The Progression of Intracerebral Hemorrhage (ICH) is Related to the Expression of Integrin Β1 (ITGB1)

The Progression of Intracerebral Hemorrhage (ICH) is Related to the Expression of Integrin Β1 (ITGB1) | 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 The Progression of Intracerebral Hemorrhage (ICH) is Related to the Expression of Integrin Β1 (ITGB1) Hai-Yang Ma, Yan Xu, Chun-You Qiao, Yi Peng, Qi Ding, Li-Zhong Wang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-45466/v3 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Mar, 2021 Read the published version in Chinese Neurosurgical Journal → Version 3 posted You are reading this latest preprint version Show more versions Abstract Background: Intracerebral hemorrhage (ICH) is fatal and detrimental to quality of life. Clinically, options for monitoring are often limited, potentially missing subtle neurological changes. Integrin β 1 (ITGB1) and β 3 (ITGB3) are the main components of integrin family receptors, which regulate the formation and stability of blood vessels. This study explored the relationship between expression of ITGB1, ITGB3 in intracerebral hemorrhage (ICH) to analyze their functional and clinical relevance. Methods: The expression of ITGB1 and ITGB3 in ICH was accomplished by immunohistochemical (IHC) staining and western blotting (WB) analysis, respectively. Results: Furthermore, the results demonstrated that ITGB1 was expressed in ICH tissues, but ITGB3 was not expressed in ICH tissues. Conclusions: In summary, the clinical progression of ICH was related to the expression of ITGB1. ITGB1 may be a potential biomarker and contribute to the treatment of ICH. Neurosurgery Biomarker Immunohistochemical staining Inflammation Integrins Intracerebral hemorrhage Western blotting Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Intracerebral hemorrhage (ICH) is a critical type of cerebrovascular disease with a high incidence. 1 Although the treatment strategy of ICH in acute phase has become more and more standardized, it is still limited to hematoma clearance and symptomatic supportive treatment. 2, 3 At present, there is no effective treatment for the loss of neurological function caused by cerebral hemorrhage, so the mortality and disability rate are still high in the past ten years. 4 It is known that the injury mechanism of intracerebral hemorrhage indicated that the composition, structure and distribution of extracellular matrix (ECM) have changed. 5 In addition, the destruction of blood-brain barrier (BBB) and the formation of brain edema are closely related to ECM. 6, 7 Previous studies have confirmed that ECM is involved in the formation, development, repair and regeneration of embryos and various tissues and organs. 8, 9 The integrins are a superfamily of cell adhesion receptors that bind to ECM ligands, cell-surface ligands, and soluble ligands. 10 ECM participates in the interaction between cells through the mediation of integrins. 11 Once the nervous system is injured, integrins can affect nerve regeneration by promoting the survival of neurons, regulating the length of nerve axons, and participating in the directional migration and differentiation of nerve cells. 12 Moreover, integrins are involved in almost every stage of cancer progression from primary tumor to metastasis through their role in signaling molecules, mechanical transducers, and key components of cell migration mechanisms. 13 Integrin β 1 (ITGB1) and β 3 (ITGB3) are the main components of integrin family receptors, which regulate the formation and stability of blood vessels. 14-16 However, whether the injury of ICH leads to the alteration of ITGB1 and ITGB3 has not been confirmed. The purpose of this study was to explore the mechanism of tissue and neural function recovery by analyzing the expression of ITGB1 and ITGB3 in ICH patients. This would provide new clues and ideas for the development and treatment strategies of basic and clinical exploration of ICH. Methods Tissue slides collection The tissues of 12 patients with ICH and the matched normal tissue slides were collected. Meanwhile, pathological characteristics of these samples were obtained, including age, smoking history, drinking history, diabetes history, systolic blood pressure, diastolic blood pressure, fasting blood glucose, and lipid levels. All patients in this study signed informed consents. Immunohistochemical (IHC) staining Firstly, the tissue slides were placed at 65℃ for 30 min, then dewaxed with xylene and washed with alcohol. Then the tissue slides were repaired by citrate buffer, cooled to room temperature and soaked in 1×PBST buffer (1×PBS + 0.1% Tween 20) for 5 min. Next, the tissue slides were sealed with 3% H 2 O 2 and 5% serum for 15 min, respectively. Following, they were incubated with anti-ITGB1 antibody (1:200, abcam, Cat # ab8991) and anti-ITGB3 antibody (1:200, abcam, Cat # ab119992) overnight at 4℃, respectively. The slides were washed with 1×PBST buffer solution for 5 min/3 times, and then the secondary antibody HRP Goat Anti-Rabbit IgG (1:200, abcam, Cat #ab111909) was added and placed at 37℃ for 1h. After washing the slides at the end of the secondary antibody reaction with 1×PBST buffer, the slides were dyed for 5 min with DAB solution, and then staining was terminated by washing with H 2 O. After that, it was re-dyed with hematoxylin for 15 s, and finally sealed with neutral gum. IHC scores were determined by staining percentage scores (classified as: 1 (1%-24%), 2 (25%-49%), 3 (50%-74%), 4 (75%-100%)) and staining intensity scores (scored as 0: signal less color, 1: brown, 2: light yellow, 3: dark brown). To distinguish between high and low expression, the median was selected as cut off-value to reduce the impact of outliers. All tissue microarray chips were pictured with microscopic and viewed with Image Scope and Case Viewer. Animal model construction In this study, 12 clean grade Sprague-Dawley (SD) rats (250-3008, 8-10 weeks old), half male and half female, were reared in SPF animal room. The animals were kept in cage with alternating light and dark for 12 h, keeping the feeding temperature at 20°C and humidity at 50-60%. The experiment was divided into two parts: in the first part, 12 rats were numbered one by one and randomly divided into sham operation group (n = 3) and cerebral hemorrhage group (n = 9). The animal model of cerebral hemorrhage group was injected with collagenase (0.2 U/ulVII collagenase) prepared by 2.5 μL normal saline, and the sham operation group was injected with 2.5 μL normal saline. The rats were killed at 4 days, 7 days and 21 days after the establishment of the model, respectively. 3 rats were randomly selected at each time point, and one in the control group was killed. Then, western blotting (WB) was used to detect the expression of ITGB1 and ITGB3 in the brain tissue of rats with hemorrhagic stroke. Western blotting (WB) analysis The experiment was divided into control (CON) and ICH group. The rats in the ICH group were sacrificed 4 days, 7 days and 21 days after modeling, and the expressions of ITGB1 and ITGB3 in the ICH tissues of the rats with hemorrhagic stroke were detected by WB. Firstly, the proteins were extracted with cell lysate, and detected by BCA protein detection kit (HyClone-Pierce). The 10-µg protein was separated by SDS-PAGE (Invitrogen) and transferred to the PVDF membrane, then sealed at room temperature for 1 h with TBST solution. After that, the membrane was first incubated with primary antibodies (ITGB1, 1:1000, abcam, Cat # ab8991; ITGB3, abcam, Cat # 1:1000, ab119992; GAPDH, 1:3000, Bioworld, AP0063) at 37℃ for 2 h. Following, the membrane was incubated with HRP-conjugated (goat anti-rabbit, 1:3000, Beyotime，Cat # A0208；goat anti-mouse, 1:3000, Beyotime, Cat # A0216) at room temperature for 1 h. Finally, Millipore Immobilon Western Chemiluminescent HRP Substrate kit (Millipore, Cat # RPN2232) was used for color rendering and Chemiluminescent imager (GE, Cat # AI600) observation. Results Comparison of general clinical data of patients with stroke The expression of ITGB1 and ITGB3 in tissue samples of patients with ICH was detected by IHC staining. As illustrated in the Figure 1, indicating that positive cell line exhibited increased staining intensity for ITGB1. By contrast, ITGB3 was not detected in all samples. Moreover, Figure 2 suggested that ITGB1 was expressed in different parts of ICH tissues. In addition, the positive rate of ITGB1 in ICH patient tissue samples was illustrated in Figure 3. Accordingly, the expression of ITGB1 was relatively high in ICH tissues. Differences of ITGB1 and ITGB3 protein expression in animal models The alteration of ITGB1 and ITGB3 expression in cerebral tissue of rats with hemorrhagic stroke were detected by WB. Compared with the control group, the protein expression level of ITGB3 in the brain tissue of the No. 3 rats in the ICH group increased significantly 4 days after the establishment of the model (p < 0.05). After 21d, ITGB1 protein expression levels of the No. 1 and No. 3 rats in the ICH group were significantly increased, whereas ITGB3 protein was not detected after 7 d (Figure 4). Discussion ICH remains a cause of significant morbidity and mortality and is associated with severe long-term disability. 2 In addition, its incidence is 24.6 per 100000 person-years, and the related incidence is increasing as the population ages. 17 Despite this, ICH is the last form of stroke without specific therapy. Treatment of ICH ranges from best medical therapy to approaches involving several different surgical techniques, most of which are at different levels of experimental state. 18 A lack of definitive evidence-based recommendations to guide the care of patients with ICH has led to significant heterogeneity in current clinical practice. The ECM, a non-cellular 3D macromolecular network composed of diverse fibrous ECM proteins, proteoglycans, and glycoproteins, provides not only a physical scaffold to structure the 3D microenvironment but also signals a variety of cellular responses. 19, 20 In particular, ECM-derived signals are transported to the cytoplasm through integrins that directly recognize components of the ECM, resulting in cytological alterations. 6, 11 Therefore, the stimulation of ECM protein-derived signals by integrins makes it possible to accurately regulate the specificity of cells. ICH causes inflammation characterized by leukocyte recruitment and elevated levels of cytokines. 21 Specific leukocyte populations, including neutrophils, T cells and inflammatory monocytes, promote secondary injury in intracerebral hemorrhage models. 22 Previous study demonstrated that integrin complex plays a significant role in cellular interactions with interstitial collagen that are involved in matrix remodeling such as is seen during morphogenesis and wound healing. 23 Hammond et al., suggested that blocking the function of α-4 integrin led to the decrease of leukocyte recruitment and the improvement of motor function after ICH. 24 Dardiotis et al., found that genetic polymorphism in the ITGAV and ITGB8 that may alter the structure or function of integrins may render individuals more susceptible to ICH. 25 At present study, we indicated that ITGB1 was expressed in ICH tissues, but ITGB3 was not detectable in ICH tissues. The clinical progress of ICH is related to the expression of ITGB1, which was also proved by animal model. Conclusions Our results indicate that the clinical progression of ICH is related to the expression of ITGB1, which may be a potential biomarker of ICH. This would provide new clues and ideas for the development and treatment strategies of ICH. Abbreviations BBB: blood-brain barrier CON: control DMEM: Dulbecco’s modified Eagle’s medium ECM: extracellular matrix FBS: Fetal bovine serum ICH: intracerebral hemorrhage IHC: immunohistochemical ITGB: integrin β SD: Sprague-Dawley WB: western blotting Declarations STATEMENT OF NON-DUPLICATION: We certify that our manuscript is a unique submission and is not being considered for publication by any other source in any medium. FUNDING: This work was supported by the Beijing-Tianjin-Hebei Collaborative innovation community construction project (No. 18247788D). The sponsor had no role in the design or conduct of this research. AUTHORS’ CONTRIBUTIONS: HYM and YX: Performed the experiments, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft. CYQ and YP: Contributed materials/analysis tools, Formal analysis, Data curation. QD, LZW, JFY and YH: Methodology, Software. FD : Conceived and designed the experiments, Funding acquisition, Methodology, Project, Supervision, Writing - review & editing. All authors read and approved the final manuscript. ETHICS APPROVAL AND CONSENT TO PARTICIPATE: The institutional review committee of Beijing Tiantan Hospital approved the research program (No. 18247788D). This study adhered to good clinical practice and ethical principles described in the Declaration of Helsinki, and was approved by the IRB of the authors’ institution. Written informed consent was obtained from all participants or their legally authorized representative. Additionally, all animal experimental procedures were approved by the Beijing Tiantan Hospital, Capital Medical University and were performed in accordance with National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. CONSENT FOR PUBLICATION: Not applicable. CONFLICTS OF INTERESTS: All authors declare that they have no any conflict of interests. ACKNOWLEDGEMENTS: None. DATA STATEMENT: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Ikram, M.A., R.G. Wieberdink, and P.J. Koudstaal, International epidemiology of intracerebral hemorrhage. Curr Atheroscler Rep, 2012. 14 (4): p. 300-6. Ziai, W.C. and J.R. Carhuapoma, Intracerebral Hemorrhage. Continuum (Minneap Minn), 2018. 24 (6): p. 1603-1622. Cordonnier, C., et al., Intracerebral haemorrhage: current approaches to acute management. Lancet, 2018. 392 (10154): p. 1257-1268. Morotti, A. and J.N. Goldstein, Diagnosis and Management of Acute Intracerebral Hemorrhage. Emerg Med Clin North Am, 2016. 34 (4): p. 883-899. Keep, R.F., Y. Hua, and G. Xi, Intracerebral haemorrhage: mechanisms of injury and therapeutic targets. Lancet Neurol, 2012. 11 (8): p. 720-31. Edwards, D.N. and G.J. Bix, Roles of blood-brain barrier integrins and extracellular matrix in stroke. Am J Physiol Cell Physiol, 2019. 316 (2): p. C252-C263. Michinaga, S. and Y. Koyama, Pathogenesis of brain edema and investigation into anti-edema drugs. Int J Mol Sci, 2015. 16 (5): p. 9949-75. Birch, H.L., Extracellular Matrix and Ageing. Subcell Biochem, 2018. 90 : p. 169-190. Muncie, J.M. and V.M. Weaver, The Physical and Biochemical Properties of the Extracellular Matrix Regulate Cell Fate. Curr Top Dev Biol, 2018. 130 : p. 1-37. Takada, Y., X. Ye, and S. Simon, The integrins. Genome Biol, 2007. 8 (5): p. 215. Brizzi, M.F., G. Tarone, and P. Defilippi, Extracellular matrix, integrins, and growth factors as tailors of the stem cell niche. Curr Opin Cell Biol, 2012. 24 (5): p. 645-51. Campbell, I.D. and M.J. Humphries, Integrin structure, activation, and interactions. Cold Spring Harb Perspect Biol, 2011. 3 (3). Hamidi, H. and J. Ivaska, Every step of the way: integrins in cancer progression and metastasis. Nat Rev Cancer, 2018. 18 (9): p. 533-548. Lu, Z., et al., Implications of the differing roles of the beta1 and beta3 transmembrane and cytoplasmic domains for integrin function. Elife, 2016. 5 . Clegg, D.O., et al., Integrins in the development, function and dysfunction of the nervous system. Front Biosci, 2003. 8 : p. d723-50. Rossier, O., et al., Integrins beta1 and beta3 exhibit distinct dynamic nanoscale organizations inside focal adhesions. Nat Cell Biol, 2012. 14 (10): p. 1057-67. Fomchenko, E.I., et al., Management of Subdural Hematomas: Part I. Medical Management of Subdural Hematomas. Curr Treat Options Neurol, 2018. 20 (8): p. 28. Sembill, J.A., H.B. Huttner, and J.B. Kuramatsu, Impact of Recent Studies for the Treatment of Intracerebral Hemorrhage. Curr Neurol Neurosci Rep, 2018. 18 (10): p. 71. Singh, P., C. Carraher, and J.E. Schwarzbauer, Assembly of fibronectin extracellular matrix. Annu Rev Cell Dev Biol, 2010. 26 : p. 397-419. Frantz, C., K.M. Stewart, and V.M. Weaver, The extracellular matrix at a glance. J Cell Sci, 2010. 123 (Pt 24): p. 4195-200. Dziedzic, T., et al., Intracerebral hemorrhage triggers interleukin-6 and interleukin-10 release in blood. Stroke, 2002. 33 (9): p. 2334-5. Sansing, L.H., et al., Neutrophil depletion diminishes monocyte infiltration and improves functional outcome after experimental intracerebral hemorrhage. Acta Neurochir Suppl, 2011. 111 : p. 173-8. Park, H.K. and D.J. Jo, Polymorphisms of integrin, alpha 6 contribute to the development and neurologic symptoms of intracerebral hemorrhage in korean population. J Korean Neurosurg Soc, 2011. 50 (4): p. 293-8. Hammond, M.D., et al., alpha4 integrin is a regulator of leukocyte recruitment after experimental intracerebral hemorrhage. Stroke, 2014. 45 (8): p. 2485-7. Dardiotis, E., et al., Integrins AV and B8 Gene Polymorphisms and Risk for Intracerebral Hemorrhage in Greek and Polish Populations. Neuromolecular Med, 2017. 19 (1): p. 69-80. Cite Share Download PDF Status: Published Journal Publication published 01 Mar, 2021 Read the published version in Chinese Neurosurgical Journal → Version 3 posted You are reading this latest preprint version Show more versions 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. 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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-45466","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":7878932,"identity":"4dbebb67-8162-4782-9377-9d25f3e1a9fb","order_by":0,"name":"Hai-Yang Ma","email":"","orcid":"","institution":"Beijing Tiantan Hospital, Capital Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hai-Yang","middleName":"","lastName":"Ma","suffix":""},{"id":7878933,"identity":"8c878188-fff0-427f-aa5f-ee772e78030c","order_by":1,"name":"Yan Xu","email":"","orcid":"","institution":"Beijing Tiantan Hospital, Capital Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Xu","suffix":""},{"id":7878934,"identity":"fa9c65c3-5b45-4c71-8ce8-105db56b0977","order_by":2,"name":"Chun-You Qiao","email":"","orcid":"","institution":"ZhangJiakou First Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chun-You","middleName":"","lastName":"Qiao","suffix":""},{"id":7878935,"identity":"44fb553f-51ff-4a8a-a564-047e19272153","order_by":3,"name":"Yi Peng","email":"","orcid":"","institution":"ZhangJiakou First Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Peng","suffix":""},{"id":7878936,"identity":"6f7b23f8-81f0-4781-8e09-29ebe03bfbf2","order_by":4,"name":"Qi Ding","email":"","orcid":"","institution":"ZhangJiakou First Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Ding","suffix":""},{"id":7878937,"identity":"c6afe97b-55fe-4cb3-a46b-ac8e8ea39bb2","order_by":5,"name":"Li-Zhong Wang","email":"","orcid":"","institution":"ZhangJiakou First Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li-Zhong","middleName":"","lastName":"Wang","suffix":""},{"id":7878938,"identity":"30fefc24-1ab7-4f6b-8fd5-e3e0bfadce63","order_by":6,"name":"Jun-Fei Yan","email":"","orcid":"","institution":"ZhangJiakou First Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jun-Fei","middleName":"","lastName":"Yan","suffix":""},{"id":7878939,"identity":"c8c045e0-3c02-45ab-877a-802bc8ca6f18","order_by":7,"name":"Yuan Hou","email":"","orcid":"","institution":"ZhangJiakou First Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Hou","suffix":""},{"id":7878940,"identity":"62ea90ec-7466-4d61-baf2-315550f4b028","order_by":8,"name":"Fei Di","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIie3PMQrCMBSA4RcC7VLqWgf1CoGCi4J4k2Spi4ggOHfqqLObR6g3CATbRXRtcWnxArpl6GBTcW0zCuYfkgzvIwmAyfSLCVQAVQc7RJzqEUyAUgLgcNAkvCagiEeBawk3xagoZcXi/FHwMhIrsMU5biN9gTGpH8bie0A4i8QGnCDI2ggRvcT7ENoQFnrOuINgWzYkT5/axIKGZI7mLeovHg18/3BZrjm9LljU9Rf3JtBLToeDXZqeSrmdsL0tklbybX7k9YosAEtnXDUbhWqrdOdNJpPpn3oDJZ5YAlHkLeoAAAAASUVORK5CYII=","orcid":"","institution":"Beijing Tiantan Hospital, Capital Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Di","suffix":""}],"badges":[],"createdAt":"2020-07-18 11:49:18","currentVersionCode":3,"declarations":"","doi":"10.21203/rs.3.rs-45466/v3","doiUrl":"https://doi.org/10.21203/rs.3.rs-45466/v3","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s41016-021-00234-4","type":"published","date":"2021-03-01T15:05:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":5047945,"identity":"49d716ba-cca9-4157-9ba5-ed396ca82c1f","added_by":"auto","created_at":"2021-01-18 14:17:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":861013,"visible":true,"origin":"","legend":"The expression of ITGB1 and ITGB3 in ICH was accomplished by IHC staining. The magnification is 200×.","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-45466/v3/65f0c0d73b2b54aa128d669e.jpg"},{"id":5047946,"identity":"87136395-1b1b-4766-9d9a-b88c5a2b1bda","added_by":"auto","created_at":"2021-01-18 14:17:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1387051,"visible":true,"origin":"","legend":"The expression of ITGB1 in ICH was detected by IHC staining.","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-45466/v3/38f11f2d0d024b9053fa80a6.jpg"},{"id":5047834,"identity":"1463973c-3a41-4cc7-8cab-c5ea76c2ca6e","added_by":"auto","created_at":"2021-01-18 14:14:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":48147,"visible":true,"origin":"","legend":"The expression of ITGB1 (positive cell staining rate) in 12 tissue slides was showed.","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-45466/v3/36dc2e2f7173d5e11f556c08.jpg"},{"id":5047947,"identity":"f4735428-6a85-4278-9c66-48b2a8025dc6","added_by":"auto","created_at":"2021-01-18 14:17:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1574790,"visible":true,"origin":"","legend":"Differences of ITGB1 and ITGB3 protein expression in animal models at day 4, 7, and 21.","description":"","filename":"RevisedFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-45466/v3/453987cd6ee88f0fdd60c1a9.png"},{"id":13647071,"identity":"737dc81b-4cef-4ae4-bdb0-92203deea791","added_by":"auto","created_at":"2021-09-17 09:26:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1040300,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-45466/v3/37da2457-b3b0-4c70-9c97-acb7211e102c.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eThe Progression of Intracerebral Hemorrhage (ICH) is Related to the Expression of Integrin Β1 (ITGB1)\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eIntracerebral hemorrhage (ICH) is a critical type of cerebrovascular disease with a high incidence.\u003csup\u003e1\u0026nbsp;\u003c/sup\u003eAlthough the treatment strategy of ICH in acute phase has become more and more standardized, it is still limited to hematoma clearance and symptomatic supportive treatment.\u003csup\u003e2, 3\u003c/sup\u003e At present, there is no effective treatment for the loss of neurological function caused by cerebral hemorrhage, so the mortality and disability rate are still high in the past ten years.\u003csup\u003e4\u003c/sup\u003e It is known that the injury mechanism of intracerebral hemorrhage indicated that the composition, structure and distribution of extracellular matrix (ECM) have changed.\u003csup\u003e5\u003c/sup\u003e In addition, the destruction of blood-brain barrier (BBB) and the formation of brain edema are closely related to ECM.\u003csup\u003e6, 7\u003c/sup\u003ePrevious studies have confirmed that ECM is involved in the formation, development, repair and regeneration of embryos and various tissues and organs.\u003csup\u003e8, 9\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe integrins are a superfamily of cell adhesion receptors that bind to ECM ligands, cell-surface ligands, and soluble ligands.\u003csup\u003e10\u003c/sup\u003e ECM participates in the interaction between cells through the mediation of integrins.\u003csup\u003e11\u003c/sup\u003e Once the nervous system is injured, integrins can affect nerve regeneration by promoting the survival of neurons, regulating the length of nerve axons, and participating in the directional migration and differentiation of nerve cells.\u003csup\u003e12\u003c/sup\u003e Moreover, integrins are involved in almost every stage of cancer progression from primary tumor to metastasis through their role in signaling molecules, mechanical transducers, and key components of cell migration mechanisms.\u003csup\u003e13\u003c/sup\u003e Integrin\u0026nbsp;\u0026beta; 1 (ITGB1) and \u0026beta; 3 (ITGB3) are the main components of integrin family receptors, which regulate the formation and stability of blood vessels.\u003csup\u003e14-16\u003c/sup\u003e However, whether the injury of ICH leads to the alteration of ITGB1 and ITGB3 has not been confirmed.\u003c/p\u003e\n\u003cp\u003eThe purpose of this study was to explore the mechanism of tissue and neural function recovery by analyzing the expression of ITGB1 and ITGB3 in ICH patients. This would provide new clues and ideas for the development and treatment strategies of basic and clinical exploration of ICH.\u003c/p\u003e\n"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eTissue slides collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tissues of 12 patients with ICH and the matched normal tissue slides were collected. Meanwhile, pathological characteristics of these samples were obtained, including age, smoking history, drinking history, diabetes history, systolic blood pressure, diastolic blood pressure, fasting blood glucose, and lipid levels. All patients in this study signed informed consents.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical (IHC) staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirstly, the tissue slides were placed at 65℃ for 30 min, then dewaxed with xylene and washed with alcohol. Then the tissue slides were repaired by citrate buffer, cooled to room temperature and soaked in 1\u0026times;PBST buffer (1\u0026times;PBS + 0.1% Tween 20) for 5 min. Next, the tissue slides were sealed with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eand 5% serum for 15 min, respectively. Following, they were incubated with anti-ITGB1 antibody (1:200, abcam, Cat # ab8991) and anti-ITGB3 antibody (1:200, abcam, Cat # ab119992) overnight at 4℃, respectively. The slides were washed with 1\u0026times;PBST buffer solution for 5 min/3 times, and then the secondary antibody HRP Goat Anti-Rabbit IgG (1:200, abcam, Cat #ab111909) was added and placed at 37℃ for 1h. After washing the slides at the end of the secondary antibody reaction with 1\u0026times;PBST buffer, the slides were dyed for 5 min with DAB solution, and then staining was terminated by washing with H\u003csub\u003e2\u003c/sub\u003eO. After that, it was re-dyed with hematoxylin for 15 s, and finally sealed with neutral gum.\u003c/p\u003e\n\u003cp\u003eIHC scores were determined by\u0026nbsp;staining\u0026nbsp;percentage scores (classified as: 1 (1%-24%), 2 (25%-49%), 3 (50%-74%), 4 (75%-100%))\u0026nbsp;and staining intensity scores (scored as 0: signal less color, 1: brown, 2: light yellow, 3: dark brown).\u0026nbsp;To distinguish between high and low expression, the median was selected as cut off-value to reduce the impact of outliers.\u0026nbsp;All tissue microarray chips were pictured with microscopic\u0026nbsp;and viewed with Image Scope and Case Viewer.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal model construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, 12 clean grade Sprague-Dawley (SD) rats (250-3008, 8-10 weeks old), half male and half female, were reared in SPF animal room. The animals were kept in cage with alternating light and dark for 12 h, keeping the feeding temperature at 20\u0026deg;C and humidity at 50-60%. The experiment was divided into two parts: in the first part, 12 rats were numbered one by one and randomly divided into sham operation group (n = 3) and cerebral hemorrhage group (n = 9). The animal model of cerebral hemorrhage group was injected with collagenase (0.2 U/ulVII collagenase) prepared by 2.5\u0026nbsp;\u0026mu;L\u0026nbsp;normal saline, and the sham operation group was injected with 2.5\u0026nbsp;\u0026mu;L\u0026nbsp;normal saline. The rats were killed at 4 days, 7 days and 21 days after the establishment of the model, respectively. 3 rats were randomly selected at each time point, and one in the control group was killed. Then, western blotting (WB) was used to detect the expression of ITGB1 and ITGB3 in the brain tissue of rats with hemorrhagic stroke.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting (WB) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiment was divided into control (CON) and ICH group. The rats in the ICH group were sacrificed 4 days, 7 days and 21 days after modeling, and the expressions of ITGB1 and ITGB3 in the ICH tissues of the rats with hemorrhagic stroke were detected by WB. Firstly, the proteins were extracted with cell lysate, and detected by BCA protein detection kit (HyClone-Pierce). The 10-\u0026micro;g protein was separated by SDS-PAGE (Invitrogen) and transferred to the PVDF membrane, then sealed at room temperature for 1 h with TBST solution. After that, the membrane was first incubated with primary antibodies (ITGB1, 1:1000, abcam, Cat # ab8991; ITGB3, abcam, Cat # 1:1000, ab119992; GAPDH, 1:3000, Bioworld, AP0063) at 37℃ for 2 h. Following, the membrane was incubated with HRP-conjugated (goat anti-rabbit, 1:3000, Beyotime，Cat # A0208；goat anti-mouse, 1:3000, Beyotime, Cat # A0216) at room temperature for 1 h. Finally, Millipore Immobilon Western Chemiluminescent HRP Substrate kit (Millipore, Cat # RPN2232) was used for color rendering and Chemiluminescent imager (GE, Cat # AI600) observation.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eComparison of general clinical data of patients with stroke\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of ITGB1 and ITGB3 in tissue samples of patients with ICH was detected by IHC staining. As illustrated in the Figure 1, indicating that positive cell line exhibited increased staining intensity for ITGB1. By contrast, ITGB3 was not detected in all samples. Moreover, Figure 2 suggested that ITGB1 was expressed in different parts of ICH tissues. In addition, the positive rate of ITGB1 in ICH patient tissue samples was illustrated in Figure 3. Accordingly, the expression of ITGB1 was relatively high in ICH tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferences of ITGB1 and ITGB3 protein expression in animal models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe alteration of ITGB1 and ITGB3 expression in cerebral tissue of rats with hemorrhagic stroke were detected by WB. Compared with the control group, the protein expression level of ITGB3 in the brain tissue of the No. 3 rats in the ICH group increased significantly 4 days after the establishment of the model (p \u0026lt; 0.05). After 21d, ITGB1 protein expression levels of the No. 1 and No. 3 rats in the ICH group were significantly increased, whereas ITGB3 protein was not detected after 7 d (Figure 4).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eICH remains a cause of significant morbidity and mortality and is associated with severe long-term disability.\u003csup\u003e2\u003c/sup\u003e In addition, its incidence is 24.6 per 100000 person-years, and the related incidence is increasing as the population ages.\u003csup\u003e17\u003c/sup\u003e Despite this, ICH is the last form of stroke without specific therapy. Treatment of ICH ranges from best medical therapy to approaches involving several different surgical techniques, most of which are at different levels of experimental state.\u003csup\u003e18\u003c/sup\u003e A lack of definitive evidence-based recommendations to guide the care of patients with ICH has led to significant heterogeneity in current clinical practice.\u003c/p\u003e\n\u003cp\u003eThe ECM, a non-cellular 3D macromolecular network composed of diverse fibrous ECM proteins, proteoglycans, and glycoproteins, provides not only a physical scaffold \u0026nbsp;to structure the 3D microenvironment but also signals a variety of cellular responses.\u003csup\u003e19, 20\u003c/sup\u003e In particular, ECM-derived signals are transported to the cytoplasm through integrins that directly recognize components of the ECM, resulting in cytological alterations.\u003csup\u003e6, 11\u003c/sup\u003e Therefore, the stimulation of ECM protein-derived signals by integrins makes it possible to accurately regulate the specificity of cells.\u003c/p\u003e\n\u003cp\u003eICH\u0026nbsp;causes inflammation characterized by leukocyte recruitment and elevated levels of cytokines.\u003csup\u003e21\u003c/sup\u003e Specific leukocyte populations, including neutrophils, T cells and inflammatory monocytes, promote secondary injury in intracerebral hemorrhage models.\u003csup\u003e22\u003c/sup\u003e Previous study demonstrated that integrin complex plays a significant role in cellular interactions with interstitial collagen that are involved in matrix remodeling such as is seen during morphogenesis and wound healing.\u003csup\u003e23\u003c/sup\u003e Hammond et al., suggested that blocking the function of \u0026alpha;-4 integrin led to the decrease of leukocyte recruitment and the improvement of motor function after ICH.\u003csup\u003e24\u003c/sup\u003e Dardiotis et al., found that genetic polymorphism in the ITGAV and ITGB8 that may alter the structure or function of integrins may render individuals more susceptible to ICH.\u003csup\u003e25\u003c/sup\u003e At present study, we indicated that ITGB1 was expressed in ICH tissues, but ITGB3 was not detectable in ICH tissues. The clinical progress of ICH is related to the expression of ITGB1, which was also proved by animal model.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur results indicate that the clinical progression of ICH is related to the expression of ITGB1, which may be a potential biomarker of ICH. This would provide new clues and ideas for the development and treatment strategies of ICH.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBBB: blood-brain barrier\u003c/p\u003e\n\u003cp\u003eCON: control\u003c/p\u003e\n\u003cp\u003eDMEM: Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium\u003c/p\u003e\n\u003cp\u003eECM: extracellular matrix\u003c/p\u003e\n\u003cp\u003eFBS: Fetal bovine serum\u003c/p\u003e\n\u003cp\u003eICH: intracerebral hemorrhage\u003c/p\u003e\n\u003cp\u003eIHC: immunohistochemical\u003c/p\u003e\n\u003cp\u003eITGB: integrin \u0026beta;\u003c/p\u003e\n\u003cp\u003eSD: Sprague-Dawley\u003c/p\u003e\n\u003cp\u003eWB: western blotting\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSTATEMENT OF NON-DUPLICATION:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe certify that our manuscript is a unique submission and is not being considered for publication by any other source in any medium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Beijing-Tianjin-Hebei Collaborative innovation community construction project (No. 18247788D). The sponsor had no role in the design or conduct of this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS\u0026rsquo; CONTRIBUTIONS:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHYM and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eYX:\u003c/strong\u003e Performed the experiments, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft. \u003cstrong\u003eCYQ and YP:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eContributed materials/analysis tools, Formal analysis, Data curation. \u003cstrong\u003eQD, LZW, JFY and\u003c/strong\u003e \u003cstrong\u003eYH:\u003c/strong\u003e Methodology, Software. \u003cstrong\u003eFD\u003c/strong\u003e: Conceived and designed the experiments, Funding acquisition, Methodology, Project, Supervision, Writing - review \u0026amp; editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong style=\"text-align: inherit;\"\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe institutional review committee of Beijing Tiantan Hospital approved the research program (No. 18247788D). This study adhered to good clinical practice and ethical principles described in the Declaration of Helsinki, and was approved by the IRB of the authors\u0026rsquo; institution. Written informed consent was obtained from all participants or their legally authorized representative. Additionally, all animal experimental procedures were approved by the Beijing Tiantan Hospital, Capital Medical University and were performed in accordance with National Institutes of Health\u0026rsquo;s Guide for the Care and Use of Laboratory Animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTERESTS:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no any conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA STATEMENT:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eIkram, M.A., R.G. 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Medical Management of Subdural Hematomas.\u003c/em\u003e Curr Treat Options Neurol, 2018. \u003cstrong\u003e20\u003c/strong\u003e(8): p. 28.\u003c/li\u003e\n\u003cli\u003eSembill, J.A., H.B. Huttner, and J.B. Kuramatsu, \u003cem\u003eImpact of Recent Studies for the Treatment of Intracerebral Hemorrhage.\u003c/em\u003e Curr Neurol Neurosci Rep, 2018. \u003cstrong\u003e18\u003c/strong\u003e(10): p. 71.\u003c/li\u003e\n\u003cli\u003eSingh, P., C. Carraher, and J.E. Schwarzbauer, \u003cem\u003eAssembly of fibronectin extracellular matrix.\u003c/em\u003e Annu Rev Cell Dev Biol, 2010. \u003cstrong\u003e26\u003c/strong\u003e: p. 397-419.\u003c/li\u003e\n\u003cli\u003eFrantz, C., K.M. Stewart, and V.M. Weaver, \u003cem\u003eThe extracellular matrix at a glance.\u003c/em\u003e J Cell Sci, 2010. \u003cstrong\u003e123\u003c/strong\u003e(Pt 24): p. 4195-200.\u003c/li\u003e\n\u003cli\u003eDziedzic, T., et al., \u003cem\u003eIntracerebral hemorrhage triggers interleukin-6 and interleukin-10 release in blood.\u003c/em\u003e Stroke, 2002. \u003cstrong\u003e33\u003c/strong\u003e(9): p. 2334-5.\u003c/li\u003e\n\u003cli\u003eSansing, L.H., et al., \u003cem\u003eNeutrophil depletion diminishes monocyte infiltration and improves functional outcome after experimental intracerebral hemorrhage.\u003c/em\u003e Acta Neurochir Suppl, 2011. \u003cstrong\u003e111\u003c/strong\u003e: p. 173-8.\u003c/li\u003e\n\u003cli\u003ePark, H.K. and D.J. Jo, \u003cem\u003ePolymorphisms of integrin, alpha 6 contribute to the development and neurologic symptoms of intracerebral hemorrhage in korean population.\u003c/em\u003e J Korean Neurosurg Soc, 2011. \u003cstrong\u003e50\u003c/strong\u003e(4): p. 293-8.\u003c/li\u003e\n\u003cli\u003eHammond, M.D., et al., \u003cem\u003ealpha4 integrin is a regulator of leukocyte recruitment after experimental intracerebral hemorrhage.\u003c/em\u003e Stroke, 2014. \u003cstrong\u003e45\u003c/strong\u003e(8): p. 2485-7.\u003c/li\u003e\n\u003cli\u003eDardiotis, E., et al., \u003cem\u003eIntegrins AV and B8 Gene Polymorphisms and Risk for Intracerebral Hemorrhage in Greek and Polish Populations.\u003c/em\u003e Neuromolecular Med, 2017. \u003cstrong\u003e19\u003c/strong\u003e(1): p. 69-80.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biomarker, Immunohistochemical staining, Inflammation, Integrins, Intracerebral hemorrhage, Western blotting","lastPublishedDoi":"10.21203/rs.3.rs-45466/v3","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-45466/v3","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Intracerebral hemorrhage (ICH) is fatal and detrimental to quality of life. Clinically, options for monitoring are often limited, potentially missing subtle neurological changes. Integrin β 1 (ITGB1) and β 3 (ITGB3) are the main components of integrin family receptors, which regulate the formation and stability of blood vessels. This study explored the relationship between expression of ITGB1, ITGB3 in intracerebral hemorrhage (ICH) to analyze their functional and clinical relevance. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e The expression of ITGB1 and ITGB3 in ICH was accomplished by immunohistochemical (IHC) staining and western blotting (WB) analysis, respectively. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Furthermore, the results demonstrated that ITGB1 was expressed in ICH tissues, but ITGB3 was not expressed in ICH tissues. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e In summary, the clinical progression of ICH was related to the expression of ITGB1. ITGB1 may be a potential biomarker and contribute to the treatment of ICH.\u003c/p\u003e","manuscriptTitle":"The Progression of Intracerebral Hemorrhage (ICH) is Related to the Expression of Integrin Β1 (ITGB1)","msid":"","msnumber":"","nonDraftVersions":[{"code":3,"date":"2021-01-18 14:14:41","doi":"10.21203/rs.3.rs-45466/v3","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":2,"date":"2020-10-21 17:47:54","doi":"10.21203/rs.3.rs-45466/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2020-07-30 17:59:22","doi":"10.21203/rs.3.rs-45466/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bd537c8b-0efa-4f48-a897-ca7bf28bd592","owner":[],"postedDate":"January 18th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":1945702,"name":"Neurosurgery"}],"tags":[],"updatedAt":"2021-03-07T15:07:28+00:00","versionOfRecord":{"articleIdentity":"rs-45466","link":"https://doi.org/10.1186/s41016-021-00234-4","journal":{"identity":"chinese-neurosurgical-journal","isVorOnly":false,"title":"Chinese Neurosurgical Journal"},"publishedOn":"2021-03-01 15:05:39","publishedOnDateReadable":"March 1st, 2021"},"versionCreatedAt":"2021-01-18 14:14:41","video":"","vorDoi":"10.1186/s41016-021-00234-4","vorDoiUrl":"https://doi.org/10.1186/s41016-021-00234-4","workflowStages":[]},"version":"v3","identity":"rs-45466","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-45466","identity":"rs-45466","version":["v3"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}