 for a total of 3 doses/3 consecutive intervals by intravenous injection)/5 (ASS\u0026thinsp;+\u0026thinsp;combined ceftriaxone and ADMSC) and spinal cord tissues were harvested by day 28. Circulatory levels of TNF-α/IL-6 at days 7 and 28, and these two parameters in spinal fluid at day 28 were lowest in group 1, highest in group 2, significantly lower in group 5 than in groups 3/4, and significantly lower in group 3 than in group 4 (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The day-28 bacterial colony formation unit (\u003cem\u003eCFU\u003c/em\u003e) in vertebral bone and circulatory WBC counts at the time points of days 7/14/28, and the protein expressions of upstream (TRL-2/TLR-4/MYD88/TRAF6/IKKα/IKKβ /IKBβ/p-NF-κB) and downstream (IL-1β/IL-6/TNF-α/IFN-γ/iNOS) inflammatory signalings displayed a similar pattern of inflammatory biomarkers in spinal fluid among the groups (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). By day 28, the bone injury score/bone marrow density/ratio of bone volume (BV) to the bone tissue volume (TV)/ratio of bone surface (BS) to BV/ratio of BS to bone TV/trabecular number exhibited an opposite, whereas the trabecular space exhibited an alike pattern of inflammatory biomarkers among the groups (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Combined ceftriaxone and ADMSCs therapy offered an additional benefit on protecting the vertebral bone/spinal cord against ASI damage.\u003c/p\u003e","manuscriptTitle":"Additional benefits of combined ceftriaxone and adipose-derived mesenchymal stem cells on revamping the outcomes in rodent after acute spinal infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-24 13:14:08","doi":"10.21203/rs.3.rs-5310715/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-18T19:18:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-18T17:47:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-14T21:00:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"302538798058705297202147533898540195676","date":"2024-11-08T09:04:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"150277508924925202038824373611140581607","date":"2024-11-06T11:45:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-23T23:39:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-23T23:35:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-22T13:42:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Histology","date":"2024-10-22T09:54:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-histology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hijo","sideBox":"Learn more about [Journal of Molecular Histology](https://www.springer.com/journal/10735)","snPcode":"10735","submissionUrl":"https://submission.springernature.com/new-submission/10735/3","title":"Journal of Molecular Histology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5b248d85-f024-49b0-91a2-cec7a51ad03b","owner":[],"postedDate":"October 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-13T16:01:34+00:00","versionOfRecord":{"articleIdentity":"rs-5310715","link":"https://doi.org/10.1007/s10735-024-10344-9","journal":{"identity":"journal-of-molecular-histology","isVorOnly":false,"title":"Journal of Molecular Histology"},"publishedOn":"2025-01-06 15:56:58","publishedOnDateReadable":"January 6th, 2025"},"versionCreatedAt":"2024-10-24 13:14:08","video":"","vorDoi":"10.1007/s10735-024-10344-9","vorDoiUrl":"https://doi.org/10.1007/s10735-024-10344-9","workflowStages":[]},"version":"v1","identity":"rs-5310715","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5310715","identity":"rs-5310715","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}