Pathophysiological Mechanisms of Chronic Subdural Hematoma: Insights from Histopathological, Immunological, and Imaging Analyses Pathophysiological Mechanisms of CSDH

Pathophysiological Mechanisms of Chronic Subdural Hematoma: Insights from Histopathological, Immunological, and Imaging Analyses Pathophysiological Mechanisms of CSDH | 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 Pathophysiological Mechanisms of Chronic Subdural Hematoma: Insights from Histopathological, Immunological, and Imaging Analyses Pathophysiological Mechanisms of CSDH Renkun Zhang, Qiuyu Lu, Yuhao Tan, Yu Zhou, Zifu Li, Lei Zhang, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7242529/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective To further explore the pathophysiological mechanisms of the chronic subdural hematoma (CSDH) through the simultaneous histopathological, immunological, embolic-agent-detecting, and imaging analyses of the dura and the outer membrane of the hematoma (OMH). Method Specimens were collected from 5 patients who underwent burr hole drainage craniectomy (BHD) of CSDH and 5 patients who underwent middle meningeal artery embolization (MMAE) + BHD at the Neurovascular Center of Shanghai Changhai Hospital from March 2022 to December 2022. The dura and OMH removed intraoperatively were sent for histopathological, immunological, and embolic-agent-detecting analyses (detecting the tantalum content). The preoperative MRI of the patients were studied to observe the dura and OMH and measure the width of the linearly enhanced tissue on the T1 enhanced sequence. Results The dura and OMH were closely connected under the light microscope. the dura mainly consisted of collagen fibers, and blood vessels were normal. The OMH is rich in blood vessels and uneven in thickness, and collagen fibers were scattered, with inflammatory cells infiltrated, new blood vessels formed, and red blood cell exudation, and had higher VEGF, VEGFR − 1, and VEGFR − 2 contents than dura. Penetrating vessels were at the dura - OMH junction. The MMAE + BHD group's OMH had higher tantalum content than BHD’s. All patients had linear enhancement in the T1 enhanced sequence. The MRI enhanced sequence's linear enhancement averaged 1.71 mm in thickness, the dura plus OMH averaged 1.60 mm, and the dura averaged 0.5 mm. Conclusions This study identified penetrating dura-OMH vessels and detected tantalum in OMH after MMAE. High VEGF and CD31 expressions were observed in OMH’s neovascular endothelium. These findings support the inflammatory-angiogenic theory of CSDH pathogenesis and provide anatomical evidence for MMAE mechanisms. The detected angiogenic biomarkers suggest potential therapeutic value of anti-angiogenic drugs for CSDH. Chronic subdural hematoma Dura Outer membrane Pathophysiological mechanism VEGF CD31 Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Chronic subdural hematoma (CSDH) is one of the most common neurosurgical diseases. The incidence of CSDH, which is especially prevalent among aging population, ranges from 1.72 to 20.6 per 100,000 persons per year. ( 1 – 3 ) The most usual procedures for CSDH treatment include single or multiple burr hole drainage craniectomy (BHD), but approximately 10 ~ 20% of surgically treated patients experience postoperative recurrence necessitating reoperation. ( 2 , 4 – 6 ) The middle meningeal artery embolization (MMAE), which can effectively reduce the recurrence rate and adverse events compared with conventional management, represents one of the latest additions to the therapeutic arsenal of cerebrovascular specialists in treating CSDH. ( 2 , 7 – 11 ) The inflammation and angiogenesis were considered as the key factors in the development of CSDH. ( 12 ) Many drugs targeting different elements of this cycle were being actively investigated as potential therapeutic agents. ( 13 – 16 ) The currently prevailing viewpoint considered CSDH as a cycle of hemorrhage, hyperproliferation, fragility and re-hemorrhage. ( 17 ) There was a growth of fragile neovessels with poorly developed endothelial cell junctions containing thrombomodulin, which was one of the factors that prevent the blood in the CSDH from clotting. These vessels were thought to be the source of repeated multifocal bleeding. ( 3 , 18 – 22 ) Hence, CSDH was currently recognized as an inflammatory-angiogenic disease. ( 21 ) The purpose of our study is to demonstrate this theory by further exploring pathophysiological mechanisms of CSDH through the histopathological, immunological, embolic-agent-detecting and imaging analyses of the dura and the outer membrane of the hematoma (OMH) simultaneously. METHODS Ethical information This study was conducted in accordance with the principles of the Helsinki Declaration. Approval of the Institutional Review Board of Changhai Hospital was obtained in December 2020 (CHEC2020-155). Written informed consent was obtained from all study participants. The study presented in this manuscript did not fall under the scope of established reporting guidelines (such as CONSORT, STROBE, PRISMA, ARRIVE, etc.) as it is a comprehensive pathophysiological analysis combining histopathological, immunological, and imaging data, rather than a clinical trial, observational study, systematic review, or animal experiment specifically covered by these standards. Clinical trial number: not applicable. Specimen collection From March 2022 to December 2022, we collected specimens from 10 patients at the Neurovascular Center of Shanghai Changhai Hospital. Among them, 5 patients underwent BHD, and the other 5 patients underwent MMAE + BHD. The MMAE was embolized with Onyx (Medtronic, America). In the surgical process of all patients, when the relevant tissues were removed, the dura and OMH were resected as a whole. The specific randomization and surgical methods were provided in Supplementary materials. Pathological and immunological analysis The dura and OMH removed intraoperatively from each patient were sent for histopathological and immunological analyses. Specific experimental methods were described in Supplementary materials. In order to conduct qualitative and quantitative analyses on the vascular conditions of the dura and OMH, we carried out CD31 immunohistochemistry (IHC) and immunofluorescence (IF) staining. We defined the vascular density as the number of blood vessels within the tissue per unit area. On the same tissue section, the regions with the highest vascular density in both the dura and the OMH were selected respectively. Then, using the CaseViewer software, six regions were chosen within these areas. This software can automatically calculate the area of these regions. Meanwhile, the number of vessels in these regions was counted manually. Thus, the vascular density was calculated as: \(\:vascular\:density=\frac{Number\:of\:vessels}{Area\:of\:tℎe\:region}\:\) (counts/mm 2 ) Embolic agent detecting In the dura and OMH of the five patients who underwent MMAE + BHD, the tantalum which was the main content of Onyx was detected by inductance coupled plasma mass spectrometry. Specific experimental methods were listed in Supplementary materials. Imaging analysis In order to further clarify the imaging features of the dura and OMH, we studied the T1 sequence, T2 sequence and T1 enhanced sequence of the preoperative MRI of the patient. The dura and OMH were observed and the width of the linearly enhanced tissue in the T1 enhanced sequence was measured. Statistical analysis Prism GraphPad 9.0 and R software (4.0.3) were used for statistics and mapping. For comparison between the two groups of data, independent sample T test was used if the distribution was normal, and non-parametric test was used if the distribution was not normal. Single factor analysis of variance was used for comparison among groups, and multiple T test was used for comparison among groups. Measurement data are expressed as mean ± standard deviation (X ± S); The statistical difference standard was P < 0.05. RESULTS Pathological analysis The structures of the dura and OMH under the light microscope Under the light microscope, dura and OMH were closely connected, with the surface of dura being smooth and white in appearance, while OMH shown red staining due to extensive hemorrhage and exudate, with varying thickness (Fig. 1 A, B). Post-fixation, the dura assumed a milky white color, and the OMH presented a yellow-brown hue, being distinctly distinguishable from the dura in terms of structure. Under lateral view, although the dura and the OMH were tightly connected, there existed a clear demarcation line between them, signifying their distinct tissue characteristics (Fig. 1 C). With a gentle manipulation using microscopic forceps, the dura and the OMH could be readily separated. Moreover, within the local region of the OMH, irregularly arranged vascular structures were observable (Fig. 1 D). The histopathological structures of dura and OMH The overall images of the HE staining and Masson staining of the dura and OMH were presented in the Supplementary Materials. Figure 1 E, I, M presented the magnified local views of the HE staining. The dura was replete with collagen fibers, which were arranged in two distinct layers. The outer layer of the dura was characterized by a transverse distribution pattern, with sporadic cells being discernible and no distinct blood vessels in evidence. In contrast, the inner layer of the dura demonstrated a longitudinal arrangement, where scattered infiltrations of inflammatory cells were noted, and the vascular structures were dispersed in a sporadic fashion. When compared to the dura, the collagen fibers within the OMH were distributed in a scattered manner. A significant number of inflammatory cells were evident, infiltrating the area. Among them, a multitude of newly formed blood vessels with varying diameters were observable, accompanied by a large quantity of exuded red blood cells. This indicated the presence of intense inflammatory responses, active angiogenesis, and erythrocyte leakage within the OMH. Figures 1 F, J, N showcased the magnified local views of the Masson staining. The Masson staining further served to distinguish the dura and OMH. The collagen fibers were stained blue, the red blood cells were stained red, and the cell nuclei were stained blue-black. In comparison to the dura, although the OMH also had collagen fibers as its primary structural component, the fiber density was considerably lower. Moreover, the collagen fiber structure within the OMH was disorganized and chaotic. A large number of newly formed blood vessels with diverse diameters were visible on it, and the exudation of red blood cells was prominent. Cell types comprising dura and OMH The dura consisted of collagen fibers, among which the cell bodies of fibroblasts were discernible. Concurrently, scattered blood vessels were present on the inner aspect of the dura, which are mainly composed of endothelial cells. Occasionally, lymphocytes could be found along the inner edge of the dura. In the OMH, a large number of newly formed blood vessels were composed of endothelial cells, and there were scattered fibroblasts. In contrast to the cellular composition of the dura, the OMH exhibited a pronounced infiltration of inflammatory cells, principally comprising lymphocytes, macrophages, and eosinophils(Fig. 1 E, M) . Vascular characteristics of dura and OMH According to the HE and Masson staining outcomes, the blood vessels within the dura were principally located on its inner side, adjacent to OMH. These vessels exhibited normal diameters and compositions, with the endothelial and smooth muscle cells (appearing red on Masson staining) being clearly distinguishable. There were no exuded red blood cells around the blood vessels. In contrast to the dura, the OMH demonstrated a marked increase in the number of blood vessels, lacking a discernible distribution pattern. The vessel diameters were heterogeneous, and the inner lumens were obviously widened. The blood vessels were formed by a single, thin layer of endothelial cells that lacked tight intercellular junctions. Concurrently, the gaps between the endothelial cells were significantly widened, and the basement membrane structure was absent, along with a lack of smooth muscle cells. Around the blood vessels, a significant amount of red blood cells had leaked out through the wide gaps among the endothelial cells (Fig. 1 I, J). Under CD31 IHC and IF staining, a large number of newly formed blood vessels composed merely of a single layer of endothelial cells could be seen in OMH (Fig. 1 G,H,K,L). The vascular density of the OMH (853.3 ± 5.289 counts/mm 2 ) was significantly higher than that of the dura (130.7 ± 4.014 counts/mm 2 ), and the difference was statistically significant, with P < 0.05. The vascular density of the OMH of patient B (853.3 ± 5.289 counts/mm 2 ) was also significantly higher than that of patient A (375.7 ± 4.958 counts/mm 2 ), and the difference was statistically significant, with P < 0.05 (Fig. 1 O,P). Penetrating vascular between dura and OMH Pathological analysis HE and Masson staining of the junction area between dura and OMH showed penetrating vascular communication between dura and OMH (Fig. 2 A,B,D,E). The penetrating vessels are morphologically similar to those in the OMH region and consist of a single layer of weak endothelial cells without smooth muscle. Embolic agent detecting Tantalum content standard curve: Y = 141.693X + 54.021(Fig. 2 C). Tantalum content of OMH in the MMAE + BHD group was 1.678 ± 0.06127ug/g, and that in the BHD group was 0.1000 ± 0.01304ug/g. Tantalum content of OMH in the MMAE + BHD group was significantly higher than that in the BHD group, with statistical difference (P < 0.05) (Fig. 2 F). Expression of dura and OMH angiogenesis related factors OMH showed a large number of neovascularization, and we further explored the factors leading to angiogenesis, focusing on IHC and IF staining of VEGF, VEGFR-1 and VEGFR-2. The results showed that the contents of VEGF, VEGFR-1 and VEGFR-2 in OMH were significantly higher than those in dura (Fig. 3 A ~ C). The results of Mean Fluorescence Intensity (MFI) showed that compared with dura, the contents of VEGF, VEGFR-1 and VEGFR-2 in OMH were significantly increased, with statistical significance (P < 0.05) (Fig. 3 ). Imaging features of dura and OMH All patients showed varying degrees of significant linear enhancement on the T1 enhancement sequence, showing dura and OMH structures. At the same time, we measured the thickness of development on the enhanced sequence, meninges and OMH HE stained sections. The average thickness of linear enhancement on the MRI enhanced sequence was 1.71mm, while the average thickness of dura plus OMH was 1.60 mm, and the average thickness of dura was 0.5mm (Fig. 4 ). Discussion This study provided histopathological evidence of penetrating vessels between the dura and OMH, characterized by their spiral trajectory towards the OMH. These vessels were identified as potential arterial suppliers for CSDH development. Through quantitative analysis of tantalum distribution in MMAE + BHD patients, we demonstrated significantly higher tantalum concentrations in OMH in the MMAE + BHD group compared to those in the BHD group, indicating functional vascular channels between MMA and OMH. MRI enhancement analysis revealed linear enhancement (average 1.71 mm) corresponding to combined dural-OMH structures, validated by histological measurements ( average 1.60 mm). In 1987, Roland, J. found the image of a spiral penetrating artery originating from the anastomotic network of secondary superficial artery in the microangiogram of falx cerebri. ( 23 ) According to Maksim Shapiro’s theory published in 2021, the dura could be divided into periosteal layer (PL), meningeal layer (ML), border cell layer (BCL). ( 17 ) Theoretically, in the relatively avascular ML, there were penetrating vessels from the secondary anastomotic network of PL (20 ~ 40µm) to the capillary network within the BZL layer (10µm), which was consistent with our finding of the penetrating vessels between the dura mater and OMH. In addition, the analysis of the results of HE staining, Masson staining, IHC and IF shown that OMH exhibits a large number of neovascularizations, red blood cell leakage, and inflammatory cell infiltration, which was consistent with other studies on the pathological characteristics of CSDH. ( 18 – 22 ) Through quantitative analysis of tantalum distribution in MMAE + BHD patients, we demonstrated significantly higher tantalum concentrations in OMH in the MMAE + BHD group compared to those in the BHD group. Given that tantalum was a component of onyx and could be visualized under X - rays, this result directly confirmed the existence of a channel between the dura and OMH. This channel was most likely the penetrating artery, thus providing a breakthrough basis for the treatment of CSDH by MMAE. In addition, by imaging analysis, it was confirmed that the linear enhancement of CSDH under T1 enhancement was formed by both OMH and the dura mater, providing imaging evidence for the inflammatory changes and rich blood vessels in OMH. Our multimodal analysis established OMH as the pathophysiological epicenter of CSDH progression, characterized by unique angiogenic activation and inflammatory microenvironment. The demonstrated MMA-OMH vascular connectivity provided mechanistic validation for MMAE, while VEGF/CD31 overexpression of OMH proposed testable targets for pharmacological intervention. Based on these research findings, we proposed the potential value of anti-angiogenic drugs in the treatment of CSDH. In the future, it is urgent to conduct more controlled clinical trials to rigorously evaluate the drugs used for the non-surgical treatment of CSDH, especially those targeting the inflammatory-angiogenic pathogenic mechanism, so as to promote the development and progress of the CSDH treatment field. Conclusion This study identified penetrating dura-OMH vessels and detected tantalum in OMH after MMAE. High VEGF and CD31 expressions were observed in OMH’s neovascular endothelium. These findings support the inflammatory-angiogenic theory of CSDH pathogenesis and provide anatomical evidence for MMAE mechanisms. This study proposed the potential value of anti-angiogenic drugs and emphasized the urgency of more controlled clinical trials to promote CSDH treatment development. Declarations Disclosures: The authors have no personal, financial, or institutional conflict of interest in any of the drugs, materials, or devices described in this article. Funding: This research was supported by "Rising Stars of Medical Talents" Youth Development Program of Shanghai Municipal Health Commission (2023-62); by the “Changfeng” Talent Program of Changhai Hospital (2024-1); and by the “GuHai” Program of Changhai Hospital (GH145-05). Author Contribution Renkun Zhang, Qiuyu Lu and Yuhao Tan wrote the main manuscript text and prepared figures 1-4. Yu Zhou, Zifu Li, Lei Zhang, Qinghai Huang, Yi Xu, Yongwei Zhang, Rui Zhao, Qiang Li, Pengfei Yang, Qiao Zuo and Jianmin Liu reviewed the manuscript. References Rauhala M, Luoto TM, Huhtala H, Iverson GL, Niskakangas T, Öhman J et al (2019) The incidence of chronic subdural hematomas from 1990 to 2015 in a defined Finnish population. J Neurosurg 132(4):1147–1157 Feghali J, Yang W, Huang J (2020) Updates in Chronic Subdural Hematoma: Epidemiology, Etiology, Pathogenesis, Treatment, and Outcome. 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Surg Radiol Anat 9(1):43–49 Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterialforReview.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7242529","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":531732871,"identity":"058678a3-5af9-458c-98a1-fa2a88ed12db","order_by":0,"name":"Renkun Zhang","email":"","orcid":"","institution":"Changhai Hospital, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Renkun","middleName":"","lastName":"Zhang","suffix":""},{"id":531732873,"identity":"99f917d1-a712-42de-be60-4f74d6ccb8dd","order_by":1,"name":"Qiuyu Lu","email":"","orcid":"","institution":"Changhai Hospital, Naval Medical 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1","display":"","copyAsset":false,"role":"figure","size":32211003,"visible":true,"origin":"","legend":"\u003cp\u003ePathology images of dura and OMH. A,B: Gross view of dura (A) and OMH (B) immediately removed intraoperatively. C,D: Lateral view of dura and OMH after 4% paraformaldehyde fixation (C) and vascular network on OMH surface (D). E~H: dura HE staining (E), MS staining (F), CD31 staining (G), CD31 fluorescence staining (H) images under light microscope, magnification:10*40 . I,J: Images stained by HE (E) and MS (F) at the interface of dura and OMH under light microscope, magnification:10*40 . K~N: images of OMH HE staining (M), MS staining (N), CD31 staining (K), CD31 fluorescence staining (L) under light microscope, magnification:10*40. O: Comparison of blood vessel density between dura and OMH in the same patient. P: Comparison of blood vessel density between OMH in different patients (a,b) d:dura; n:neomembrance; ****: p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7242529/v1/8a9c1cb45e2042d6b694ac4f.png"},{"id":94155112,"identity":"a1d1bd6c-91aa-4de2-9734-f55f9fd6b5ef","added_by":"auto","created_at":"2025-10-23 02:44:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":20633147,"visible":true,"origin":"","legend":"\u003cp\u003ePenetrating vascular HE staining (A,B) and MS staining (D,E), with dura on the left and OMH on the right, magnification:10*40. Red arrows show the blood vessels that pass through dura and OMH. C: Standard curve of tantalum content. F:OMH tantalum content, Normal: simple burr hole drainage craniectomy, MMAE: middle meningeal artery embolization + burr hole drainage craniectomy, Ta: tantalum. ****: p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7242529/v1/b37447259dfd7a74cf552af6.png"},{"id":94156213,"identity":"c6edb9cf-1d64-436b-ba07-f1f28b00a5cc","added_by":"auto","created_at":"2025-10-23 02:52:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24587814,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of VEGF(A),VEGFR-1(B),VEGFR-2(C) histochemical staining and fluorescence staining (each fluorescence staining was fitted with DAPI staining) and MFI of dura and OMH. In histochemical staining, brown was positive. In each fluorescent staining, red is positive. MFI: Mean Fluorescence Intensity; ****: p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7242529/v1/27994597b9af1b4493ccce1e.png"},{"id":94156211,"identity":"c12f03eb-1cbc-44ff-b33a-c2f0d44a09a2","added_by":"auto","created_at":"2025-10-23 02:52:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6358459,"visible":true,"origin":"","legend":"\u003cp\u003eA~D: MRI images of chronic subdural hematoma. A is T1-weighted, B is T2-weighted, C is T1 enhanced, and D is local amplification of T1 enhanced. D linear enhancement width: 1.61mm, 1.74mm, 1.88mm, 1.64mm, 1.63mm, 1.76mm. E: HE staining of dura and OMH , dura+OMH width: 1.7608mm, 1.5283mm, 1.5326mm, 1.6520mm, 1.5324mm, 1.5860mm.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7242529/v1/0088404e54d057952485de1e.png"},{"id":95528050,"identity":"0c2a7b9c-af74-405e-a46d-957fe7bbf6e6","added_by":"auto","created_at":"2025-11-10 10:15:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":76548981,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7242529/v1/6b42562c-c43a-4a71-9dfa-1da7de851e0b.pdf"},{"id":94155102,"identity":"672a8682-5019-425b-b0e0-de4a6abe39e3","added_by":"auto","created_at":"2025-10-23 02:44:59","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16760,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialforReview.docx","url":"https://assets-eu.researchsquare.com/files/rs-7242529/v1/02fe223d16ebb80bf4d518b4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pathophysiological Mechanisms of Chronic Subdural Hematoma: Insights from Histopathological, Immunological, and Imaging Analyses Pathophysiological Mechanisms of CSDH","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eChronic subdural hematoma (CSDH) is one of the most common neurosurgical diseases. The incidence of CSDH, which is especially prevalent among aging population, ranges from 1.72 to 20.6 per 100,000 persons per year.\u003csup\u003e(\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/sup\u003e The most usual procedures for CSDH treatment include single or multiple burr hole drainage craniectomy (BHD), but approximately 10\u0026thinsp;~\u0026thinsp;20% of surgically treated patients experience postoperative recurrence necessitating reoperation. \u003csup\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/sup\u003e The middle meningeal artery embolization (MMAE), which can effectively reduce the recurrence rate and adverse events compared with conventional management, represents one of the latest additions to the therapeutic arsenal of cerebrovascular specialists in treating CSDH.\u003csup\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe inflammation and angiogenesis were considered as the key factors in the development of CSDH.\u003csup\u003e(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/sup\u003e Many drugs targeting different elements of this cycle were being actively investigated as potential therapeutic agents.\u003csup\u003e(\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e)\u003c/sup\u003e The currently prevailing viewpoint considered CSDH as a cycle of hemorrhage, hyperproliferation, fragility and re-hemorrhage.\u003csup\u003e(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u003c/sup\u003e There was a growth of fragile neovessels with poorly developed endothelial cell junctions containing thrombomodulin, which was one of the factors that prevent the blood in the CSDH from clotting. These vessels were thought to be the source of repeated multifocal bleeding.\u003csup\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/sup\u003e Hence, CSDH was currently recognized as an inflammatory-angiogenic disease.\u003csup\u003e(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe purpose of our study is to demonstrate this theory by further exploring pathophysiological mechanisms of CSDH through the histopathological, immunological, embolic-agent-detecting and imaging analyses of the dura and the outer membrane of the hematoma (OMH) simultaneously.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEthical information\u003c/h2\u003e\u003cp\u003e This study was conducted in accordance with the principles of the Helsinki Declaration. Approval of the Institutional Review Board of Changhai Hospital was obtained in December 2020 (CHEC2020-155). Written informed consent was obtained from all study participants. The study presented in this manuscript did not fall under the scope of established reporting guidelines (such as CONSORT, STROBE, PRISMA, ARRIVE, etc.) as it is a comprehensive pathophysiological analysis combining histopathological, immunological, and imaging data, rather than a clinical trial, observational study, systematic review, or animal experiment specifically covered by these standards. Clinical trial number: not applicable.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSpecimen collection\u003c/h3\u003e\n\u003cp\u003eFrom March 2022 to December 2022, we collected specimens from 10 patients at the Neurovascular Center of Shanghai Changhai Hospital. Among them, 5 patients underwent BHD, and the other 5 patients underwent MMAE\u0026thinsp;+\u0026thinsp;BHD. The MMAE was embolized with Onyx (Medtronic, America). In the surgical process of all patients, when the relevant tissues were removed, the dura and OMH were resected as a whole. The specific randomization and surgical methods were provided in Supplementary materials.\u003c/p\u003e\n\u003ch3\u003ePathological and immunological analysis\u003c/h3\u003e\n\u003cp\u003eThe dura and OMH removed intraoperatively from each patient were sent for histopathological and immunological analyses. Specific experimental methods were described in Supplementary materials.\u003c/p\u003e\u003cp\u003eIn order to conduct qualitative and quantitative analyses on the vascular conditions of the dura and OMH, we carried out CD31 immunohistochemistry (IHC) and immunofluorescence (IF) staining. We defined the vascular density as the number of blood vessels within the tissue per unit area. On the same tissue section, the regions with the highest vascular density in both the dura and the OMH were selected respectively. Then, using the CaseViewer software, six regions were chosen within these areas. This software can automatically calculate the area of these regions. Meanwhile, the number of vessels in these regions was counted manually. Thus, the vascular density was calculated as:\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:vascular\\:density=\\frac{Number\\:of\\:vessels}{Area\\:of\\:tℎe\\:region}\\:\\)\u003c/span\u003e\u003c/span\u003e\u003cem\u003e(counts/mm\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003ch3\u003eEmbolic agent detecting\u003c/h3\u003e\n\u003cp\u003eIn the dura and OMH of the five patients who underwent MMAE\u0026thinsp;+\u0026thinsp;BHD, the tantalum which was the main content of Onyx was detected by inductance coupled plasma mass spectrometry. Specific experimental methods were listed in Supplementary materials.\u003c/p\u003e\n\u003ch3\u003eImaging analysis\u003c/h3\u003e\n\u003cp\u003eIn order to further clarify the imaging features of the dura and OMH, we studied the T1 sequence, T2 sequence and T1 enhanced sequence of the preoperative MRI of the patient. The dura and OMH were observed and the width of the linearly enhanced tissue in the T1 enhanced sequence was measured.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003ePrism GraphPad 9.0 and R software (4.0.3) were used for statistics and mapping. For comparison between the two groups of data, independent sample T test was used if the distribution was normal, and non-parametric test was used if the distribution was not normal. Single factor analysis of variance was used for comparison among groups, and multiple T test was used for comparison among groups. Measurement data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (X\u0026thinsp;\u0026plusmn;\u0026thinsp;S); The statistical difference standard was P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003ePathological analysis\u003c/h2\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003eThe structures of the dura and OMH under the light microscope\u003c/h2\u003e\u003cp\u003eUnder the light microscope, dura and OMH were closely connected, with the surface of dura being smooth and white in appearance, while OMH shown red staining due to extensive hemorrhage and exudate, with varying thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Post-fixation, the dura assumed a milky white color, and the OMH presented a yellow-brown hue, being distinctly distinguishable from the dura in terms of structure. Under lateral view, although the dura and the OMH were tightly connected, there existed a clear demarcation line between them, signifying their distinct tissue characteristics (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). With a gentle manipulation using microscopic forceps, the dura and the OMH could be readily separated. Moreover, within the local region of the OMH, irregularly arranged vascular structures were observable (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eThe histopathological structures of dura and OMH\u003c/h2\u003e\u003cp\u003eThe overall images of the HE staining and Masson staining of the dura and OMH were presented in the Supplementary Materials. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, I, M presented the magnified local views of the HE staining. The dura was replete with collagen fibers, which were arranged in two distinct layers. The outer layer of the dura was characterized by a transverse distribution pattern, with sporadic cells being discernible and no distinct blood vessels in evidence. In contrast, the inner layer of the dura demonstrated a longitudinal arrangement, where scattered infiltrations of inflammatory cells were noted, and the vascular structures were dispersed in a sporadic fashion. When compared to the dura, the collagen fibers within the OMH were distributed in a scattered manner. A significant number of inflammatory cells were evident, infiltrating the area. Among them, a multitude of newly formed blood vessels with varying diameters were observable, accompanied by a large quantity of exuded red blood cells. This indicated the presence of intense inflammatory responses, active angiogenesis, and erythrocyte leakage within the OMH. Figures\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, J, N showcased the magnified local views of the Masson staining. The Masson staining further served to distinguish the dura and OMH. The collagen fibers were stained blue, the red blood cells were stained red, and the cell nuclei were stained blue-black. In comparison to the dura, although the OMH also had collagen fibers as its primary structural component, the fiber density was considerably lower. Moreover, the collagen fiber structure within the OMH was disorganized and chaotic. A large number of newly formed blood vessels with diverse diameters were visible on it, and the exudation of red blood cells was prominent.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCell types comprising dura and OMH\u003c/h2\u003e\u003cp\u003eThe dura consisted of collagen fibers, among which the cell bodies of fibroblasts were discernible. Concurrently, scattered blood vessels were present on the inner aspect of the dura, which are mainly composed of endothelial cells. Occasionally, lymphocytes could be found along the inner edge of the dura. In the OMH, a large number of newly formed blood vessels were composed of endothelial cells, and there were scattered fibroblasts. In contrast to the cellular composition of the dura, the OMH exhibited a pronounced infiltration of inflammatory cells, principally comprising lymphocytes, macrophages, and eosinophils(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, M) .\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eVascular characteristics of dura and OMH\u003c/h2\u003e\u003cp\u003eAccording to the HE and Masson staining outcomes, the blood vessels within the dura were principally located on its inner side, adjacent to OMH. These vessels exhibited normal diameters and compositions, with the endothelial and smooth muscle cells (appearing red on Masson staining) being clearly distinguishable. There were no exuded red blood cells around the blood vessels. In contrast to the dura, the OMH demonstrated a marked increase in the number of blood vessels, lacking a discernible distribution pattern. The vessel diameters were heterogeneous, and the inner lumens were obviously widened. The blood vessels were formed by a single, thin layer of endothelial cells that lacked tight intercellular junctions. Concurrently, the gaps between the endothelial cells were significantly widened, and the basement membrane structure was absent, along with a lack of smooth muscle cells. Around the blood vessels, a significant amount of red blood cells had leaked out through the wide gaps among the endothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, J).\u003c/p\u003e\u003cp\u003eUnder CD31 IHC and IF staining, a large number of newly formed blood vessels composed merely of a single layer of endothelial cells could be seen in OMH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG,H,K,L). The vascular density of the OMH (853.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.289 counts/mm\u003csup\u003e2\u003c/sup\u003e) was significantly higher than that of the dura (130.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.014 counts/mm\u003csup\u003e2\u003c/sup\u003e), and the difference was statistically significant, with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The vascular density of the OMH of patient B (853.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.289 counts/mm\u003csup\u003e2\u003c/sup\u003e) was also significantly higher than that of patient A (375.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.958 counts/mm\u003csup\u003e2\u003c/sup\u003e), and the difference was statistically significant, with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO,P).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003ePenetrating vascular between dura and OMH\u003c/h2\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003ePathological analysis\u003c/h2\u003e\u003cp\u003eHE and Masson staining of the junction area between dura and OMH showed penetrating vascular communication between dura and OMH (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B,D,E). The penetrating vessels are morphologically similar to those in the OMH region and consist of a single layer of weak endothelial cells without smooth muscle.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eEmbolic agent detecting\u003c/h2\u003e\u003cp\u003eTantalum content standard curve: Y\u0026thinsp;=\u0026thinsp;141.693X\u0026thinsp;+\u0026thinsp;54.021(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Tantalum content of OMH in the MMAE\u0026thinsp;+\u0026thinsp;BHD group was 1.678\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06127ug/g, and that in the BHD group was 0.1000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01304ug/g. Tantalum content of OMH in the MMAE\u0026thinsp;+\u0026thinsp;BHD group was significantly higher than that in the BHD group, with statistical difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eExpression of dura and OMH angiogenesis related factors\u003c/h2\u003e\u003cp\u003eOMH showed a large number of neovascularization, and we further explored the factors leading to angiogenesis, focusing on IHC and IF staining of VEGF, VEGFR-1 and VEGFR-2. The results showed that the contents of VEGF, VEGFR-1 and VEGFR-2 in OMH were significantly higher than those in dura (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026thinsp;~\u0026thinsp;C). The results of Mean Fluorescence Intensity (MFI) showed that compared with dura, the contents of VEGF, VEGFR-1 and VEGFR-2 in OMH were significantly increased, with statistical significance (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eImaging features of dura and OMH\u003c/h2\u003e\u003cp\u003eAll patients showed varying degrees of significant linear enhancement on the T1 enhancement sequence, showing dura and OMH structures. At the same time, we measured the thickness of development on the enhanced sequence, meninges and OMH HE stained sections. The average thickness of linear enhancement on the MRI enhanced sequence was 1.71mm, while the average thickness of dura plus OMH was 1.60 mm, and the average thickness of dura was 0.5mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provided histopathological evidence of penetrating vessels between the dura and OMH, characterized by their spiral trajectory towards the OMH. These vessels were identified as potential arterial suppliers for CSDH development. Through quantitative analysis of tantalum distribution in MMAE\u0026thinsp;+\u0026thinsp;BHD patients, we demonstrated significantly higher tantalum concentrations in OMH in the MMAE\u0026thinsp;+\u0026thinsp;BHD group compared to those in the BHD group, indicating functional vascular channels between MMA and OMH. MRI enhancement analysis revealed linear enhancement (average 1.71 mm) corresponding to combined dural-OMH structures, validated by histological measurements ( average 1.60 mm).\u003c/p\u003e\u003cp\u003eIn 1987, Roland, J. found the image of a spiral penetrating artery originating from the anastomotic network of secondary superficial artery in the microangiogram of falx cerebri.\u003csup\u003e(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e)\u003c/sup\u003e According to Maksim Shapiro\u0026rsquo;s theory published in 2021, the dura could be divided into periosteal layer (PL), meningeal layer (ML), border cell layer (BCL).\u003csup\u003e(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u003c/sup\u003e Theoretically, in the relatively avascular ML, there were penetrating vessels from the secondary anastomotic network of PL (20\u0026thinsp;~\u0026thinsp;40\u0026micro;m) to the capillary network within the BZL layer (10\u0026micro;m), which was consistent with our finding of the penetrating vessels between the dura mater and OMH. In addition, the analysis of the results of HE staining, Masson staining, IHC and IF shown that OMH exhibits a large number of neovascularizations, red blood cell leakage, and inflammatory cell infiltration, which was consistent with other studies on the pathological characteristics of CSDH.\u003csup\u003e(\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThrough quantitative analysis of tantalum distribution in MMAE\u0026thinsp;+\u0026thinsp;BHD patients, we demonstrated significantly higher tantalum concentrations in OMH in the MMAE\u0026thinsp;+\u0026thinsp;BHD group compared to those in the BHD group. Given that tantalum was a component of onyx and could be visualized under X - rays, this result directly confirmed the existence of a channel between the dura and OMH. This channel was most likely the penetrating artery, thus providing a breakthrough basis for the treatment of CSDH by MMAE. In addition, by imaging analysis, it was confirmed that the linear enhancement of CSDH under T1 enhancement was formed by both OMH and the dura mater, providing imaging evidence for the inflammatory changes and rich blood vessels in OMH.\u003c/p\u003e\u003cp\u003eOur multimodal analysis established OMH as the pathophysiological epicenter of CSDH progression, characterized by unique angiogenic activation and inflammatory microenvironment. The demonstrated MMA-OMH vascular connectivity provided mechanistic validation for MMAE, while VEGF/CD31 overexpression of OMH proposed testable targets for pharmacological intervention. Based on these research findings, we proposed the potential value of anti-angiogenic drugs in the treatment of CSDH. In the future, it is urgent to conduct more controlled clinical trials to rigorously evaluate the drugs used for the non-surgical treatment of CSDH, especially those targeting the inflammatory-angiogenic pathogenic mechanism, so as to promote the development and progress of the CSDH treatment field.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study identified penetrating dura-OMH vessels and detected tantalum in OMH after MMAE. High VEGF and CD31 expressions were observed in OMH\u0026rsquo;s neovascular endothelium. These findings support the inflammatory-angiogenic theory of CSDH pathogenesis and provide anatomical evidence for MMAE mechanisms. This study proposed the potential value of anti-angiogenic drugs and emphasized the urgency of more controlled clinical trials to promote CSDH treatment development.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eDisclosures:\u003c/h2\u003e\u003cp\u003eThe authors have no personal, financial, or institutional conflict of interest in any of the drugs, materials, or devices described in this article.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis research was supported by \"Rising Stars of Medical Talents\" Youth Development Program of Shanghai Municipal Health Commission (2023-62); by the \u0026ldquo;Changfeng\u0026rdquo; Talent Program of Changhai Hospital (2024-1); and by the \u0026ldquo;GuHai\u0026rdquo; Program of Changhai Hospital (GH145-05).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRenkun Zhang, Qiuyu Lu and Yuhao Tan wrote the main manuscript text and prepared figures 1-4. Yu Zhou, Zifu Li, Lei Zhang, Qinghai Huang, Yi Xu, Yongwei Zhang, Rui Zhao, Qiang Li, Pengfei Yang, Qiao Zuo and Jianmin Liu reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRauhala M, Luoto TM, Huhtala H, Iverson GL, Niskakangas T, \u0026Ouml;hman J et al (2019) The incidence of chronic subdural hematomas from 1990 to 2015 in a defined Finnish population. J Neurosurg 132(4):1147\u0026ndash;1157\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeghali J, Yang W, Huang J (2020) Updates in Chronic Subdural Hematoma: Epidemiology, Etiology, Pathogenesis, Treatment, and Outcome. World Neurosurg 141:339\u0026ndash;345\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKolias AG, Chari A, Santarius T, Hutchinson PJ (2014) Chronic subdural haematoma: modern management and emerging therapies. Nat Rev Neurol 10(10):570\u0026ndash;578\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee KS (2019) How to Treat Chronic Subdural Hematoma? Past and Now. J Korean Neurosurg Soc 62(2):144\u0026ndash;152\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGazzeri R, Laszlo A, Faiola A, Colangeli M, Comberiati A, Bolognini A et al (2020) Clinical investigation of chronic subdural hematoma: Relationship between surgical approach, drainage location, use of antithrombotic drugs and postoperative recurrence. Clin Neurol Neurosurg 191:105705\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlmenawer SA, Farrokhyar F, Hong C, Alhazzani W, Manoranjan B, Yarascavitch B et al (2014) Chronic subdural hematoma management: a systematic review and meta-analysis of 34,829 patients. Ann Surg 259(3):449\u0026ndash;457\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIronside N, Nguyen C, Do Q, Ugiliweneza B, Chen CJ, Sieg EP et al (2021) Middle meningeal artery embolization for chronic subdural hematoma: a systematic review and meta-analysis. J Neurointerv Surg 13(10):951\u0026ndash;957\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZuo Q, Ni W, Yang P, Gu Y, Yu Y, Yang H et al (2023) Managing non-acute subdural hematoma using liquid materials: a Chinese randomized trial of middle meningeal artery treatment (MAGIC-MT)-protocol. Trials 24(1):586\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDavies JM, Knopman J, Mokin M, Hassan AE, Harbaugh RE, Khalessi A et al (2024) Adjunctive Middle Meningeal Artery Embolization for Subdural Hematoma. N Engl J Med 391(20):1890\u0026ndash;1900\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu J, Ni W, Zuo Q, Yang H, Peng Y, Lin Z et al (2024) Middle Meningeal Artery Embolization for Nonacute Subdural Hematoma. N Engl J Med 391(20):1901\u0026ndash;1912\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFiorella D, Monteith SJ, Hanel R, Atchie B, Boo S, McTaggart RA et al (2024) Embolization of the Middle Meningeal Artery for Chronic Subdural Hematoma. N Engl J Med\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEdlmann E, Giorgi-Coll S, Whitfield PC, Carpenter KLH, Hutchinson PJ (2017) Pathophysiology of chronic subdural haematoma: inflammation, angiogenesis and implications for pharmacotherapy. J Neuroinflammation 14(1):108\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQiu S, Zhuo W, Sun C, Su Z, Yan A, Shen L (2017) Effects of atorvastatin on chronic subdural hematoma: A systematic review. Med (Baltim) 96(26):e7290\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBerghauser Pont LM, Dirven CM, Dippel DW, Verweij BH, Dammers R (2012) The role of corticosteroids in the management of chronic subdural hematoma: a systematic review. Eur J Neurol 19(11):1397\u0026ndash;1403\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIorio-Morin C, Blanchard J, Richer M, Mathieu D (2016) Tranexamic Acid in Chronic Subdural Hematomas (TRACS): study protocol for a randomized controlled trial. Trials 17(1):235\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang J, Gao C, Dong J, Zhang J, Jiang R (2020) Drug treatment of chronic subdural hematoma. Expert Opin Pharmacother 21(4):435\u0026ndash;444\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShapiro M, Walker M, Carroll KT, Levitt MR, Raz E, Nossek E et al (2021) Neuroanatomy of cranial dural vessels: implications for subdural hematoma embolization. J Neurointerv Surg 13(5):471\u0026ndash;477\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHaines DE (1991) On the question of a subdural space. Anat Rec 230(1):3\u0026ndash;21\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHaines DE, Harkey HL, al-Mefty O (1993) The subdural space: a new look at an outdated concept. Neurosurgery 32(1):111\u0026ndash;120\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eInglis K (1946) Subdural haemorrhage, cysts and false membranes; illustrating the influence of intrinsic factors in disease when development of the body is normal. Brain 69(3):157\u0026ndash;194\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu X, Wang D, Han Z, Wang B, Gao W, Fan Y et al (2021) A novel rat model of chronic subdural hematoma: Induction of inflammation and angiogenesis in the subdural space mimicking human-like features of progressively expanding hematoma. Brain Res Bull 172:108\u0026ndash;119\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJafari N, Gesner L, Koziol JM, Rotoli G, Hubschmann OR (2017) The Pathogenesis of Chronic Subdural Hematomas: A Study on the Formation of Chronic Subdural Hematomas and Analysis of Computed Tomography Findings. World Neurosurg 107:376\u0026ndash;381\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoland J, Bernard C, Bracard S, Czorny A, Floquet J, Race JM et al (1987) Microvascularization of the intracranial dura mater. Surg Radiol Anat 9(1):43\u0026ndash;49\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chronic subdural hematoma, Dura, Outer membrane, Pathophysiological mechanism, VEGF, CD31","lastPublishedDoi":"10.21203/rs.3.rs-7242529/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7242529/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eTo further explore the pathophysiological mechanisms of the chronic subdural hematoma (CSDH) through the simultaneous histopathological, immunological, embolic-agent-detecting, and imaging analyses of the dura and the outer membrane of the hematoma (OMH).\u003c/p\u003e\u003ch2\u003eMethod\u003c/h2\u003e\u003cp\u003eSpecimens were collected from 5 patients who underwent burr hole drainage craniectomy (BHD) of CSDH and 5 patients who underwent middle meningeal artery embolization (MMAE)\u0026thinsp;+\u0026thinsp;BHD at the Neurovascular Center of Shanghai Changhai Hospital from March 2022 to December 2022. The dura and OMH removed intraoperatively were sent for histopathological, immunological, and embolic-agent-detecting analyses (detecting the tantalum content). The preoperative MRI of the patients were studied to observe the dura and OMH and measure the width of the linearly enhanced tissue on the T1 enhanced sequence.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe dura and OMH were closely connected under the light microscope. the dura mainly consisted of collagen fibers, and blood vessels were normal. The OMH is rich in blood vessels and uneven in thickness, and collagen fibers were scattered, with inflammatory cells infiltrated, new blood vessels formed, and red blood cell exudation, and had higher VEGF, VEGFR \u0026minus;\u0026thinsp;1, and VEGFR \u0026minus;\u0026thinsp;2 contents than dura. Penetrating vessels were at the dura - OMH junction. The MMAE\u0026thinsp;+\u0026thinsp;BHD group's OMH had higher tantalum content than BHD\u0026rsquo;s. All patients had linear enhancement in the T1 enhanced sequence. The MRI enhanced sequence's linear enhancement averaged 1.71 mm in thickness, the dura plus OMH averaged 1.60 mm, and the dura averaged 0.5 mm.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study identified penetrating dura-OMH vessels and detected tantalum in OMH after MMAE. High VEGF and CD31 expressions were observed in OMH\u0026rsquo;s neovascular endothelium. These findings support the inflammatory-angiogenic theory of CSDH pathogenesis and provide anatomical evidence for MMAE mechanisms. The detected angiogenic biomarkers suggest potential therapeutic value of anti-angiogenic drugs for CSDH.\u003c/p\u003e","manuscriptTitle":"Pathophysiological Mechanisms of Chronic Subdural Hematoma: Insights from Histopathological, Immunological, and Imaging Analyses Pathophysiological Mechanisms of CSDH","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-23 02:44:54","doi":"10.21203/rs.3.rs-7242529/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":"8db4c21f-867e-47ac-a8ae-87803f2820f4","owner":[],"postedDate":"October 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-09T15:38:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-23 02:44:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7242529","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7242529","identity":"rs-7242529","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}