Mitochondrial viability in neurogenic bladder urothelium after sigmoidocolocystoplasty. Implications for persistent vesicoureteral reflux

Mitochondrial viability in neurogenic bladder urothelium after sigmoidocolocystoplasty. Implications for persistent vesicoureteral reflux | 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 Mitochondrial viability in neurogenic bladder urothelium after sigmoidocolocystoplasty. Implications for persistent vesicoureteral reflux Kazuto Suda, Rumi Arii, Hongzhao Ma, Takamasa Suzuki, Soichi Shibuya, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4842425/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Aug, 2024 Read the published version in Pediatric Surgery International → Version 1 posted 7 You are reading this latest preprint version Abstract Purpose We investigated whether inflammatory cell infiltration (ICI), fibrosis, and mitochondrial viability of the neurogenic bladder urothelium are involved in the mechanism of persistent vesicoureteral reflux (VUR) after sigmoidocolocystoplasty (SCP). Methods Bladder biopsies obtained 1994–2023 from 62 neurogenic bladder patients were examined by hematoxylin and eosin for ICI, Masson’s trichrome for fibrosis, and immunofluorescence for urothelial growth differentiation factor 15 (GDF15; a mitochondrial stress-responsive cytokine) (positive/negative) and heat shock protein 60 (HSP60; a mitochondrial matrix marker) (strong = > 50%/weak = < 50%) expression. GDF15+/weak HSP60 indicated compromised mitochondrial viability. Cystometry measured neobladder compliance/capacity. Results Mean ages (years) at SCP and bladder biopsies were 9.4 ± 4.6 and 14.2 ± 7.1, respectively. VUR was present in 38/62 patients (51 ureters) at SCP and resolved with SCP alone in 4/38 patients, with SCP and ureteroneocystostomy in 17/38, and persisted in 17/38. Fibrosis was significantly denser in GDF15+ (n = 24)/weak HSP60 (n = 31) compared with GDF15- (n = 38)/strong HSP60 (n = 31) ( p < 0.001 and p < 0.01, respectively). Differences in ICI were significant for GDF15 + versus GDF15- ( p < 0.05) but not for HSP60. Patients with VUR after SCP had higher incidence of GDF15+/weak HSP60 compared with cases without VUR ( p < 0.05 and p < 0.001, respectively). Conclusion Viability of mitochondria appears to be compromised with possible etiologic implications for VUR persisting after SCP. Neurogenic bladder Sigmoidocolocystoplasty Vesicoureteral reflux Mitochondria Growth differentiation factor 15 Heat shock protein 60. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Bladder augmentation cystoplasty (BAC) is performed using segments of gastrointestinal tract (GIT), notably sigmoid colon (sigmoidocolocystoplasty: SCP) to improve bladder compliance and capacity in patients with neurogenic bladder [ 1 , 2 ]. As a consequence of this improvement in bladder compliance after BAC, vesicoureteral reflux (VUR) has been reported to disappear in 66.7% of cases [ 3 ]. Nevertheless, VUR can persist postoperatively, requiring subsequent intervention such as vesicoureteroneostomy. Theories for persistence involving chronic inflammation due to regular physical contact with foreign material during clean intermittent catheterization or indwelling catheterization after BAC, or nitrosamines from urinary tract infection (UTI) or bacteriuria [ 4 , 5 ] have been postulated as well as histopathologic evidence that bladder urothelial interstitial and muscular layer fibrosis and suburothelial inflammatory cell infiltration (ICI) present in patients with neurogenic bladder and a mouse model for neurogenic bladder induced by spinal cord injury model [ 6 – 10 ] can persist after BAC. However, the mechanism by which VUR persists postoperatively involving histopathologic findings in neurogenic bladder urothelial morphology or remodeling of post-BAC urothelium resulting in compromised urothelium after BAC remain largely unknown. The authors hypothesized whether mitochondrial dysfunction may be implicated as mitochondria are important organelles responsible for metabolism and energy production, redox homeostasis, and components necessary for biosynthesis [ 11 , 12 ]. Lima et al. described that impaired mitochondrial function in bladder urothelium associated with chronic inflammation may successively trigger cellular degeneration, hyperplasia, and eventually malignant transformation [ 13 ]. In other words, degeneration of bladder tissue may progress even after BAC if mitochondrial function is compromised and fibrosis mediated by epithelial cells through the synthesis of extracellular matrix [ 14 ] progress. Markers of mitochondrial viability, heat shock protein 60 (HSP60; a mitochondrial matrix/inner membrane marker, known as a potential marker for detecting viable mitochondria and homeostasis of the mitochondrial protein structure [ 15 ] and growth differentiation factor 15 (GDF15; a protein belonging to the Transforming growth factor-β (TGF-β) superfamily that plays a role in the regulation of inflammatory pathways that can be upregulated when mitochondria are damaged [ 16 ] were used to determine if bladder tissue damage including fibrosis and morphologic derangement and persistent VUR after BAC were correlated with mitochondrial dysfunction. Bladder fibrosis has been reported to be dense due to high voiding pressure associated with urinary retention and denervation induced inflammation [ 17 ] in a mouse model for spinal nerve injury with restoration in function being achieved by stimulating mitochondria by antioxidant therapy [ 13 , 18 , 19 ]. Thus, suboptimal mitochondrial viability in urothelial cells could compromise urothelial morphology and contribute to structural issues that could be exacerbated by histopathologic and functional features associated with neurogenic bladder such as increased intravesical pressure. In other words, morphologic remodeling associated with urothelial stress and ICI could contribute to inducing urothelial structural inconsistency that could potentially affect the ureterovesical junction [ 20 , 21 ] and contribute to VUR persisting after BAC. The present study was designed to investigate whether expression of HSP60 and GDF15 in neurogenic bladder urothelium after SCP correlated with bladder histopathologic damage, fibrosis, or persistent VUR. Materials and methods Study design, patient selection, specimen and data collection The present study was designed as a retrospective study of patients with neurogenic bladder due to spina bifida treated by myelomeningocele repair immediately after birth who had SCP between 1990 and 2018 (n = 62) for the indications described in a previous report from the authors’ institute [ 22 ]. All SCP were performed by a team of board-certified staff pediatric surgeons. Bladder specimens obtained from the native bladder remnant around the anastomosis avoiding the bladder trigone [ 10 ] at post-SCP follow-up cystoscopy from each patient between 1994 and 2023 were investigated by H&E staining, Masson’s trichrome (MT) staining, and immunofluorescence. Cystography was performed less than 6 months before routine bladder biopsies for screening of bladder capacity, bladder compliance (mL/cmH 2 O), bladder shape, and presence of VUR in the present study. Patients having SCP and ureteroneocystostomy at the same time (n = 17) were excluded from the assessment of correlations between data and VUR in the present study. Thus, bladder compliance (mL/cmH2O) before/after SCP, preoperative native bladder data, and postoperative neobladder data were evaluated by cystometry and contrast cystography and all perioperative/postoperative management was protocolized [ 10 ]. Histopathologic analyses Sections were taken blindly from bladder specimens embedded in paraffin. H&E staining was performed to assess ICI according to a four-point scale; none, mild ( 50 inflammatory cells per 400x field) and eosinophil infiltration classified as present or absent, as detailed in previous reports [ 23 , 24 ]. Typical images of H&E staining for mild/moderate/severe ICI and eosinophil infiltration are shown in Figs. 1 A-D, respectively. “None” was not identified for any specimens in this study. MT staining was performed to estimate the enhancement of interstitial collagen and the replacement of muscle fibers with fibrous connective tissue. Density of fibrosis was defined using a color deconvolution algorithm to measure the mean intensity of fiber deposition (grey value). Images were digitalized using Fiji/ImageJ software according to a previously published method [ 25 ]. Representative images of MT staining for mild and severe fibrosis are shown in Fig. 2 , respectively. Immunofluorescence was performed on slides using the following primary antibodies according to a previously described technique [ 26 ] and manufacturer protocols: E-cadherin (BD Bioscience, 1:200) to detect urothelial surface adhesion molecules [ 27 ], HSP60 (ab46798, Abcam, 1:200); Bindi et al., Pediatr Surg Int, 2021), and GDF15 (HPA011191, Sigma-Aldrich, 1:200); Arinaga-Hino et al., Sci Rep, 2022). All images were acquired with a BZ-X 710 microscope (KEYENCE, Japan). HSP60 expression was quantified using the ImageJ software Region of Interest (ROI) Manager according to a previous report [ 28 ]. Expression of HSP60 was “strong” if > 50% of the cytoplasm of E-cadherin positive urothelial cells was positive and ‘weak’ if < 50%. The detection of GDF15 was classified as positive or negative. Statistical analyses Differences between two groups and three groups were tested using the unpaired t-test, or ANOVA test, the Chi-square test (n > 5) or Fisher's exact test (n ≤ 5), or Pearson’s correlation test. All statistical analyses were two-sided, and all data were expressed as mean ± standard deviation. A p -value of 0.05 or less was considered statistically significant. Ethical approval This study was approved by the Juntendo University School of Medicine Institutional Review Board and complies with the Declaration of Helsinki of 2008 (Institutional review board number: H20-0397). Results Male to female ratio was 38:24. Mean age at SCP was 9.4±4.6 years; mean age at routine follow-up bladder biopsies was 14.2±7.1 years. At SCP, VUR was present in 38/62 patients (61.3%; 51 ureters) and absent in 24/62 patients. VUR ranged from grades I to V as follows: right (n=15/38 patients; grades I=9, II=2, III=0, IV=3, V=1; 15 ureters), left (n=10/38 patients; grades I=1, II=1, III=5, IV=1, V=2; 10 ureters), and bilateral (n=13/38 patients; right grades I=3, II=0, III=4, IV=5, V=1; left grades I=2, II=1, III=7, IV=2, V=1; 26 ureters) (Fig. 3). After SCP, VUR resolved after SCP alone in 4/38 patients, after SCP and ureteroneocystostomy in 17/38 patients (Cohen procedure=14 and Politano-Leadbetter procedure=3) (Fig. 3) and persisted in 17/38 patients. Grades of persistent VUR ranged from I to V distributed as follows: right (n=8/17; grades I=5, II=3, III=0, IV=0, V=0; 8 ureters), left (n=5/17; grades I=0, II=1, III=3, IV=0, V=1; 5 ureters), and bilateral (n=4/17; right grades I=2, II=0, III=0, IV=2, V=0; left grades I=2, II=0, III=2, IV=0, V=0; 8 ureters). Of these, VUR improved in 3 patients, remained unchanged in 13 patients, and worsened in 1 patient. At the time of writing, persistent VUR had resolved spontaneously in 7 patients, 9 patients required ureteroneocystostomy, and 1 patient is being observed conservatively because there has been no deterioration in renal function. All 17 persistent VUR cases had VUR at SCP and all patients without VUR at SCP remained free from VUR after SCP. Correlations between data and VUR were compared using three groups classified according to the change in VUR status at and after SCP; VUR(-→-) (n=24), VUR(+→-) (n=4), and VUR (+→+) (n=17) (Fig. 3). On comparing data at SCP with data after SCP, there was no correlation between age at SCP, native bladder capacity before SCP, neobladder capacity after SCP, and increased neobladder capacity with respect to persistent VUR (Figs. 4A-D). While preoperative compliance was statistically higher in VUR(-→-) compared with VUR(+→-) and VUR(+→+) ( p <0.05 and p <0.01, respectively) (Fig. 4E), no correlation was seen between postoperative compliance with respect to persistent VUR (Fig. 4F). Mean density of fibrosis (digitalized intensity of MT staining) was 198.1±12.1. Mean ages at SCP/biopsy, neobladder capacity after SCP, increased capacity of the neobladder, postoperative bladder compliance, or the incidence of VUR did not correlate with fibrosis (Table 1). Immunofluorescence for GDF15 was positive (GDF15+) in 24/62 patients and negative (GDF15-) in 38/62 patients (Fig. 5A). HSP60 expression was strong in 31/62 patients and weak in 31/62 patients (Fig. 5B). Neither GDF15 nor HSP60 correlated with mean ages at SCP/biopsy, increased neobladder capacity, neobladder capacity after SCP, or postoperative compliance (Table 1). However, fibrosis was significantly denser in GDF15+ patients compared with GDF15- patients ( p <0.001) (Fig. 5C) and in weak HSP60 patients compared with strong HSP60 patients ( p <0.01) (Fig. 5D). There were significantly more GDF15+ patients in the VUR(+→+) group compared with the VUR(-→-) group ( p <0.05) (Fig. 6A). HSP60 was significantly weaker in the VUR(+→+) group compared with the VUR(-→-) group ( p <0.001) (Figs. 6B-C). There were no specific correlations between GDF15 and HSP60 with respect to individual grades of persistent VUR. Table 2 summarizes data for ICI and eosinophil infiltration. Density of fibrosis did not correlate with ICI or eosinophil infiltration. However, severe ICI was noted in 66.7% of GDF15+ patients compared with 39.5% of GDF15- patients and 58.1% of weak HSP60 patients compared with 41.9% of strong HSP60 patients. Differences were significant for GDF15+ ( p <0.05) but not for HSP60 ( p =0.30). Eosinophil infiltration was present in 54.2% of GDF15+ patients compared with 42.1% of GDF15- and 58.1% of weak HSP60 patients compared with 35.5% of strong HSP60; both were not statistically significant ( p =0.86 and p =0.08, respectively). Discussion The present study demonstrated for the first time that mitochondrial damage in urothelium proven by GDF15+ and weak HSP60 may be implicated in mechanisms causing ICI, fibrosis, and persistent VUR after SCP. This provides novel insight into the evaluation of urothelial damage-induced bladder morphologic remodeling in neurogenic bladder that is independent of the fibrosis. In other words, the density of fibrosis did not correlate with signs of inflammation or functional parameters or the incidence of persistent VUR, despite fibrosis being a common criterion of bladder tissue damage in general. This is of particular interest for the management of neurogenic bladder patients after BAC. Liu et al. reported that mitochondrial morphological and functional abnormalities in mice due to genetic ablation of a gene, Peroxisome proliferator-activated receptor γ, were associated with aberrant urothelial differentiation that can cause lack of structural integrity of the urothelium and also impair urothelial regeneration after bacteria-induced inflammation [29]. Furthermore, abnormal urothelial structure involving the ureterovesical junction in neurogenic bladder and genetic ablation of uroplakin III, a gene for terminally differentiated urothelial cells in a mouse model, are associated with the development of VUR [20, 21, 30]. Collectively, a correlation between aberrant expression of GDF15 and HSP60 in urothelial cells and persistent VUR in the present study may be the outcome of morphologic remodeling based on compromised urothelium. To date, no-one has reported on correlations between GDF15, HSP60, and urothelium, including neurogenic bladder urothelium. Of note, compliance pre-SCP was lower in the VUR(+→-) group and the VUR(+→+) group compared with the VUR(-→-) group indicating a correlation between VUR and neuropathic features of neurogenic bladder, but in patients with persistent VUR after SCP, there was no correlation with postoperative compliance observed. This finding is relevant to the hypothesis of the present study concerning the potential effect of compromised mitochondria viability on the neobladder after SCP as shown by the high rates of GDF15+/weak HSP60 present in mitochondria from patients with persistent VUR. The fact that no differences were found between the VUR(-→-) and VUR(+→-) groups is of academic interest as both groups did not have VUR after SCP and both would be expected to have the same mitochondrial viability. There were also no statistically significant differences between the VUR(+→+) group and the VUR(+→-) group possibly due to the small number of cases in the VUR(+→-) group (n=4). Further research is warranted to clarify these findings. Other limitations include not designating specific sites for follow-up biopsies and lack of comparison with pre-SCP native bladders. At the authors’ institution, during SCP, the apex of the bladder is excised circumferentially along a line approximately 2cm caudal to the upper limit of the bladder trigone. As a result, the histology of the colon cap may be more appropriate for evaluating factors that correlate with functional parameters of the entire neo-bladder after SCP. Interestingly, GDF15 rather than HSP60 was found to be correlated with severe ICI in neurologic bladder urothelium in the present study, which is reasonable as GDF15, is a stress-induced cytokine, belonging to the TGF-β superfamily, closely associated with impaired mitochondrial function [16] as well as data that up-regulation of TGF-β may play an important role in fibrosis mechanisms in the bladder [31]. Furthermore, Costa et al. mentioned that GDF15 from urine sample analysis could be a novel technique for accurate prediction of bladder cancer as an epigenetic biomarker [32], suggesting its potential for detecting activated carcinogenic signaling. However, it is unknown whether the expression of GDF15 in neurogenic bladder patients after SCP may be a useful biomarker for malignant transformation and it is also unclear from the present study whether mitochondrial damage in urothelial cells is time-dependent, as all bladder specimens in the present study were obtained after SCP. Mitochondria status may find application to distinguish patients with neurogenic bladder at risk for urothelial derangement as an indication for more intensive follow-up. Another potential application of the findings of the present study may be improving mitochondrial function for therapeutic reasons such as the antioxidant therapy mentioned earlier reported to mitigate urothelial damage in neurogenic bladder due to spinal cord injury in a mouse model [18]. In conclusion, the present study demonstrated that inflammatory signs, fibrosis, and persistence of VUR after SCP are associated with urothelial cell mitochondrial damage. Declarations Acknowledgments The authors wish to thank the Laboratory of Molecular and Biochemical Research, Research Support Center, Juntendo University Graduate School of Medicine, for technical assistance. This study was supported by Japan Society for the Promotion of Science KAKENHI grants (23K08725) and a University President's Grant for Young Researchers, Juntendo University Grant Numbers YP23-34 and YP23-126. Authors’ contributions KS and RA designed the study. SS, HK, and AY provided conceptual advice. KS, RA, HM, and TS analyzed data. KS wrote the manuscript. GL revised the manuscript as a native English speaker. Conflicts of Interest The authors declare that they have no conflicts of interest. References Koeck I, Burkhard FC, Monastyrskaya K (2016) Activation of common signaling pathways during remodeling of the heart and the bladder. Biochem Pharmacol 102:7–19 Cheng KC, Kan CF, Chu PS, Man CW, Wong BT, Ho LY et al (2015) Augmentation cystoplasty: Urodynamic and metabolic outcomes at 10-year follow-up. 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Supplementary Files Table1.docx Table2.docx Cite Share Download PDF Status: Published Journal Publication published 13 Aug, 2024 Read the published version in Pediatric Surgery International → Version 1 posted Editorial decision: Accepted 02 Aug, 2024 Reviews received at journal 02 Aug, 2024 Reviewers agreed at journal 02 Aug, 2024 Reviewers invited by journal 02 Aug, 2024 Editor assigned by journal 02 Aug, 2024 Submission checks completed at journal 02 Aug, 2024 First submitted to journal 01 Aug, 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4842425","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":335135034,"identity":"487421e5-2700-4062-9457-498c14a287e7","order_by":0,"name":"Kazuto Suda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBACCYYEBgYeBhtmCJcNQhkQoSUNqIWZNC2HGVC04AWS7enPJN7UnGc3uN1/8HFBmQ0Df/sBhuICPFqked6YSc45dpvZ4M5hZuMZ59IYJM4kMBjPwKNFTiKHTZqHDajlRjKbNG8b0IU3GBiMefBqSX8mzfPvHEgL+2+QFnlCWqQlEsyAhh8A28IM0mJASItkzxtjy7l9ycySN5KNpXnOpfEYnklswOsXiePpD2+8+WaXzHcj8eFnnjIbObnjh48Z4wsxGEiGMYBOYmwzJkIHgx0yh/kxMVpGwSgYBaNgxAAAIMhEMyWhm4IAAAAASUVORK5CYII=","orcid":"","institution":"Juntendo University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Kazuto","middleName":"","lastName":"Suda","suffix":""},{"id":335135035,"identity":"93e943c2-7a81-4fa6-9acd-8b1a435d42eb","order_by":1,"name":"Rumi Arii","email":"","orcid":"","institution":"Juntendo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Rumi","middleName":"","lastName":"Arii","suffix":""},{"id":335135036,"identity":"baec0698-cd06-4b8e-9d24-63a648d79348","order_by":2,"name":"Hongzhao Ma","email":"","orcid":"","institution":"Juntendo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hongzhao","middleName":"","lastName":"Ma","suffix":""},{"id":335135037,"identity":"3a4c1719-e6ca-4ed2-b3b1-a36fb4669470","order_by":3,"name":"Takamasa Suzuki","email":"","orcid":"","institution":"Juntendo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Takamasa","middleName":"","lastName":"Suzuki","suffix":""},{"id":335135038,"identity":"e22b57dd-9022-4da1-8e30-27f949ac6133","order_by":4,"name":"Soichi Shibuya","email":"","orcid":"","institution":"Juntendo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Soichi","middleName":"","lastName":"Shibuya","suffix":""},{"id":335135039,"identity":"833955f1-ab8b-47ee-b462-4751efe82f12","order_by":5,"name":"Hiroyuki Koga","email":"","orcid":"","institution":"Juntendo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Koga","suffix":""},{"id":335135040,"identity":"60e00b28-a920-45a5-b1f9-c1d7083cbc10","order_by":6,"name":"Geoffrey J Lane","email":"","orcid":"","institution":"Juntendo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Geoffrey","middleName":"J","lastName":"Lane","suffix":""},{"id":335135041,"identity":"7a3b295c-f496-40fd-9c8c-48944d2a06e1","order_by":7,"name":"Atsuyuki Yamataka","email":"","orcid":"","institution":"Juntendo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Atsuyuki","middleName":"","lastName":"Yamataka","suffix":""}],"badges":[],"createdAt":"2024-08-01 13:13:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4842425/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4842425/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00383-024-05803-z","type":"published","date":"2024-08-13T15:56:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":64146575,"identity":"0983ec63-b0d5-485d-b1d0-07f9a960ba11","added_by":"auto","created_at":"2024-09-08 19:59:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1138925,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInflammatory cell and eosinophil infiltration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B, and C) Typical imaging of inflammatory cell infiltration when mild (A), moderate (B) and severe (C). Inflammatory cells are indicated by arrows. \u0026nbsp;Scale bar: 50μm.\u003c/p\u003e\n\u003cp\u003e(D) Eosinophil infiltration is indicated by arrows. Scale bar: 25μm.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4842425/v1/34012511cda988ae902297b9.jpg"},{"id":64145577,"identity":"9814033b-184f-4602-983d-10ae77d057aa","added_by":"auto","created_at":"2024-09-08 19:51:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":600725,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFibrosis in neurogenic bladder\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative images of MT staining showing mild (left) and severe (right) fibrosis quantified by FIJI/ImageJ software. Scale bar: 50μm.\u003c/p\u003e","description":"","filename":"FIgure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4842425/v1/5dce5ba17020ffee1652b9d6.jpg"},{"id":64145581,"identity":"90481e10-33bb-4051-b145-2a89bf9cd8b5","added_by":"auto","created_at":"2024-09-08 19:51:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":585769,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy flowchart\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4842425/v1/5e768198faacdc46a9fe30e8.jpg"},{"id":64145575,"identity":"ec985b2e-f830-45d5-89d2-052f0622993e","added_by":"auto","created_at":"2024-09-08 19:51:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":943761,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelations between selected data and VUR with respect to SCP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Mean age at SCP versus VUR.\u003c/p\u003e\n\u003cp\u003e(B) Capacity of preoperative bladder versus VUR.\u003c/p\u003e\n\u003cp\u003e(C) Capacity of postoperative bladder versus VUR.\u003c/p\u003e\n\u003cp\u003e(D) Increase in neobladder capacity versus VUR.\u003c/p\u003e\n\u003cp\u003e(E) Preoperative compliance versus VUR. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e(F) Postoperative compliance versus VUR.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4842425/v1/9eb65d91e0e1c337813b02b2.jpg"},{"id":64145580,"identity":"9d1d5873-533b-435a-9bc6-38174955f8f2","added_by":"auto","created_at":"2024-09-08 19:51:17","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1096720,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFibrosis versus GDF15/HSP60\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of GDF15+ (top) and GDF15- (bottom). GDF15 in urothelium is shown with respect to E-cadherin positive urothelial cells and DAPI staining. Yellow arrows indicate GDF15+ cells. Scale bar: 50μm.\u003c/p\u003e\n\u003cp\u003e(B) Representative images of strong HSP60 (top) and weak HSP60 (bottom) in urothelium shown with respect to E-cadherin-positive urothelial cells and DAPI staining. Scale bar: 50μm.\u003c/p\u003e\n\u003cp\u003e(C) Density of fibrosis quantified by FIJI/ImageJ software after MT staining versus GDF15. ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e(D) Density of fibrosis quantified by FIJI/ImageJ software after MT staining versus HSP60. **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4842425/v1/f8c7c66e365d5cf9c2bd95e9.jpg"},{"id":64145579,"identity":"28d583fc-22f0-4b35-bad2-950cbeab7da8","added_by":"auto","created_at":"2024-09-08 19:51:17","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":730479,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVUR versus GDF15 and VUR versus HSP60\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003e(B) ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e(C) Scatter plot showing quantified HSP60/E-cadherin (%) in urothelium versus VUR for each patient.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4842425/v1/b06a86175b980507d50fd8c1.jpg"},{"id":64146579,"identity":"35d00f30-439f-4dc1-8770-623b92fac15e","added_by":"auto","created_at":"2024-09-08 19:59:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5514776,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4842425/v1/344a9f9b-bdbf-4702-b6ac-c7644081aa10.pdf"},{"id":64146576,"identity":"b7c062a6-5d39-4072-b14c-db319028e874","added_by":"auto","created_at":"2024-09-08 19:59:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":60108,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4842425/v1/07badb91ac0a4da43bf1bf4b.docx"},{"id":64145574,"identity":"43fe3b5c-6bf1-4825-a35f-cd4414d72198","added_by":"auto","created_at":"2024-09-08 19:51:17","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":54915,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4842425/v1/329afcae314da9914dc4fe76.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mitochondrial viability in neurogenic bladder urothelium after sigmoidocolocystoplasty. Implications for persistent vesicoureteral reflux","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBladder augmentation cystoplasty (BAC) is performed using segments of gastrointestinal tract (GIT), notably sigmoid colon (sigmoidocolocystoplasty: SCP) to improve bladder compliance and capacity in patients with neurogenic bladder [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. As a consequence of this improvement in bladder compliance after BAC, vesicoureteral reflux (VUR) has been reported to disappear in 66.7% of cases [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Nevertheless, VUR can persist postoperatively, requiring subsequent intervention such as vesicoureteroneostomy. Theories for persistence involving chronic inflammation due to regular physical contact with foreign material during clean intermittent catheterization or indwelling catheterization after BAC, or nitrosamines from urinary tract infection (UTI) or bacteriuria [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] have been postulated as well as histopathologic evidence that bladder urothelial interstitial and muscular layer fibrosis and suburothelial inflammatory cell infiltration (ICI) present in patients with neurogenic bladder and a mouse model for neurogenic bladder induced by spinal cord injury model [\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] can persist after BAC. However, the mechanism by which VUR persists postoperatively involving histopathologic findings in neurogenic bladder urothelial morphology or remodeling of post-BAC urothelium resulting in compromised urothelium after BAC remain largely unknown.\u003c/p\u003e \u003cp\u003eThe authors hypothesized whether mitochondrial dysfunction may be implicated as mitochondria are important organelles responsible for metabolism and energy production, redox homeostasis, and components necessary for biosynthesis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Lima et al. described that impaired mitochondrial function in bladder urothelium associated with chronic inflammation may successively trigger cellular degeneration, hyperplasia, and eventually malignant transformation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In other words, degeneration of bladder tissue may progress even after BAC if mitochondrial function is compromised and fibrosis mediated by epithelial cells through the synthesis of extracellular matrix [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] progress. Markers of mitochondrial viability, heat shock protein 60 (HSP60; a mitochondrial matrix/inner membrane marker, known as a potential marker for detecting viable mitochondria and homeostasis of the mitochondrial protein structure [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and growth differentiation factor 15 (GDF15; a protein belonging to the Transforming growth factor-β (TGF-β) superfamily that plays a role in the regulation of inflammatory pathways that can be upregulated when mitochondria are damaged [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] were used to determine if bladder tissue damage including fibrosis and morphologic derangement and persistent VUR after BAC were correlated with mitochondrial dysfunction.\u003c/p\u003e \u003cp\u003eBladder fibrosis has been reported to be dense due to high voiding pressure associated with urinary retention and denervation induced inflammation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] in a mouse model for spinal nerve injury with restoration in function being achieved by stimulating mitochondria by antioxidant therapy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thus, suboptimal mitochondrial viability in urothelial cells could compromise urothelial morphology and contribute to structural issues that could be exacerbated by histopathologic and functional features associated with neurogenic bladder such as increased intravesical pressure. In other words, morphologic remodeling associated with urothelial stress and ICI could contribute to inducing urothelial structural inconsistency that could potentially affect the ureterovesical junction [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and contribute to VUR persisting after BAC.\u003c/p\u003e \u003cp\u003eThe present study was designed to investigate whether expression of HSP60 and GDF15 in neurogenic bladder urothelium after SCP correlated with bladder histopathologic damage, fibrosis, or persistent VUR.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design, patient selection, specimen and data collection\u003c/h2\u003e \u003cp\u003eThe present study was designed as a retrospective study of patients with neurogenic bladder due to spina bifida treated by myelomeningocele repair immediately after birth who had SCP between 1990 and 2018 (n\u0026thinsp;=\u0026thinsp;62) for the indications described in a previous report from the authors\u0026rsquo; institute [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. All SCP were performed by a team of board-certified staff pediatric surgeons. Bladder specimens obtained from the native bladder remnant around the anastomosis avoiding the bladder trigone [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] at post-SCP follow-up cystoscopy from each patient between 1994 and 2023 were investigated by H\u0026amp;E staining, Masson\u0026rsquo;s trichrome (MT) staining, and immunofluorescence. Cystography was performed less than 6 months before routine bladder biopsies for screening of bladder capacity, bladder compliance (mL/cmH\u003csub\u003e2\u003c/sub\u003eO), bladder shape, and presence of VUR in the present study.\u003c/p\u003e \u003cp\u003ePatients having SCP and ureteroneocystostomy at the same time (n\u0026thinsp;=\u0026thinsp;17) were excluded from the assessment of correlations between data and VUR in the present study. Thus, bladder compliance (mL/cmH2O) before/after SCP, preoperative native bladder data, and postoperative neobladder data were evaluated by cystometry and contrast cystography and all perioperative/postoperative management was protocolized [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHistopathologic analyses\u003c/h2\u003e \u003cp\u003eSections were taken blindly from bladder specimens embedded in paraffin. H\u0026amp;E staining was performed to assess ICI according to a four-point scale; none, mild (\u0026lt;\u0026thinsp;10 inflammatory cells per 400x field), moderate (10\u0026ndash;50 inflammatory cells per 400x field), severe (\u0026gt;\u0026thinsp;50 inflammatory cells per 400x field) and eosinophil infiltration classified as present or absent, as detailed in previous reports [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Typical images of H\u0026amp;E staining for mild/moderate/severe ICI and eosinophil infiltration are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D, respectively. \u0026ldquo;None\u0026rdquo; was not identified for any specimens in this study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMT staining was performed to estimate the enhancement of interstitial collagen and the replacement of muscle fibers with fibrous connective tissue. Density of fibrosis was defined using a color deconvolution algorithm to measure the mean intensity of fiber deposition (grey value). Images were digitalized using Fiji/ImageJ software according to a previously published method [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Representative images of MT staining for mild and severe fibrosis are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunofluorescence was performed on slides using the following primary antibodies according to a previously described technique [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and manufacturer protocols: E-cadherin (BD Bioscience, 1:200) to detect urothelial surface adhesion molecules [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], HSP60 (ab46798, Abcam, 1:200); Bindi et al., Pediatr Surg Int, 2021), and GDF15 (HPA011191, Sigma-Aldrich, 1:200); Arinaga-Hino et al., Sci Rep, 2022). All images were acquired with a BZ-X 710 microscope (KEYENCE, Japan). HSP60 expression was quantified using the ImageJ software Region of Interest (ROI) Manager according to a previous report [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Expression of HSP60 was \u0026ldquo;strong\u0026rdquo; if\u0026thinsp;\u0026gt;\u0026thinsp;50% of the cytoplasm of E-cadherin positive urothelial cells was positive and \u0026lsquo;weak\u0026rsquo; if\u0026thinsp;\u0026lt;\u0026thinsp;50%. The detection of GDF15 was classified as positive or negative.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eDifferences between two groups and three groups were tested using the unpaired t-test, or ANOVA test, the Chi-square test (n\u0026thinsp;\u0026gt;\u0026thinsp;5) or Fisher's exact test (n\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;5), or Pearson\u0026rsquo;s correlation test. All statistical analyses were two-sided, and all data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. A \u003cem\u003ep\u003c/em\u003e-value of 0.05 or less was considered statistically significant.\u003c/p\u003e \u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Juntendo University School of Medicine Institutional Review Board and complies with the Declaration of Helsinki of 2008 (Institutional review board number: H20-0397).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eMale to female ratio was 38:24. \u0026nbsp;Mean age at SCP was 9.4\u0026plusmn;4.6 years; mean age at routine\u0026nbsp;follow-up bladder biopsies was 14.2\u0026plusmn;7.1 years. \u0026nbsp; At SCP, VUR was present in 38/62 patients (61.3%; 51 ureters) and absent in 24/62 patients. VUR ranged from grades I to V as follows: right (n=15/38 patients; grades I=9, II=2, III=0, IV=3, V=1; 15 ureters), left (n=10/38 patients; grades I=1, II=1, III=5, IV=1, V=2; 10 ureters), and bilateral (n=13/38 patients; right grades I=3, II=0, III=4, IV=5, V=1; left grades I=2, II=1, III=7, IV=2, V=1; 26 ureters)\u0026nbsp;(Fig. 3). \u0026nbsp;After\u0026nbsp;SCP, VUR resolved after SCP alone in 4/38 patients, after SCP and ureteroneocystostomy in 17/38 patients (Cohen procedure=14 and Politano-Leadbetter procedure=3) (Fig. 3) and persisted in 17/38 patients. \u0026nbsp; Grades of persistent VUR ranged from I to V distributed as follows: right (n=8/17; grades I=5, II=3, III=0, IV=0, V=0; 8 ureters), left (n=5/17; grades I=0, II=1,\u0026nbsp;III=3, IV=0, V=1; 5 ureters), and bilateral (n=4/17; right grades I=2, II=0, III=0, IV=2, V=0; left grades I=2, II=0, III=2, IV=0, V=0; 8 ureters). \u0026nbsp;Of these, VUR improved in 3 patients, remained unchanged in 13 patients, and worsened in 1 patient. \u0026nbsp;At the time of writing, persistent VUR had resolved spontaneously in 7 patients, 9 patients required ureteroneocystostomy, and 1 patient is being observed conservatively because there has been no deterioration in renal function. \u0026nbsp;All 17 persistent VUR cases had VUR at SCP and all patients without VUR at SCP remained free from VUR after SCP.\u003c/p\u003e\n\u003cp\u003eCorrelations between data and VUR were compared using three groups classified according to the change in VUR status at and after SCP; VUR(-\u0026rarr;-) (n=24), VUR(+\u0026rarr;-) (n=4), and VUR (+\u0026rarr;+) (n=17) (Fig. 3). \u0026nbsp;On comparing data at SCP with data after SCP, there was no correlation between age at SCP, native bladder capacity before SCP, neobladder capacity after SCP, and increased neobladder capacity with respect to persistent VUR (Figs. 4A-D). \u0026nbsp;While preoperative compliance was statistically higher in VUR(-\u0026rarr;-) compared with VUR(+\u0026rarr;-) and VUR(+\u0026rarr;+) (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, respectively) (Fig. 4E), no correlation was seen between postoperative compliance with respect to persistent VUR (Fig. 4F).\u003c/p\u003e\n\u003cp\u003eMean\u0026nbsp;density of fibrosis (digitalized intensity of MT staining) was 198.1\u0026plusmn;12.1. \u0026nbsp;Mean ages at SCP/biopsy, neobladder capacity after SCP, increased capacity of the neobladder, postoperative bladder compliance, or the incidence of VUR did not correlate with fibrosis (Table 1). \u0026nbsp;Immunofluorescence for GDF15 was positive (GDF15+) in 24/62 patients and negative\u0026nbsp;(GDF15-) in 38/62 patients (Fig. 5A). \u0026nbsp;HSP60 expression was strong in 31/62 patients and weak in 31/62 patients (Fig. 5B). \u0026nbsp;Neither GDF15 nor HSP60 correlated with mean ages at SCP/biopsy, increased neobladder capacity, neobladder capacity after SCP, or postoperative\u0026nbsp;compliance (Table 1). \u0026nbsp;However, fibrosis\u0026nbsp;was significantly denser in GDF15+ patients compared with GDF15- patients (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001) (Fig. 5C) and in weak HSP60 patients compared with strong HSP60 patients (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01) (Fig. 5D). \u0026nbsp; There were significantly more GDF15+ patients in the VUR(+\u0026rarr;+) group compared with the VUR(-\u0026rarr;-) group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) (Fig. 6A). \u0026nbsp;HSP60 was significantly weaker in the VUR(+\u0026rarr;+) group compared with the VUR(-\u0026rarr;-) group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001) (Figs. 6B-C). \u0026nbsp; There were no specific correlations between GDF15 and HSP60 with\u0026nbsp;respect to individual grades of persistent VUR.\u003c/p\u003e\n\u003cp\u003eTable 2 summarizes data for ICI and eosinophil infiltration.\u0026nbsp;\u0026nbsp;Density of fibrosis did not correlate with ICI or eosinophil infiltration. \u0026nbsp;However, severe ICI was noted in 66.7% of GDF15+ patients compared with 39.5% of GDF15- patients and 58.1% of weak HSP60 patients compared with 41.9% of strong HSP60 patients. \u0026nbsp; Differences were significant for GDF15+ (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) but not for HSP60 (\u003cem\u003ep\u003c/em\u003e=0.30). \u0026nbsp;Eosinophil infiltration was present in 54.2% of GDF15+ patients compared with 42.1% of GDF15- and 58.1% of weak HSP60 patients compared with 35.5% of strong HSP60; both were not statistically significant (\u003cem\u003ep\u003c/em\u003e=0.86 and \u003cem\u003ep\u003c/em\u003e=0.08, respectively).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study demonstrated for the first time that mitochondrial damage in urothelium proven by GDF15+ and weak HSP60 may be implicated in mechanisms causing ICI, fibrosis, and persistent VUR after SCP. This provides novel insight into the evaluation of urothelial damage-induced bladder morphologic remodeling in neurogenic bladder that is independent of the fibrosis. In other words, the density of fibrosis did not correlate with signs of inflammation or functional parameters or the incidence of persistent VUR, despite fibrosis being a common criterion of bladder tissue damage in general. This is of particular interest for the management of neurogenic bladder patients after BAC. \u003c/p\u003e\n\u003cp\u003eLiu et al. reported that mitochondrial morphological and functional abnormalities in mice due to genetic ablation of a gene, Peroxisome proliferator-activated receptor \u0026gamma;, were associated with aberrant urothelial differentiation that can cause lack of structural integrity of the urothelium and also impair urothelial regeneration after bacteria-induced inflammation [29]. Furthermore, abnormal urothelial structure involving the ureterovesical junction in neurogenic bladder and genetic ablation of uroplakin III, a gene for terminally differentiated urothelial cells in a mouse model, are associated with the development of VUR [20, 21, 30]. Collectively, a correlation between aberrant expression of GDF15 and HSP60 in urothelial cells and persistent VUR in the present study may be the outcome of morphologic remodeling based on compromised urothelium.\u003c/p\u003e\n\u003cp\u003eTo date, no-one has reported on correlations between GDF15, HSP60, and urothelium, including neurogenic bladder urothelium. Of note, compliance pre-SCP was lower in the VUR(+\u0026rarr;-) group and the VUR(+\u0026rarr;+) group compared with the VUR(-\u0026rarr;-) group indicating a correlation between VUR and neuropathic features of neurogenic bladder, but in patients with persistent VUR after SCP, there was no correlation with postoperative compliance observed. This finding is relevant to the hypothesis of the present study concerning the potential effect of compromised mitochondria viability on the neobladder after SCP as shown by the high rates of GDF15+/weak HSP60 present in mitochondria from patients with persistent VUR. The fact that no differences were found between the VUR(-\u0026rarr;-) and VUR(+\u0026rarr;-) groups is of academic interest as both groups did not have VUR after SCP and both would be expected to have the same mitochondrial viability. There were also no statistically significant differences between the VUR(+\u0026rarr;+) group and the VUR(+\u0026rarr;-) group possibly due to the small number of cases in the VUR(+\u0026rarr;-) group (n=4). Further research is warranted to clarify these findings. Other limitations include not designating specific sites for follow-up biopsies and lack of comparison with pre-SCP native bladders. At the authors\u0026rsquo; institution, during SCP, the apex of the bladder is excised circumferentially along a line approximately 2cm caudal to the upper limit of the bladder trigone. As a result, the histology of the colon cap may be more appropriate for evaluating factors that correlate with functional parameters of the entire neo-bladder after SCP. \u003c/p\u003e\n\u003cp\u003eInterestingly, GDF15 rather than HSP60 was found to be correlated with severe ICI in neurologic bladder urothelium in the present study, which is reasonable as GDF15, is a stress-induced cytokine, belonging to the TGF-\u0026beta; superfamily, closely associated with impaired mitochondrial function [16] as well as data that up-regulation of TGF-\u0026beta; may play an important role in fibrosis mechanisms in the bladder [31]. Furthermore, Costa et al. mentioned that GDF15 from urine sample analysis could be a novel technique for accurate prediction of bladder cancer as an epigenetic biomarker [32], suggesting its potential for detecting activated carcinogenic signaling. However, it is unknown whether the expression of GDF15 in neurogenic bladder patients after SCP may be a useful biomarker for malignant transformation and it is also unclear from the present study whether mitochondrial damage in urothelial cells is time-dependent, as all bladder specimens in the present study were obtained after SCP. \u003c/p\u003e\n\u003cp\u003eMitochondria status may find application to distinguish patients with neurogenic bladder at risk for urothelial derangement as an indication for more intensive follow-up. Another potential application of the findings of the present study may be improving mitochondrial function for therapeutic reasons such as the antioxidant therapy mentioned earlier reported to mitigate urothelial damage in neurogenic bladder due to spinal cord injury in a mouse model [18]. \u003c/p\u003e\n\u003cp\u003eIn conclusion, the present study demonstrated that inflammatory signs, fibrosis, and persistence of VUR after SCP are associated with urothelial cell mitochondrial damage.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank the Laboratory of Molecular and Biochemical Research, Research Support Center, Juntendo University Graduate School of Medicine, for technical assistance. \u0026nbsp;This study was supported by Japan Society for the Promotion of Science KAKENHI grants (23K08725) and\u0026nbsp;a University President's Grant for Young Researchers, Juntendo University Grant Numbers YP23-34 and YP23-126.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKS and RA designed the study. \u0026nbsp;SS, HK, and AY provided conceptual advice. \u0026nbsp;KS, RA, HM, and TS analyzed data. \u0026nbsp;KS wrote the manuscript. \u0026nbsp;GL revised the manuscript as a native English speaker.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKoeck I, Burkhard FC, Monastyrskaya K (2016) Activation of common signaling pathways during remodeling of the heart and the bladder. Biochem Pharmacol 102:7\u0026ndash;19\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng KC, Kan CF, Chu PS, Man CW, Wong BT, Ho LY et al (2015) Augmentation cystoplasty: Urodynamic and metabolic outcomes at 10-year follow-up. Int J Urol 22(12):1149\u0026ndash;1154\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlsowayan O, Barham A, Alqarni N, Alnazari M, Alsakharni M, Ewida T, Trbay M, Elderwy AA, Alhazmi H, Neel KF (2015) Outcomes of a minimally invasive surgical approach to manage persistent high-grade vesicoureteric reflux post successful augmentation cystoplasty of patients with non-compliant bladder. J Pediatr Urol Apr 11(2):60\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuda K, Matsumoto Y, Ochi T, Koga H, Lane GJ, Hattori N et al (2022) Successful engraftment of bladder organoids in de-epithelialized mouse colon. Pediatr Surg Int 39(1):14\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBraasch MR, Griffith TS, Cooper CS, Austin JC (2011) Description of a novel murine model for ileocystoplasty and early histologic changes. ScientificWorldJournal 11:1325\u0026ndash;1331\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun XG, Li YX, Ji LF, Xu JL, Chen WX, Wang RY (2022) Outcomes of seromuscular bladder augmentation compared with standard bladder augmentation in the treatment of children with neurogenic bladder. World J Clin Cases 10(23):8115\u0026ndash;8123\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Huang J, Chen L, Ren W, Cai W (2022) Human umbilical cord mesenchymal stem cells contribute to the reconstruction of bladder function after acute spinal cord injury via p38 mitogen-activated protein kinase/nuclear factor-kappa B pathway. Bioengineered 13(3):4844\u0026ndash;4856\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKispal ZF, Kardos D, Jilling T, Kereskai L, Isaacs M, Balogh DL et al (2015) Long-term histological and mucin alterations in the neobladder mucosa following urinary bladder augmentation or substitution with gastrointestinal segment. J Pediatr Urol 11(6):349e1\u0026ndash;349e6\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVajda P, Kaiser L, Magyarlaki T, Farkas A, Vastyan AM, Pinter AB (2002) Histological findings after colocystoplasty and gastrocystoplasty. J Urol 168(2):698\u0026ndash;701 discussion\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyano T, Yamataka A, Iwashita K, Morioka A, Lane GJ, Kobayashi H et al (2000) Histology of the neobladder mucosa after sigmoidocolocystoplasty. J Pediatr Surg 35(1):104\u0026ndash;108\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang CC, Liu HY, Hsu TW, Lee WC (2022) Updates on the Pivotal Roles of Mitochondria in Urothelial Carcinoma. Biomedicines 10(10)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAuger C, Alhasawi A, Contavadoo M, Appanna VD (2015) Dysfunctional mitochondrial bioenergetics and the pathogenesis of hepatic disorders. Front Cell Dev 3:40\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLima TRR, de Oliveira Lima E, Delafiori J, Ramos Catharino R, Viana de Camargo JL, Pereira LC (2022) Molecular signatures associated with diuron exposure on rat urothelial mitochondria. Toxicol Mech Methods 32(8):628\u0026ndash;635\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaskin L, Howard PS, Macarak E (1993) Effect of physical forces on bladder smooth muscle and urothelium. J Urol 150(2 Pt 2):601\u0026ndash;607\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBindi E, Alganabi M, Biouss G, Liu J, Li B, Miyake H et al (2021) Hepatic oxidative injury: role of mitochondrial dysfunction in necrotizing enterocolitis. Pediatr Surg Int 37(3):325\u0026ndash;332\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArinaga-Hino T, Ide T, Akiba J, Suzuki H, Kuwahara R, Amano K et al (2022) Growth differentiation factor 15 as a novel diagnostic and therapeutic marker for autoimmune hepatitis. Sci Rep 12(1):8759\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Li S, Wang Y, Shang A (2022) Functional, morphological and molecular characteristics in a novel rat model of spinal sacral nerve injury-surgical approach, pathological process and clinical relevance. Sci Rep 12(1):10026\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKullmann AF, Truschel ST, Wolf-Johnston AS, McDonnell BM, Lynn AM, Kanai AJ et al (2019) Acute spinal cord injury is associated with mitochondrial dysfunction in mouse urothelium. Neurourol Urodyn 38(6):1551\u0026ndash;1559\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKullmann FA, McDonnell BM, Wolf-Johnston AS, Kanai AJ, Shiva S, Chelimsky T et al (2019) Stress-induced autonomic dysregulation of mitochondrial function in the rat urothelium. Neurourol Urodyn 38(2):572\u0026ndash;581\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eda Silva CMP, Vasconcelos MMA, Lima EM, de Bessa JJ, Reis OAF, Filgueiras M et al (2023) Dynamic and static ultrasound features predictive of vesicoureteral reflux and renal damage in children and adolescents with neurogenic bladder. Int Braz J Urol 49(6):700\u0026ndash;715\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalayeh K, Fowlkes JB, Schultz WW, Sack BS (2020) Ureterovesical junction deformation during urine storage in the bladder and the effect on vesicoureteral reflux. J Biomech 113:110123\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayashi Y, Nishimura E, Shimizu S, Miyano G, Okawada M, Nagae I et al (2017) Sigmoidocolocystoplasty for neurogenic bladder reviewed after 20 years. J Pediatr Surg 52(12):2070\u0026ndash;2073\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJhang JF, Hsu YH, Jiang YH, Ho HC, Kuo HC (2021) Clinical Relevance of Bladder Histopathological Findings and Their Impact on Treatment Outcomes among Patients with Interstitial Cystitis/Bladder Pain Syndrome: An Investigation of the European Society for the Study of Interstitial Cystitis Histopathological Classification. J Urol 205(1):226\u0026ndash;235\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang YH, Jhang JF, Hsu YH, Kuo HC (2022) Usefulness of Urinary Biomarkers for Assessing Bladder Condition and Histopathology in Patients with Interstitial Cystitis/Bladder Pain Syndrome. Int J Mol Sci 23(19)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrowe AR, Yue W (2019) Semi-quantitative Determination of Protein Expression using Immunohistochemistry Staining and Analysis: An Integrated Protocol. Bio Protoc 9(24)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuda K, Okabe A, Matsuo J, Chuang LSH, Li Y, Jangphattananont N et al (2024) Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer. Cancer Res Commun 4(2):279\u0026ndash;292\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZwaans BMM, Carabulea AL, Bartolone SN, Ward EP, Chancellor MB, Lamb LE (2021) Voiding defects in acute radiation cystitis driven by urothelial barrier defect through loss of E-cadherin, ZO-1 and Uroplakin III. Sci Rep 11(1):19277\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarangi U, Singh MK, Abhijnya KV, Reddy LP, Prasad BS, Pitke VV et al (2013) Hsp60 chaperonin acts as barrier to pharmacologically induced oxidative stress mediated apoptosis in tumor cells with differential stress response. Drug Target Insights 7:35\u0026ndash;51\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu C, Tate T, Batourina E, Truschel ST, Potter S, Adam M et al (2019) Pparg promotes differentiation and regulates mitochondrial gene expression in bladder epithelial cells. Nat Commun 10(1):4589\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu P, Deng FM, Liang FX, Hu CM, Auerbach AB, Shapiro E et al (2000) Ablation of uroplakin III gene results in small urothelial plaques, urothelial leakage, and vesicoureteral reflux. J Cell Biol 151(5):961\u0026ndash;972\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJhang JF, Birder LA, Kuo HC (2023) Pathophysiology, clinical presentation, and management of ketamine-induced cystitis. Tzu Chi Med J 35(3):205\u0026ndash;212\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosta VL, Henrique R, Danielsen SA, Duarte-Pereira S, Eknaes M, Skotheim RI et al (2010) Three epigenetic biomarkers, GDF15, TMEFF2, and VIM, accurately predict bladder cancer from DNA-based analyses of urine samples. Clin Cancer Res 16(23):5842\u0026ndash;5851\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1-2 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":"pediatric-surgery-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pesi","sideBox":"Learn more about [Pediatric Surgery International](http://link.springer.com/journal/383)","snPcode":"383","submissionUrl":"https://submission.nature.com/new-submission/383/3","title":"Pediatric Surgery International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Neurogenic bladder, Sigmoidocolocystoplasty, Vesicoureteral reflux, Mitochondria, Growth differentiation factor 15, Heat shock protein 60.","lastPublishedDoi":"10.21203/rs.3.rs-4842425/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4842425/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003ePurpose\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe investigated whether inflammatory cell infiltration (ICI), fibrosis, and mitochondrial viability of the neurogenic bladder urothelium are involved in the mechanism of persistent vesicoureteral reflux (VUR) after sigmoidocolocystoplasty (SCP).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBladder biopsies obtained 1994\u0026ndash;2023 from 62 neurogenic bladder patients were examined by hematoxylin and eosin for ICI, Masson\u0026rsquo;s trichrome for fibrosis, and immunofluorescence for urothelial growth differentiation factor 15 (GDF15; a mitochondrial stress-responsive cytokine) (positive/negative) and heat shock protein 60 (HSP60; a mitochondrial matrix marker) (strong\u0026thinsp;=\u0026thinsp;\u0026gt;\u0026thinsp;50%/weak\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;50%) expression. GDF15+/weak HSP60 indicated compromised mitochondrial viability. Cystometry measured neobladder compliance/capacity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMean ages (years) at SCP and bladder biopsies were 9.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6 and 14.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1, respectively. VUR was present in 38/62 patients (51 ureters) at SCP and resolved with SCP alone in 4/38 patients, with SCP and ureteroneocystostomy in 17/38, and persisted in 17/38. Fibrosis was significantly denser in GDF15+ (n\u0026thinsp;=\u0026thinsp;24)/weak HSP60 (n\u0026thinsp;=\u0026thinsp;31) compared with GDF15- (n\u0026thinsp;=\u0026thinsp;38)/strong HSP60 (n\u0026thinsp;=\u0026thinsp;31) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively). Differences in ICI were significant for GDF15\u0026thinsp;+\u0026thinsp;versus GDF15- (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but not for HSP60. Patients with VUR after SCP had higher incidence of GDF15+/weak HSP60 compared with cases without VUR (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively).\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eViability of mitochondria appears to be compromised with possible etiologic implications for VUR persisting after SCP.\u003c/p\u003e","manuscriptTitle":"Mitochondrial viability in neurogenic bladder urothelium after sigmoidocolocystoplasty. Implications for persistent vesicoureteral reflux","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-08 19:51:12","doi":"10.21203/rs.3.rs-4842425/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2024-08-02T09:10:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-02T09:06:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"340165079514415562491512844803786995236","date":"2024-08-02T09:05:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-02T09:02:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-02T09:02:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-02T08:07:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pediatric Surgery International","date":"2024-08-01T13:11:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pediatric-surgery-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pesi","sideBox":"Learn more about [Pediatric Surgery International](http://link.springer.com/journal/383)","snPcode":"383","submissionUrl":"https://submission.nature.com/new-submission/383/3","title":"Pediatric Surgery International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d92303c7-96c7-48d9-8b83-dbce2573ed48","owner":[],"postedDate":"September 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-08T19:51:12+00:00","versionOfRecord":{"articleIdentity":"rs-4842425","link":"https://doi.org/10.1007/s00383-024-05803-z","journal":{"identity":"pediatric-surgery-international","isVorOnly":false,"title":"Pediatric Surgery International"},"publishedOn":"2024-08-13 15:56:57","publishedOnDateReadable":"August 13th, 2024"},"versionCreatedAt":"2024-09-08 19:51:12","video":"","vorDoi":"10.1007/s00383-024-05803-z","vorDoiUrl":"https://doi.org/10.1007/s00383-024-05803-z","workflowStages":[]},"version":"v1","identity":"rs-4842425","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4842425","identity":"rs-4842425","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}