Expression of TNF-α, VEGF-A and microvessel density in cerebral alveolar echinococcosis and their correlation with perilesional brain edema

Expression of TNF-α, VEGF-A and microvessel density in cerebral alveolar echinococcosis and their correlation with perilesional brain edema | 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 Expression of TNF-α, VEGF-A and microvessel density in cerebral alveolar echinococcosis and their correlation with perilesional brain edema Wuerken Wumier, Alimasi Abulizi, Pengfei Wu, Najiahai Jinsihan, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4256751/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Jan, 2025 Read the published version in Acta Parasitologica → Version 1 posted 5 You are reading this latest preprint version Abstract Alveolar echinococcosis (AE) is an infrequent zoonosis caused by Echinococcus multilocularis with a high degree of disability and mortality. Metastatic cerebral alveolar echinococcosis (CAE) is very rare and the lesions could lead to severe perilesional brain edema (PLBE) and subsequent uncontrollable intracranial hypertension. In this study, we sought to determine the expression of edema-associated factors in CAE lesions and their associations with PLBE. We retrospectively evaluated the clinical data of 18 CAE patients who received craniotomy. Severity of PLBE was described by edema index (EI). Archived specimens were processed for immunohistochemistry to detect tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor A (VEGF-A) and microvessel density (MVD) in CAE lesions. Expression intensity of CAE lesions was quantified by integral optical density (IOD) or count and was compared to the control group. The results showed TNF-α and VEGF-A were significantly expressed in CAE lesions ( p < 0.001), their levels were positively correlated with PLBE (TNF-α: r = 0.701, p = 0.001; VEGF-A: r = 0.803, p < 0.001). The MVD of CAE lesions had a similar expression with normal brain tissue, and it was positively correlated with PLBE and VEGF-A (PLBE: r = 0.849, p < 0.001; VEGF-A: r = 0.687, p = 0.002). In conclusion, we speculated the upregulation of TNF-α and VEGF-A induced the formation of PLBE. Besides, though there was no extra increase of MVD, it was still regulated by VEGF-A and provided a better anatomical basis for the formation of PLBE and further promoted it. Cerebral alveolar echinococcosis TNF-α VEGF-A Microvessel density Brain edema Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Alveolar echinococcosis (AE) is a potentially lethal zoonotic disease caused by infecting Echinococcus multilocularis[1]. This parasite is widely distributed over the northern hemisphere and it is typically maintained in the definitive hosts like canids[2]. Humans develop into accidental intermediate hosts through ingesting infective ova and an increasing number of AE cases in western, northern and eastern Europe, as well as in central Asia were reported in the past 20 years[3]. The most affected organ is the liver and the parasite caused tumor-like malignant lesions could metastasize to distance via lymphatic or various hematogenous pathways[4, 5]. Metastatic cerebral alveolar echinococcosis (CAE) is very rare and only 1–3% of infected patients could be observed[5, 6]. Therefore, this cerebral occupying disease is easy to be neglected. In published research on CAE, obvious perilesional brain edema (PLBE) was confirmed through magnetic resonance imaging (MRI), and clinical findings showed chiefly intracranial hypertension even could lead to a severe cerebral hernia[7–9]. Therefore, PLBE is now a major factor that governs clinical management, but there are no possible causes of edema or related formation explain associated with CAE have been reported so far. The progression and outcome of brain edema could be affected by the intricate relationship between the blood-brain barrier (BBB) and tumor necrosis factor alpha (TNF-α)[10, 11]. In recent years, TNF-α was confirmed to intact BBB by a receptor-mediated transport system that is influenced by central nervous system (CNS) trauma, inflammation and stroke[12, 13]. In typical hepatic AE lesions, TNF-α had also been proven to exist significant expression in this parasitic granulomatous lesion[14, 15], however, what role it plays in human cerebral parasitic substantive lesions and PLBE remain a question. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and angiogen, as we all know, it is essential in inducing angiogenesis and increasing vascular permeability[16]. VEGF-A is also known as the vascular permeability factor of the VEGF family and it has been regarded as a key regulator for neovascularization and PLBE formation in some brain tumors such as meningiomas or glioma[17, 18], and the angiogenic activity could be reflected in the development of novel microvessels in tumor tissue that is quantified by the intratumoral microvessel density (MVD). Considering the similarities in some biological behavior between CAE and brain tumors, the expression of VEGF-A and MVD in CAE and their correlation between PLBE are also waiting to be unveiled. Therefore, in the present study, we tried to analyze the expression of TNF-α, VEGF-A and MVD in metastatic CAE lesions, and observe their association with the PLBE. Materials and methods This research was conducted following the Helsinki Declaration and approved by the institutional research ethics committee of Xinjiang Medical University. Written informed consent was obtained for each participant. Patients and specimens From January 1st, 2012 to May 31st, 2020, 18 patients with metastatic CAE received craniotomy for resection of the target lesions in the First Affiliated Hospital of Xinjiang Medical University were analyzed in this study. All patients were excluded from other intracranial diseases. Simultaneously, 15 patients with epilepsy who received temporal lobectomy during the same period and whose tissue did not show any histologic changes were selected as the control group to contrast the content of detected cytokines with CAE lesions. All paraffin-embedded sections were acquired from pathology archives. Immunohistochemical analysis Paraffin-embedded sections from 18 patients with CAE were made histological assessment and rediagnosis through Hematoxylin-eosin stain (H&E) and Periodic Acid-Schiff stain (PAS) by pathologists (Fig. 1 ). Standard immunohistochemistry was performed. The following primary antibodies were used for staining the tissue sections: anti-VEGF-A antibody (ab183100, Abcam; 1:50, Cambridge, MA, USA), anti-TNF-α, antibody (ab9579, Abcam; 1:100), and anti-CD34 antibody (ab81289, Abcam; 1:200) for staining of endothelial cells to evaluate the MVD. The retrieval of antigen in the tissue sections was performed in 0.01 mol citrate-buffered solution (pH 6.0) employing microwave irradiation for 15 min. PBS was used to replace the primary antibodies as the negative control, specimens of granulomatous inflammation, liver cancer and kidney were used as the positive control for TNF-α, VEGF-A and CD34 respectively. Integral optical density (IOD) was used to quantify the intensity of TNF-α and VEGF-A expression. Images stained were captured with Leica DM3000 microscope (Leica Microsystems, Inc., Wetzlar, Germany): In each tissue section five areas of most prominent positive expression (hot spots) were identified at ×40 power field, and images were selected under ×400 magnification. The IOD SUM of each field was measured with image-Pro Plus v. 6.0 (Media Cybernetics, Inc., Silver Spring, MD, USA), and the optical density was corrected with the segmentation set at a constant level to allow for the detection of positive immunostaining. The mean values of IOD readings obtained from five fields were used. Any CD34 positive endothelial cell clusters distinctly separated from each other were considered as single countable microvessels and a lumen was not required to identify a vessel. Identifying five areas of the most prominent vascular density (hot spots) in the same way and microvessel counting was done under ×400 magnification. Counting was performed by two experienced pathologists blinded to clinical information and mean values of MVD obtained from five fields were used. Evaluation of neuroradiological imaging All CAE patients were evaluated using MRI before surgery. Development and severity of PLBE were described by the edema index (EI) which was defined as: (V lesion + V edema ) / V lesion . Lesion volume was estimated on gadolinium-enhanced T1-weighted images and PLBE was evaluated on FLAIR or T2-weighted images. The lesion and PLBE volume were approximated from axial, coronal, and sagittal images respectively: the maximum perpendicular diameters (a and b: X and Y axis radii) were measured on axial scans, and the extent (c: Z axis radius) was measured on coronal or sagittal scans. The ellipsoid formula (V = 4/3πabc) was used to calculate V lesion and V edema . When EI equals 1 signifies edema is absent and EI would increase with the severity of the edema. Statistical analysis Statistical analyses were conducted with IBM SPSS statistics 25.0 (IBM Corp., Armonk, NY, USA). Continuous variables were expressed as the mean ± standard (SD). Students' t-test was used for comparison between categorical groups. Correlations were accessed by 2-tailed Pearson correlation coefficient and expressed as an r value. A 2-sided p value less than 0.05 was considered statistically significant. Results Patient characteristics Table 1 shows the characteristics of patients and controls. A total of 18 patients with CAE were included in this present study (2 women and 16 men, age range 23–61 years, mean age 38.20 years, SD 9.71 years), and the lesions resected from surgery were located in the frontal lobe (4 cases, 22%), temporal lobe (4 cases, 22%), parietal lobe (3 cases, 17%), occipital lobe (2 cases, 11%), basal ganglia (3 cases, 17%), cerebellum (1 case, 5.5%) and brainstem (1 case, 5.5%) respectively. Control brain sections were from 15 patients with epilepsy without any histologic changes (2 women and 13 men, age range 22–65 years, mean age 39.67 years, SD 10.35 years). The control group was chosen under an age and sex matching principle. MRI revealed the evident PLBE in all 18 CAE patients (EI > 1), and the mean value of the EI was 12.26 ± 9.38 (1.84–42.37). Typical MRI presentations are displayed in Fig. 2 . Table 1 Patient characteristics Variable CAE Control Number of patients 18 15 Age (years) 38.20 ± 9.71 (23–61) 39.67 ± 10.35 (22–65) Sex M: 16 F: 2 M: 13 F: 2 Edema index (EI) 12.26 ± 9.38 (1.84–42.37) – Locations Frontal lobe 4 – Temporal lobe 4 Parietal lobe 3 Occipital lobe 2 Basal ganglia 3 Cerebellum 1 Brainstem 1 – – – – – – Expression of TNF-α, VEGF-A and MVD The positive expression product appeared as brown-yellow particles and we observed that the positive expressions of TNF-α, VEGF-A and MVD were mainly expressed in peripheral immune cells of lesions (Fig. 3 ). IOD analysis showed the quantification of TNF-α and VEGF-A were all significantly higher in patients with CAE ( p < 0.001). Only slightly elevated MVD counts could be observed in patients with CAE and the differences were not significant ( p = 0.866) (Table 2 ). Table 2 Expression of TNF-α, VEGF-A and MVD Variable CAE Control t value P value TNF-α (IOD) 153230.44 ± 77741.48 6588.69 ± 3759.45 7.99 <0.001 VEGFA (IOD) 154339.33 ± 73613.69 17251.16 ± 5213.28 7.88 <0.001 MVD (Count) 6.70 ± 1.78 6.60 ± 1.54 0.17 0.866 Correlation of variables with PLBE We analyzed the correlations between TNF-α, VEGFA, MVD and PLBE respectively. According to the scatter diagrams, the all their IOD values changed in the same direction with EI (Fig. 4 ), and the Pearson correlation analysis demonstrated dramatically positive correlations (TNF-α: r = 0.701, p = 0.001; VEGF-A: r = 0.803, p < 0.001; MVD: r = 0.849, p < 0.001). Simultaneously, we also found a positive correlation between VEGF-A and MVD ( r = 0.687, p = 0.002). Discussion CAE is a kind of cerebral space-occupying parasitic granuloma and with similar biological behavior to malignant tumors. The underlying mechanisms for CAE are widely considered as the rupture of a solitary cyst or embolization of cyst particles from ruptured cysts inside remote organs (mostly the liver)[19]. Cerebral lesions could grow to cause varying degrees of mass effect and the accompanying PLBE tends to cause intolerable intracranial pressure. The pathogenesis of PLBE is unknown, it seems to be a vasogenic type of edema according to the previous reports and the MRI findings we observed[7, 8]. With the rapid development of modern molecular biology, many scholars have investigated the mechanisms of brain edema caused by different diseases at the molecular level. Given the rarity of parasitic diseases of the brain, there have been few studies reported on brain edema caused by them. In our present study, we analyzed the clinical data and surgically resected specimens of 18 CAE patients, and further verified the microscopic presentations and characteristics of the CAE lesions, then we first revealed that: (1) TNF-α and VEGF-A were significantly expressed in the CAE lesions, and their levels were positively correlated with PLBE. (2) There was no higher expression of MVD in CAE lesions compared with the normal brain tissue, but it still positively correlated with VEGF-A and PLBE. In our microscopic observation, the CAE lesions are characterized by a collection of immune cells that attempt to immunologically restrain, and thoroughly surround the cystic structures of Echinococcus multilocularis. The central areas of granulomas, called caseous necrosis are essentially dead tissue surrounded by granulomatous inflammation. Immunohistochemical positive areas were mainly concentrated in the peripheral lesions. These pathological changes and their MRI characteristics are very reminiscent of the other granulomatous disease - Mycobacterium tuberculosis (MTB). Evidence indicates that TNF-α is a quite representative immune mediator in both formation and maintenance of granulomas in MTB, and mononuclear phagocytes represent the dominant cellular source of this cytokine[20]. However, the precise mechanisms of how this cytokine influences the granuloma formation are still unclear, Roach et al. suggested that TNF-α is required for the early induction of various chemokines that initiate cell recruitment, as well as for the establishment of the close apposition of Immune cells to maintain the granuloma[21]. Besides, TNF-α is predominantly important in its capacity to sustain a state of dormancy of MTB infection[22]. Amiot et al. had performed the experiments on mice carrying a deletion of TNF-α genes and confirmed that TNF-a plays an essential role in the protection mechanisms against Echinococcus multilocularis through inducing the development of efficient granulomas[23]. However, as a proinflammatory cytokine, an excess of TNF-α would promote immunopathology by interfering with cell death processes and induction of a hyper-inflammatory milieu, thus causing organ dysfunction[24]. Effects of excessive circulating inflammatory cytokines such as fever, weight loss, and tissue damage may be attributed to TNF-α.[22] These two distinct effects (protection and damage) seem to be contradictory and this contradiction may exist in CAE, of course, the specific mechanism of TNF-α in the formation of CAE lesions and whether with similarity to bacterial granuloma need further research. In addition to high expression in lesions, we also noted the expression of TNF-a is associated with different degrees of PLBE. TNF-α is considered as a pleiotropic cytokine that existed in inflammatory cascades at different phases of CNS injury[25]. The latest study identified TNF-α as a key proinflammatory cytokine that triggers the cerebral endothelium (EC) necroptosis[26], at the same time, it could also increase the permeability of blood-brain barrier (BBB) through endothelial nitric oxide synthase (eNOS) activation in endothelial cells[27]. All these accordingly contribute to vasogenic brain edema formation. Under the inflammatory environment, activated immune cells could synthesize the cell surface cytotoxic integral transmembrane form of TNF-α at the particular site of inflammation and influence the cell target by either cell-to-cell contact or local release of the soluble secretory form of TNF-α.[24] These findings suggest that TNF-α was very likely to act in the process of PLBE formation. It was regretful that perilesional brain tissues were not obtained in our experiment and the expression of TNF-α in perilesional areas is unclear. Research over the past few years has confirmed the multiple roles of VEGF-A in physiological function and pathogenic effect. In the normal healthy nervous system, VEGF-A is a crucial regulator of CNS blood vessel formation and regulates the MVD[28]. This angiogenesis mechanism also exists in the growth of tumors and granuloma[29, 30]. Interestingly, we did notice the significant expression of VEGF-A, but there is no obvious increase of MVD in CAE lesions. In our present study, the MVD of CAE lesions had a similar expression with normal brain tissue and it was mainly centered on the external lesions, which corresponded to the characteristic of low/normal blood supply reported in previous surgical reports and imageology studies[8, 31, 32]. However, the MVD still showed a positive correlation with VEGF-A, in other words, the angiogenesis mediated by VEGF-A may be weakened or suppressed to some extent, on the plus side, this limited vascular growth would reduce the further cerebral spread of the Echinococcus multilocularis. Meanwhile, the positive correlations in MVD and VEGF-A between PLBE were indicated. Low maintenance levels of VEGF-A are necessary for endothelial cell survival and the integrity of the BBB[28], however, as a vascular permeability factor at high levels, VEGF-A jeopardizes the CNS homeostasis through disruption of the BBB: stimulating the creation of interendothelial gaps, fragmentations, and fenestrations in the brain endothelium[18]. These structural changes could result in the fluid leakage from the vascular lumen into the brain parenchyma, which results in vasogenic edema and high interstitial fluid pressure (IFP). Besides, the MVD regulated by VEGF-A would provide a better anatomical basis for the effect of VEGF-A on BBB, further promote the formation of edema. A similar pro-edematous association had been reported in brain tumors like meningioma, and Ding et al. confirmed that the VEGF-A secreted by the tumor tissue enter peritumoral normal brain tissue to induce edema[33]. Furthermore, the latest study shows the VEGF-A inhibitors could relieve brain edema caused by brain tumors and reduce granuloma-associated pathologies[18, 34], which may provide a new idea for the clinical control of CAE. In this study, significant expression of TNF-α and VEGF-A in CAE lesions were found and we speculated that the upregulation of TNF-α and VEGF-A induced the formation of PLBE. Besides, though there was no extra increase of MVD in CAE lesions compared to normal brain tissue, it was still regulated by VEGF-A and provide a better anatomical basis for the formation of PLBE and further promote it. These findings may help us better realize the characteristics of cerebral parasitic granuloma and provide a new therapeutic strategy for CAE. Declarations Conflict of Interest: The authors declare no conflict of interest. Ethical Approval: This research was conducted following the Helsinki Declaration and approved by the institutional research ethics committee of Xinjiang Medical University. Written informed consent was obtained for each participant. Permission: Written informed consent was obtained from each patient and copy of the consent will be available from the corresponding author on reasonable request from the journal editor. Source of Funding: This study was funded by the State Key Laboratory of Pathogenesis, Prevention, Treatment of Central Asian High Incidence Diseases Fund (2022E01068), State Key Laboratory of Pathogenesis, Prevention, Treatment of Central Asian High Incidence Diseases Fund (SKL-HIDCA-2022-8). Authors' contributions WW and AA equally contributed to the work, ZW and SD contributed to the design of experiment and carried out research; PW contributed to data collection and analyzed data, YZ checked the validity of data, TT and AA wrote the paper. 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Harding, M. Herbath, Y. Chen, A. Rayasam, A. Ritter, B. Csoka, G. Hasko, I.P. Michael, Z. Fabry, A. Nagy, M. Sandor, VEGF-A from Granuloma Macrophages Regulates Granulomatous Inflammation by a Non-angiogenic Pathway during Mycobacterial Infection, Cell reports 27(7) (2019) 2119–2131.e6. Cite Share Download PDF Status: Published Journal Publication published 24 Jan, 2025 Read the published version in Acta Parasitologica → Version 1 posted Editorial decision: Major revisions 18 Jun, 2024 Reviewers agreed at journal 24 Apr, 2024 Reviewers invited by journal 23 Apr, 2024 Editor assigned by journal 17 Apr, 2024 First submitted to journal 12 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-4256751","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":294705861,"identity":"754b0b11-05c5-41e8-95c0-13f28e50fdc8","order_by":0,"name":"Wuerken Wumier","email":"","orcid":"","institution":"Xinjiang Medical University Affiliated First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wuerken","middleName":"","lastName":"Wumier","suffix":""},{"id":294705862,"identity":"306fe341-7976-48bc-981c-1ea08b803f16","order_by":1,"name":"Alimasi Abulizi","email":"","orcid":"","institution":"Xinjiang Medical University Affiliated First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Alimasi","middleName":"","lastName":"Abulizi","suffix":""},{"id":294705863,"identity":"4ee7ea9a-851f-45c6-97a2-e1feca4638bc","order_by":2,"name":"Pengfei Wu","email":"","orcid":"","institution":"Xinjiang Medical University Affiliated First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pengfei","middleName":"","lastName":"Wu","suffix":""},{"id":294705864,"identity":"03dc12ed-973c-43c4-8313-a1614d231291","order_by":3,"name":"Najiahai Jinsihan","email":"","orcid":"","institution":"NNMC: National Research Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Najiahai","middleName":"","lastName":"Jinsihan","suffix":""},{"id":294705865,"identity":"504f8678-d314-4d37-aee6-44950da80df1","order_by":4,"name":"Yongxin Wang","email":"","orcid":"","institution":"Xinjiang Medical University Affiliated First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yongxin","middleName":"","lastName":"Wang","suffix":""},{"id":294705866,"identity":"dcf45a89-86be-4763-95ed-bcbf5a486239","order_by":5,"name":"Serick Duysenbi","email":"","orcid":"","institution":"NNMC: National Research Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Serick","middleName":"","lastName":"Duysenbi","suffix":""},{"id":294705867,"identity":"a4646f11-f2ba-40f9-b13a-f8e84488c78f","order_by":6,"name":"Zengliang Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYFAC5uYHDAw2PPzsDURrYWwzYGBIk5HsOUC8lgYJBobDNgY3HIjUYHD+YINxwa/zPAw3GBg/fMwhRsuBgw2PZ/bd5mGc3cAsOXMbEVrMDjY2GPP23OZhljnAxsxLlJbDjA3SvD3neNgkEojVcgyohefHAR4eorXYn2FsM+ZtSOaR4DnYTJxfJPsPH37M88fO3v5488EPH4nRAgaMbWCygVj1IPCHFMWjYBSMglEw4gAAyJ42ts/ojZgAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-8411-5678","institution":"Xinjiang Medical University Affiliated First Hospital","correspondingAuthor":true,"prefix":"","firstName":"Zengliang","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-04-12 09:24:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4256751/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4256751/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11686-024-00943-7","type":"published","date":"2025-01-24T15:57:43+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55629809,"identity":"8c3122cd-8149-4aab-ad14-e6f34ee7c446","added_by":"auto","created_at":"2024-04-30 19:12:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":164920,"visible":true,"origin":"","legend":"\u003cp\u003ePathologic characteristics of CAE lesion. (A)Microscopical observation indicated the multiple cysts surrounded by necrosis and an intense granulomatous reaction infiltrated by plasmacytes, eosinophils, lymphocytes, and multinucleated giant cells under hematoxylin and eosin stain (original magnification, ×200; magnification bar, 100 μm). (B) Periodic acid-Schiff-positive, narrow, bizarre lamellar structures of thin diameters could be seen in cysts (original magnification, ×100; magnification bar, 200 μm).\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4256751/v1/0b01edaa0e77b720db755f3a.png"},{"id":55629279,"identity":"0a80bda8-eae1-417d-9093-c860725565fc","added_by":"auto","created_at":"2024-04-30 19:04:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":526511,"visible":true,"origin":"","legend":"\u003cp\u003eTypical magnetic resonance images (MRI) of CAE patient. (A) Axial T2-weighted image showing the iso-/hyperintense nodular lesion in the left insula with extensive perifocal edema. (B) Axial T1-weighted image with contrast enhancement showing this iso-/hypointense lesion with annular enhancement sign. (C) Sagittal T2-weighted image of lesion. (D) Coronal T1-weighted image with contrast enhancement of lesion.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4256751/v1/49738963cf80a488326ee5e0.png"},{"id":55629282,"identity":"8540ec55-dbdb-497c-8bae-a7aaf8a281f4","added_by":"auto","created_at":"2024-04-30 19:04:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":589385,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical positive expression in CAE and control group. Expression of TNF-α and VEGF-A is significantly higher in CAE while MVD expressed no statistical difference between control group (original magnification, ×400; magnification bar, 50 μm).\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4256751/v1/01e192bd2b18011b7beb281a.png"},{"id":55629281,"identity":"e0ded479-12de-4611-bc1d-b070492f197b","added_by":"auto","created_at":"2024-04-30 19:04:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":44402,"visible":true,"origin":"","legend":"\u003cp\u003eScatter diagrams of variables. (A) Correlation between TNF-α and EI (\u003cem\u003er\u003c/em\u003e = 0.701, \u003cem\u003ep\u003c/em\u003e= 0.001). (B) Correlation between VEGF-A and EI (\u003cem\u003er\u003c/em\u003e = 0.803, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001). (C) Correlation between MVD and EI (\u003cem\u003er\u003c/em\u003e = 0.849, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001). (D) Correlation between VEGF-A and MVD (\u003cem\u003er\u003c/em\u003e = 0.687, \u003cem\u003ep\u003c/em\u003e= 0.002).\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4256751/v1/acc09dc5ac612e9d2380e9b4.png"},{"id":74858594,"identity":"eda2b59a-942c-4677-96ed-85d3f51e2397","added_by":"auto","created_at":"2025-01-27 16:12:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2255780,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4256751/v1/161f882b-4609-43d8-9e42-00672bd383cc.pdf"}],"financialInterests":"","formattedTitle":"Expression of TNF-α, VEGF-A and microvessel density in cerebral alveolar echinococcosis and their correlation with perilesional brain edema","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlveolar echinococcosis (AE) is a potentially lethal zoonotic disease caused by infecting Echinococcus multilocularis[1]. This parasite is widely distributed over the northern hemisphere and it is typically maintained in the definitive hosts like canids[2]. Humans develop into accidental intermediate hosts through ingesting infective ova and an increasing number of AE cases in western, northern and eastern Europe, as well as in central Asia were reported in the past 20 years[3]. The most affected organ is the liver and the parasite caused tumor-like malignant lesions could metastasize to distance via lymphatic or various hematogenous pathways[4, 5]. Metastatic cerebral alveolar echinococcosis (CAE) is very rare and only 1\u0026ndash;3% of infected patients could be observed[5, 6]. Therefore, this cerebral occupying disease is easy to be neglected.\u003c/p\u003e \u003cp\u003eIn published research on CAE, obvious perilesional brain edema (PLBE) was confirmed through magnetic resonance imaging (MRI), and clinical findings showed chiefly intracranial hypertension even could lead to a severe cerebral hernia[7\u0026ndash;9]. Therefore, PLBE is now a major factor that governs clinical management, but there are no possible causes of edema or related formation explain associated with CAE have been reported so far.\u003c/p\u003e \u003cp\u003eThe progression and outcome of brain edema could be affected by the intricate relationship between the blood-brain barrier (BBB) and tumor necrosis factor alpha (TNF-α)[10, 11]. In recent years, TNF-α was confirmed to intact BBB by a receptor-mediated transport system that is influenced by central nervous system (CNS) trauma, inflammation and stroke[12, 13]. In typical hepatic AE lesions, TNF-α had also been proven to exist significant expression in this parasitic granulomatous lesion[14, 15], however, what role it plays in human cerebral parasitic substantive lesions and PLBE remain a question.\u003c/p\u003e \u003cp\u003eVascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and angiogen, as we all know, it is essential in inducing angiogenesis and increasing vascular permeability[16]. VEGF-A is also known as the vascular permeability factor of the VEGF family and it has been regarded as a key regulator for neovascularization and PLBE formation in some brain tumors such as meningiomas or glioma[17, 18], and the angiogenic activity could be reflected in the development of novel microvessels in tumor tissue that is quantified by the intratumoral microvessel density (MVD). Considering the similarities in some biological behavior between CAE and brain tumors, the expression of VEGF-A and MVD in CAE and their correlation between PLBE are also waiting to be unveiled.\u003c/p\u003e \u003cp\u003eTherefore, in the present study, we tried to analyze the expression of TNF-α, VEGF-A and MVD in metastatic CAE lesions, and observe their association with the PLBE.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eThis research was conducted following the Helsinki Declaration and approved by the institutional research ethics committee of Xinjiang Medical University. Written informed consent was obtained for each participant.\u003c/p\u003e \u003cp\u003ePatients and specimens\u003c/p\u003e \u003cp\u003eFrom January 1st, 2012 to May 31st, 2020, 18 patients with metastatic CAE received craniotomy for resection of the target lesions in the First Affiliated Hospital of Xinjiang Medical University were analyzed in this study. All patients were excluded from other intracranial diseases. Simultaneously, 15 patients with epilepsy who received temporal lobectomy during the same period and whose tissue did not show any histologic changes were selected as the control group to contrast the content of detected cytokines with CAE lesions. All paraffin-embedded sections were acquired from pathology archives.\u003c/p\u003e \u003cp\u003eImmunohistochemical analysis\u003c/p\u003e \u003cp\u003eParaffin-embedded sections from 18 patients with CAE were made histological assessment and rediagnosis through Hematoxylin-eosin stain (H\u0026amp;E) and Periodic Acid-Schiff stain (PAS) by pathologists (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Standard immunohistochemistry was performed. The following primary antibodies were used for staining the tissue sections: anti-VEGF-A antibody (ab183100, Abcam; 1:50, Cambridge, MA, USA), anti-TNF-α, antibody (ab9579, Abcam; 1:100), and anti-CD34 antibody (ab81289, Abcam; 1:200) for staining of endothelial cells to evaluate the MVD. The retrieval of antigen in the tissue sections was performed in 0.01 mol citrate-buffered solution (pH 6.0) employing microwave irradiation for 15 min. PBS was used to replace the primary antibodies as the negative control, specimens of granulomatous inflammation, liver cancer and kidney were used as the positive control for TNF-α, VEGF-A and CD34 respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIntegral optical density (IOD) was used to quantify the intensity of TNF-α and VEGF-A expression. Images stained were captured with Leica DM3000 microscope (Leica Microsystems, Inc., Wetzlar, Germany): In each tissue section five areas of most prominent positive expression (hot spots) were identified at \u0026times;40 power field, and images were selected under \u0026times;400 magnification. The IOD SUM of each field was measured with image-Pro Plus v. 6.0 (Media Cybernetics, Inc., Silver Spring, MD, USA), and the optical density was corrected with the segmentation set at a constant level to allow for the detection of positive immunostaining. The mean values of IOD readings obtained from five fields were used. Any CD34 positive endothelial cell clusters distinctly separated from each other were considered as single countable microvessels and a lumen was not required to identify a vessel. Identifying five areas of the most prominent vascular density (hot spots) in the same way and microvessel counting was done under \u0026times;400 magnification. Counting was performed by two experienced pathologists blinded to clinical information and mean values of MVD obtained from five fields were used.\u003c/p\u003e \u003cp\u003eEvaluation of neuroradiological imaging\u003c/p\u003e \u003cp\u003eAll CAE patients were evaluated using MRI before surgery. Development and severity of PLBE were described by the edema index (EI) which was defined as: (V\u003csub\u003elesion\u003c/sub\u003e + V\u003csub\u003eedema\u003c/sub\u003e) / V\u003csub\u003elesion\u003c/sub\u003e. Lesion volume was estimated on gadolinium-enhanced T1-weighted images and PLBE was evaluated on FLAIR or T2-weighted images. The lesion and PLBE volume were approximated from axial, coronal, and sagittal images respectively: the maximum perpendicular diameters (a and b: X and Y axis radii) were measured on axial scans, and the extent (c: Z axis radius) was measured on coronal or sagittal scans. The ellipsoid formula (V\u0026thinsp;=\u0026thinsp;4/3πabc) was used to calculate V\u003csub\u003elesion\u003c/sub\u003e and V\u003csub\u003eedema\u003c/sub\u003e. When EI equals 1 signifies edema is absent and EI would increase with the severity of the edema.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted with IBM SPSS statistics 25.0 (IBM Corp., Armonk, NY, USA). Continuous variables were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard (SD). Students' t-test was used for comparison between categorical groups. Correlations were accessed by 2-tailed Pearson correlation coefficient and expressed as an r value. A 2-sided \u003cem\u003ep\u003c/em\u003e value less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003ePatient characteristics\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the characteristics of patients and controls. A total of 18 patients with CAE were included in this present study (2 women and 16 men, age range 23\u0026ndash;61 years, mean age 38.20 years, SD 9.71 years), and the lesions resected from surgery were located in the frontal lobe (4 cases, 22%), temporal lobe (4 cases, 22%), parietal lobe (3 cases, 17%), occipital lobe (2 cases, 11%), basal ganglia (3 cases, 17%), cerebellum (1 case, 5.5%) and brainstem (1 case, 5.5%) respectively. Control brain sections were from 15 patients with epilepsy without any histologic changes (2 women and 13 men, age range 22\u0026ndash;65 years, mean age 39.67 years, SD 10.35 years). The control group was chosen under an age and sex matching principle. MRI revealed the evident PLBE in all 18 CAE patients (EI\u0026thinsp;\u0026gt;\u0026thinsp;1), and the mean value of the EI was 12.26\u0026thinsp;\u0026plusmn;\u0026thinsp;9.38 (1.84\u0026ndash;42.37). Typical MRI presentations are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient characteristics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of patients\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.20\u0026thinsp;\u0026plusmn;\u0026thinsp;9.71 (23\u0026ndash;61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.67\u0026thinsp;\u0026plusmn;\u0026thinsp;10.35 (22\u0026ndash;65)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM: 16 F: 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM: 13 F: 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEdema index (EI)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.26\u0026thinsp;\u0026plusmn;\u0026thinsp;9.38 (1.84\u0026ndash;42.37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocations\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFrontal lobe 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTemporal lobe 4\u003c/p\u003e \u003cp\u003eParietal lobe 3\u003c/p\u003e \u003cp\u003eOccipital lobe 2\u003c/p\u003e \u003cp\u003eBasal ganglia 3\u003c/p\u003e \u003cp\u003eCerebellum 1\u003c/p\u003e \u003cp\u003eBrainstem 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExpression of TNF-α, VEGF-A and MVD\u003c/p\u003e \u003cp\u003eThe positive expression product appeared as brown-yellow particles and we observed that the positive expressions of TNF-α, VEGF-A and MVD were mainly expressed in peripheral immune cells of lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). IOD analysis showed the quantification of TNF-α and VEGF-A were all significantly higher in patients with CAE (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Only slightly elevated MVD counts could be observed in patients with CAE and the differences were not significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.866) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExpression of TNF-α, VEGF-A and MVD\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003et value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNF-α (IOD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e153230.44\u0026thinsp;\u0026plusmn;\u0026thinsp;77741.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6588.69\u0026thinsp;\u0026plusmn;\u0026thinsp;3759.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGFA (IOD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e154339.33\u0026thinsp;\u0026plusmn;\u0026thinsp;73613.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e17251.16\u0026thinsp;\u0026plusmn;\u0026thinsp;5213.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMVD (Count)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.866\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eCorrelation of variables with PLBE\u003c/p\u003e \u003cp\u003eWe analyzed the correlations between TNF-α, VEGFA, MVD and PLBE respectively. According to the scatter diagrams, the all their IOD values changed in the same direction with EI (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), and the Pearson correlation analysis demonstrated dramatically positive correlations (TNF-α: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.701, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; VEGF-A: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.803, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; MVD: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.849, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Simultaneously, we also found a positive correlation between VEGF-A and MVD (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.687, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCAE is a kind of cerebral space-occupying parasitic granuloma and with similar biological behavior to malignant tumors. The underlying mechanisms for CAE are widely considered as the rupture of a solitary cyst or embolization of cyst particles from ruptured cysts inside remote organs (mostly the liver)[19]. Cerebral lesions could grow to cause varying degrees of mass effect and the accompanying PLBE tends to cause intolerable intracranial pressure. The pathogenesis of PLBE is unknown, it seems to be a vasogenic type of edema according to the previous reports and the MRI findings we observed[7, 8]. With the rapid development of modern molecular biology, many scholars have investigated the mechanisms of brain edema caused by different diseases at the molecular level. Given the rarity of parasitic diseases of the brain, there have been few studies reported on brain edema caused by them. In our present study, we analyzed the clinical data and surgically resected specimens of 18 CAE patients, and further verified the microscopic presentations and characteristics of the CAE lesions, then we first revealed that: (1) TNF-α and VEGF-A were significantly expressed in the CAE lesions, and their levels were positively correlated with PLBE. (2) There was no higher expression of MVD in CAE lesions compared with the normal brain tissue, but it still positively correlated with VEGF-A and PLBE.\u003c/p\u003e \u003cp\u003eIn our microscopic observation, the CAE lesions are characterized by a collection of immune cells that attempt to immunologically restrain, and thoroughly surround the cystic structures of Echinococcus multilocularis. The central areas of granulomas, called caseous necrosis are essentially dead tissue surrounded by granulomatous inflammation. Immunohistochemical positive areas were mainly concentrated in the peripheral lesions. These pathological changes and their MRI characteristics are very reminiscent of the other granulomatous disease - Mycobacterium tuberculosis (MTB). Evidence indicates that TNF-α is a quite representative immune mediator in both formation and maintenance of granulomas in MTB, and mononuclear phagocytes represent the dominant cellular source of this cytokine[20]. However, the precise mechanisms of how this cytokine influences the granuloma formation are still unclear, Roach et al. suggested that TNF-α is required for the early induction of various chemokines that initiate cell recruitment, as well as for the establishment of the close apposition of Immune cells to maintain the granuloma[21]. Besides, TNF-α is predominantly important in its capacity to sustain a state of dormancy of MTB infection[22]. Amiot et al. had performed the experiments on mice carrying a deletion of TNF-α genes and confirmed that TNF-a plays an essential role in the protection mechanisms against Echinococcus multilocularis through inducing the development of efficient granulomas[23]. However, as a proinflammatory cytokine, an excess of TNF-α would promote immunopathology by interfering with cell death processes and induction of a hyper-inflammatory milieu, thus causing organ dysfunction[24]. Effects of excessive circulating inflammatory cytokines such as fever, weight loss, and tissue damage may be attributed to TNF-α.[22] These two distinct effects (protection and damage) seem to be contradictory and this contradiction may exist in CAE, of course, the specific mechanism of TNF-α in the formation of CAE lesions and whether with similarity to bacterial granuloma need further research.\u003c/p\u003e \u003cp\u003eIn addition to high expression in lesions, we also noted the expression of TNF-a is associated with different degrees of PLBE. TNF-α is considered as a pleiotropic cytokine that existed in inflammatory cascades at different phases of CNS injury[25]. The latest study identified TNF-α as a key proinflammatory cytokine that triggers the cerebral endothelium (EC) necroptosis[26], at the same time, it could also increase the permeability of blood-brain barrier (BBB) through endothelial nitric oxide synthase (eNOS) activation in endothelial cells[27]. All these accordingly contribute to vasogenic brain edema formation. Under the inflammatory environment, activated immune cells could synthesize the cell surface cytotoxic integral transmembrane form of TNF-α at the particular site of inflammation and influence the cell target by either cell-to-cell contact or local release of the soluble secretory form of TNF-α.[24] These findings suggest that TNF-α was very likely to act in the process of PLBE formation. It was regretful that perilesional brain tissues were not obtained in our experiment and the expression of TNF-α in perilesional areas is unclear.\u003c/p\u003e \u003cp\u003eResearch over the past few years has confirmed the multiple roles of VEGF-A in physiological function and pathogenic effect. In the normal healthy nervous system, VEGF-A is a crucial regulator of CNS blood vessel formation and regulates the MVD[28]. This angiogenesis mechanism also exists in the growth of tumors and granuloma[29, 30]. Interestingly, we did notice the significant expression of VEGF-A, but there is no obvious increase of MVD in CAE lesions. In our present study, the MVD of CAE lesions had a similar expression with normal brain tissue and it was mainly centered on the external lesions, which corresponded to the characteristic of low/normal blood supply reported in previous surgical reports and imageology studies[8, 31, 32]. However, the MVD still showed a positive correlation with VEGF-A, in other words, the angiogenesis mediated by VEGF-A may be weakened or suppressed to some extent, on the plus side, this limited vascular growth would reduce the further cerebral spread of the Echinococcus multilocularis.\u003c/p\u003e \u003cp\u003eMeanwhile, the positive correlations in MVD and VEGF-A between PLBE were indicated. Low maintenance levels of VEGF-A are necessary for endothelial cell survival and the integrity of the BBB[28], however, as a vascular permeability factor at high levels, VEGF-A jeopardizes the CNS homeostasis through disruption of the BBB: stimulating the creation of interendothelial gaps, fragmentations, and fenestrations in the brain endothelium[18]. These structural changes could result in the fluid leakage from the vascular lumen into the brain parenchyma, which results in vasogenic edema and high interstitial fluid pressure (IFP). Besides, the MVD regulated by VEGF-A would provide a better anatomical basis for the effect of VEGF-A on BBB, further promote the formation of edema. A similar pro-edematous association had been reported in brain tumors like meningioma, and Ding et al. confirmed that the VEGF-A secreted by the tumor tissue enter peritumoral normal brain tissue to induce edema[33]. Furthermore, the latest study shows the VEGF-A inhibitors could relieve brain edema caused by brain tumors and reduce granuloma-associated pathologies[18, 34], which may provide a new idea for the clinical control of CAE.\u003c/p\u003e \u003cp\u003eIn this study, significant expression of TNF-α and VEGF-A in CAE lesions were found and we speculated that the upregulation of TNF-α and VEGF-A induced the formation of PLBE. Besides, though there was no extra increase of MVD in CAE lesions compared to normal brain tissue, it was still regulated by VEGF-A and provide a better anatomical basis for the formation of PLBE and further promote it. These findings may help us better realize the characteristics of cerebral parasitic granuloma and provide a new therapeutic strategy for CAE.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eConflict of Interest: The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eEthical Approval: This research was conducted following the Helsinki Declaration and approved by the institutional research ethics committee of Xinjiang Medical University. Written informed consent was obtained for each participant.\u003c/p\u003e\n\u003cp\u003ePermission: Written informed consent was obtained from each patient and copy of the consent will be available from the corresponding author on reasonable request from the journal editor.\u003c/p\u003e\u003cp\u003e \u003ch2\u003eSource of Funding:\u003c/h2\u003e \u003cp\u003eThis study was funded by the State Key Laboratory of Pathogenesis, Prevention, Treatment of Central Asian High Incidence Diseases Fund (2022E01068), State Key Laboratory of Pathogenesis, Prevention, Treatment of Central Asian High Incidence Diseases Fund (SKL-HIDCA-2022-8).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthors' contributions\u003c/h2\u003e \u003cp\u003eWW and AA equally contributed to the work, ZW and SD contributed to the design of experiment and carried out research; PW contributed to data collection and analyzed data, YZ checked the validity of data, TT and AA wrote the paper. NJ conducted all work and decided final analyses. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eGrateful acknowledgment is made to my supervisors Dr. Zengliang Wang and Dr. Serick Duysenbi who gave me considerable help through suggestions, comments and unwavering support. Besides, I deeply appreciate the funding support from the State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia Fund (2022E01068, SKL-HIDCA-2022-8).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eP. Kern, A. Menezes da Silva, O. Akhan, B. M\u0026uuml;llhaupt, K.A. Vizcaychipi, C. Budke, D.A. Vuitton, The Echinococcoses: Diagnosis, Clinical Management and Burden of Disease, Advances in parasitology 96 (2017) 259\u0026ndash;369.\u003c/li\u003e\n\u003cli\u003eP. Deplazes, L. Rinaldi, C.A. Alvarez Rojas, P.R. Torgerson, M.F. Harandi, T. Romig, D. Antolova, J.M. Schurer, S. Lahmar, G. Cringoli, J. Magambo, R.C. Thompson, E.J. Jenkins, Global Distribution of Alveolar and Cystic Echinococcosis, Advances in parasitology 95 (2017) 315\u0026ndash;493.\u003c/li\u003e\n\u003cli\u003eS. Baumann, R. Shi, W. Liu, H. Bao, J. Schmidberger, W. Kratzer, W. Li, Worldwide literature on epidemiology of human alveolar echinococcosis: a systematic review of research published in the twenty-first century, Infection 47(5) (2019) 703\u0026ndash;727.\u003c/li\u003e\n\u003cli\u003eGuidelines for treatment of cystic and alveolar echinococcosis in humans. WHO Informal Working Group on Echinococcosis, Bulletin of the World Health Organization 74(3) (1996) 231\u0026thinsp;\u0026minus;\u0026thinsp;42.\u003c/li\u003e\n\u003cli\u003eP. Kern, K. Bardonnet, E. Renner, H. Auer, Z. Pawlowski, R.W. Ammann, D.A. Vuitton, P. Kern, European echinococcosis registry: human alveolar echinococcosis, Europe, 1982\u0026ndash;2000, Emerging infectious diseases 9(3) (2003) 343-9.\u003c/li\u003e\n\u003cli\u003eD. Tappe, D. Weise, U. Ziegler, A. M\u0026uuml;ller, W. M\u0026uuml;llges, A. Stich, Brain and lung metastasis of alveolar echinococcosis in a refugee from a hyperendemic area, Journal of medical microbiology 57(Pt 11) (2008) 1420\u0026ndash;1423.\u003c/li\u003e\n\u003cli\u003eM. Tunaci, A. Tunaci, G. Engin, B. Ozkorkmaz, B. Ahishali, I. Rozanes, MRI of cerebral alveolar echinococcosis, Neuroradiology 41(11) (1999) 844-6.\u003c/li\u003e\n\u003cli\u003eO.E. Bat\u0026ccedil;ık, A. \u0026Ouml;ğrenci, O. Koban, M. Ekşi, T. Bilge, Cerebral Alveolar Echinococcosis Concomitant with Liver and Lung Lesions in a Young Adult Patient: Case Report and Literature Review, Turkiye parazitolojii dergisi 40(3) (2016) 169\u0026ndash;171.\u003c/li\u003e\n\u003cli\u003eP. Svrckova, L. Nabarro, P.L. Chiodini, H.R. 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Sandor, VEGF-A from Granuloma Macrophages Regulates Granulomatous Inflammation by a Non-angiogenic Pathway during Mycobacterial Infection, Cell reports 27(7) (2019) 2119\u0026ndash;2131.e6.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"acta-parasitologica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"actp","sideBox":"Learn more about [Acta Parasitologica](http://link.springer.com/journal/11686)","snPcode":"11686","submissionUrl":"https://submission.springernature.com/new-submission/11686/3","title":"Acta Parasitologica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cerebral alveolar echinococcosis, TNF-α, VEGF-A, Microvessel density, Brain edema","lastPublishedDoi":"10.21203/rs.3.rs-4256751/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4256751/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlveolar echinococcosis (AE) is an infrequent zoonosis caused by Echinococcus multilocularis with a high degree of disability and mortality. Metastatic cerebral alveolar echinococcosis (CAE) is very rare and the lesions could lead to severe perilesional brain edema (PLBE) and subsequent uncontrollable intracranial hypertension. In this study, we sought to determine the expression of edema-associated factors in CAE lesions and their associations with PLBE. We retrospectively evaluated the clinical data of 18 CAE patients who received craniotomy. Severity of PLBE was described by edema index (EI). Archived specimens were processed for immunohistochemistry to detect tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor A (VEGF-A) and microvessel density (MVD) in CAE lesions. Expression intensity of CAE lesions was quantified by integral optical density (IOD) or count and was compared to the control group. The results showed TNF-α and VEGF-A were significantly expressed in CAE lesions (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), their levels were positively correlated with PLBE (TNF-α: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.701, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; VEGF-A: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.803, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The MVD of CAE lesions had a similar expression with normal brain tissue, and it was positively correlated with PLBE and VEGF-A (PLBE: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.849, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; VEGF-A: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.687, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). In conclusion, we speculated the upregulation of TNF-α and VEGF-A induced the formation of PLBE. Besides, though there was no extra increase of MVD, it was still regulated by VEGF-A and provided a better anatomical basis for the formation of PLBE and further promoted it.\u003c/p\u003e","manuscriptTitle":"Expression of TNF-α, VEGF-A and microvessel density in cerebral alveolar echinococcosis and their correlation with perilesional brain edema","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-30 19:04:38","doi":"10.21203/rs.3.rs-4256751/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-06-18T11:38:10+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-04-24T05:43:01+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-23T19:49:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-17T04:45:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Parasitologica","date":"2024-04-12T05:24:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-parasitologica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"actp","sideBox":"Learn more about [Acta Parasitologica](http://link.springer.com/journal/11686)","snPcode":"11686","submissionUrl":"https://submission.springernature.com/new-submission/11686/3","title":"Acta Parasitologica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"71c85323-6265-41bb-9426-fd765cfb3f4f","owner":[],"postedDate":"April 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-27T16:06:22+00:00","versionOfRecord":{"articleIdentity":"rs-4256751","link":"https://doi.org/10.1007/s11686-024-00943-7","journal":{"identity":"acta-parasitologica","isVorOnly":false,"title":"Acta Parasitologica"},"publishedOn":"2025-01-24 15:57:43","publishedOnDateReadable":"January 24th, 2025"},"versionCreatedAt":"2024-04-30 19:04:38","video":"","vorDoi":"10.1007/s11686-024-00943-7","vorDoiUrl":"https://doi.org/10.1007/s11686-024-00943-7","workflowStages":[]},"version":"v1","identity":"rs-4256751","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4256751","identity":"rs-4256751","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}