Neutrophil Extracellular Traps Mediate the Detrimental Effects of Jugular Venous Reflux on Cerebral Ischemia–Reperfusion Injury

Neutrophil Extracellular Traps Mediate the Detrimental Effects of Jugular Venous Reflux on Cerebral Ischemia–Reperfusion Injury | 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 Neutrophil Extracellular Traps Mediate the Detrimental Effects of Jugular Venous Reflux on Cerebral Ischemia–Reperfusion Injury Yitao He, Hui Zhang, Guogao Zhang, Zhili Cai, Jian Deng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8877455/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 Background: Jugular venous reflux (JVR) impairs cerebral venous drainage and may aggravate ischemic brain injury; however, its underlying mechanisms are unclear. Neutrophil extracellular traps (NETs) are key mediators of inflammatory damage in stroke. This study investigated whether JVR exacerbates cerebral ischemia-reperfusion injury by promoting NET formation and evaluated the therapeutic potential of DNase I. Methods: Rats were allocated into Sham, BJVL alone, UCAO alone, and BJVL+UCAO groups. Chronic JVR was induced by bilateral jugular vein ligation (BJVL). One week later, transient middle cerebral artery occlusion (UCAO) was performed. Neurological deficits (Longa score), infarct volume (TTC staining), NET formation (H3Cit, MPO, CD11b), and plasma cytokines (IL-1β, IL-6, IL-10, TNF-α) were assessed. DNase I was administered post-reperfusion. In vitro, PMA-induced NETs were co-cultured with hippocampal neurons to assess cytotoxicity. Results: BJVL alone did not cause infarction but worsened UCAO-induced deficits, infarct volume, and weight loss. BJVL+UCAO increased MPO, CD11b, H3Cit, and pro-inflammatory cytokines. DNase I reduced infarct size, H3Cit, and cytokines without affecting neutrophil recruitment. In vitro, PMA-induced NETs decreased neuronal viability and increased apoptosis, effects reversed by DNase I. Conclusions: JVR aggravates cerebral ischemia-reperfusion injury by enhancing neutrophil infiltration and NET formation. DNase I mitigates injury by degrading NETs, highlighting its therapeutic potential in stroke with venous impairment. Jugular venous reflux Cerebral ischemia–reperfusion injury Neutrophil extracellular traps Middle cerebral artery occlusion Neutrophil infiltration Neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Stroke is a leading cause of death and long-term disability worldwide, with ischemic stroke constituting the majority of cases 1 , 2 . Despite advances in acute revascularization therapies, many patients still suffer from severe neurological deficits and poor functional outcomes, particularly in cases of large hemispheric infarction 3-5 . The underlying pathological mechanisms are multifactorial and extend beyond the initial arterial occlusion to include secondary inflammatory responses 6-8 , blood-brain barrier disruption 9-11 , and impaired cerebral hemodynamics 12-14 . Notably, the role of venous drainage dysfunction in exacerbating ischemic brain injury has gained increasing attention, yet its molecular and cellular mechanisms remain poorly defined. Jugular venous reflux (JVR), characterized by retrograde flow in the internal jugular veins, is known to compromise cerebral venous outflow, elevate intracranial venous pressure, and reduce cerebral perfusion. Clinically, JVR has been associated with various neurological disorders, including idiopathic intracranial hypertension 15 and transient global amnesia 16 , 17 . In the context of cerebral ischemia, impaired venous clearance may theoretically aggravate edema, impede the removal of toxic metabolites and inflammatory mediators, and thus worsen ischemic injury. However, whether and how JVR interacts with arterial ischemic events at the cellular and immune levels has not been systematically investigated, representing a significant gap in stroke pathophysiology. Emerging evidence highlights the critical role of neutrophil extracellular traps (NETs) in propagating inflammation and tissue damage in cerebral ischemia 18-21 . NETs are web-like structures composed of decondensed chromatin decorated with histones and granular proteins, released by activated neutrophils. They contribute to microvascular occlusion 22 , amplify inflammatory signaling 23 , and directly induce neuronal death 24 , 25 . NETs formation has been detected in both experimental stroke models and human stroke patients, and its inhibition has shown neuroprotective effects 18 , 20 , 26 . Nevertheless, whether venous hemodynamic disturbances such as JVR can modulate NETs dynamics in the ischemic brain remains entirely unexplored. Given that DNase I enzymatically degrades the DNA backbone of NETs and has shown efficacy in reducing injury in NETs-dependent diseases 27 , we hypothesized that JVR exacerbates cerebral ischemia–reperfusion injury by enhancing NETs formation and that DNase I could attenuate this process. To test this, we established a rat model combining chronic bilateral jugular vein ligation (BJVL) with transient middle cerebral artery occlusion (UCAO). We evaluated functional outcomes, infarct volume, NETs markers, systemic inflammation, and the therapeutic potential of DNase I. Furthermore, we validated the neurotoxic role of NETs and the protective effect of DNase I in an in vitro co-culture system. This study aims to elucidate a novel mechanism linking venous drainage impairment to immune-mediated brain injury and to propose a translational therapeutic strategy targeting NETs in stroke. Materials and Methods Animal Experiments Adult male Sprague-Dawley rats (weight 280–320 g) were housed under standard laboratory conditions (12 h light/dark cycle, with a temperature range of 21°C-24°C and humidity maintained at 50%-70%) with free access to food and water. After one week of acclimatization, rats were randomly divided into four groups: Sham, BJVL alone, UCAO alone, and BJVL + UCAO. Surgical Procedures Bilateral Jugular Vein Ligation (BJVL): Rats were anesthetized with 2% isoflurane in oxygen (flow rate 2 L/min) and placed in dorsal recumbency. A midline cervical incision (approximately 2-2.5 cm in length) was made. The sternocleidomastoid muscle was retracted laterally to expose the carotid sheath. The internal jugular vein (IJV) was isolated, doubly ligated with 6‑0 silk, and transected between ligatures. The same procedure was performed contralaterally. Sham animals underwent identical dissection without ligation. Transient Middle Cerebral Artery Occlusion (UCAO): One week after BJVL (or sham), focal cerebral ischemia was induced using the intraluminal filament method as previously established in our laboratory 28 . Rats were anesthetized as above and placed supine. A midline incision exposed the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). After placing loose sutures around the CCA and ECA, the proximal CCA was clamped. A silicon-coated monofilament was introduced into the ECA and advanced into the ICA until mild resistance indicated occlusion of the middle cerebral artery origin (approximately 18–20 mm from bifurcation). The filament was secured, and ischemia was maintained for 2 h. During ischemia, isoflurane was reduced to 1% to maintain light anesthesia. Body temperature was maintained with a heating pad. After 2 h, the filament was withdrawn to allow reperfusion. The ECA stump was sealed with gelatin sponge, and wounds were closed in layers. Drug Administration DNase I (11284932001, Roche) was administered at a dose of 5 mg/kg via tail vein injection 30 minutes after reperfusion. Subsequent daily injections were continued until the experimental endpoint. The vehicle control group received an equivalent volume of phosphate-buffered saline (PBS) following the same schedule. Postoperative Care After surgery, rats were placed in a warm, clean cage and monitored until fully recovered from anesthesia. Buprenorphine (0.05 mg/kg) was administered subcutaneously every 8–12 h for postoperative analgesia for 48 h. Animals were inspected daily for signs of pain, distress, or infection. Neurological function score (Longa Score) Neurological function was evaluated at 24 hours after UCAO surgery using the standardized Longa scoring system 29 .Rats were placed in an open field and observed for 5 minutes by two investigators blinded to group assignment. Neurological deficits were scored as follows: 0 - normal, no neurological impairment. 1 - inability to fully extend the left front paw. 2 - turning to the left when walking. 3 - leaning to the left when walking. 4 - inability to walk on its own and showing loss of consciousness. The higher the score, the more severe the neurological function deficiency. Rats with a Longa score of 2 or higher were considered to have undergone successful ischemia induction and were included in further analyses. 2,3,5-Triphenyltetrazolium chloride (TTC) staining To assess the area of cerebral infarction, TTC staining was conducted on the brain sections obtained from each group of rats. Brains were frozen briefly and sectioned into 2 mm slices. Sections were incubated in 2% TTC solution (17779, Sigma) at 37°C for 20 min in the dark, then fixed in 4% PFA. Infarct areas (unstained) and total areas were measured using ImageJ, and infarct size was expressed as percentage of total slice area. Cell Culture and Treatment 1. Isolation and Treatment of Rat Peripheral Blood Neutrophils Rat peripheral blood neutrophils were isolated using a commercial neutrophil extraction kit (Solarbio, P9200) according to the manufacturer’s instructions. Isolated neutrophils were divided into three groups and treated as follows: Vehicle group: Neutrophils were treated with DMSO at a volume equivalent to that of PMA for 4 h. PMA group: Neutrophils were stimulated with 100 nM phorbol 12‑myristate 13‑acetate (PMA) (MCE, HY-18739) for 4 h to induce neutrophil extracellular trap (NET) formation. PMA + DNase I group: Neutrophils were first treated with 100 nM PMA for 4 h, followed by incubation with 100 U/mL DNase I for 15 min to enzymatically disrupt NET structure. 2. Culture of Primary Rat Hippocampal Neurons Primary rat hippocampal neurons (iCell, RAT‑iCell‑n006) were cultured in iCell Primary Neuronal Cell Culture Medium (iCell, PriMed‑iCell‑005) and maintained at 37 °C under a humidified atmosphere of 5% CO₂, according to the supplier’s protocol. 3. Indirect Co‑culture Using Transwell System Neurons were seeded in the lower chamber of a 24‑well Transwell plate (pore size 0.4 μm; Corning). Treated neutrophils from each group were added to the corresponding upper chambers. The 0.4 μm pore size allowed diffusion of soluble factors but prevented direct cell‑cell contact. Co‑culture was maintained for 24 h before subsequent analyses. CCK‑8 Assay for Cell Viability After 24 h of co‑culture in the Transwell system, the upper chamber containing neutrophils was removed. The hippocampal neurons remaining in the lower chamber were washed with PBS and incubated with 10% CCK‑8 reagent (CellCook, T01C) in culture medium for 1.5 h at 37 °C in a 5% CO₂ incubator. Absorbance was measured at 450 nm using a Bio‑Tek microplate reader. Cell apoptosis After 24 h of co‑culture, hippocampal neurons in the lower chamber of the Transwell system were collected and washed with PBS. Cells were resuspended in binding buffer at a density of 1×10⁶ cells/mL and stained with Annexin V‑FITC (KeyGen, KGA1022) and 7-Aminoactinomycin D (7-AAD) (Invitrogen, 00‑6993‑50) for 15 min at room temperature in the dark. Apoptotic cells were analyzed using a flow cytometer within 1 h. Western blot Proteins were extracted from hippocampal tissue or primary rat hippocampal neurons cells with RIPA buffer (P0013B, Beyotime) and quantified using BCA Protein Assay Kit (BL521A, Biosharp). Equal amounts of protein were separated by SDS‑PAGE, transferred to PVDF membranes, and blocked with 5% milk. Membranes were incubated overnight at 4 °C with primary antibodies against MPO (proteintech, 22225-1-AP), CD11b (abcam, ab133357), H3Cit (abcam, ab281584), GAPDH (60004-1-lg, Proteintech) and H3 (proteintech, 17168-1-AP), followed by HRP‑conjugated secondary antibodies for 1 h. Signals were detected by ECL and quantified using ImageJ, normalized to GAPDH or H3. Enzyme -linked immunosorbent assay (ELISA) Plasma levels of TNF‑α, IL‑6, IL-10 and IL‑1β were measured using commercial rat ELISA kits (4A Biotech: TNF‑α CRE0003, IL‑6 CRE0005, IL‑10 CRE0007, IL‑1β CRE0006) according to the manufacturer’s instructions. Briefly, samples and standards were added to pre‑coated plates and incubated for 90 min at 37 °C. After washing, biotin‑labeled detection antibodies were added and incubated for 60 min. Following another wash, streptavidin‑HRP was added for 30 min. Color was developed with TMB substrate for 15 min in the dark, and the reaction was stopped with stop solution. Absorbance was measured at 450 nm using a microplate reader. Cytokine concentrations were calculated from standard curves. All samples were run in duplicate. Statistical analysis Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism version 8.0. Comparisons between two groups were conducted using Student’s t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test. A p-value of less than 0.05 was considered statistically significant. Results Jugular venous reflux exacerbates neurological deficits and infarct volume after cerebral ischemia-reperfusion injury As shown in Fig. 1B, body weight was significantly reduced in rats subjected to UCAO alone compared with sham‑operated animals. Although BJVL alone did not cause significant weight loss, it markedly potentiated the weight reduction observed after UCAO when the two procedures were combined. Neurological function was assessed using the Longa scoring system at 24 h post‑reperfusion. Rats in the UCAO‑alone group exhibited clear neurological deficits, whereas BJVL‑alone rats showed no apparent impairment. Notably, the combination of BJVL and UCAO resulted in significantly more severe neurological dysfunction than UCAO alone (Fig. 1C), indicating that chronic jugular venous reflux exacerbates ischemia‑induced functional disability. To evaluate structural brain damage, TTC staining was performed. While no infarct was detected in the BJVL‑alone group, UCAO alone induced a well‑defined cortical‑subcortical infarction. Importantly, the infarct volume was significantly larger in the BJVL+UCAO group compared with the UCAO‑alone group (Fig. 1D, E). These results collectively demonstrate that jugular venous reflux not only worsens post‑ischemic neurological performance but also augments the extent of cerebral infarction, suggesting a synergistic detrimental effect of venous drainage impairment on ischemic brain injury. Jugular venous reflux enhances neutrophil infiltration and net formation in the ischemic hippocampus The infiltration of neutrophils into the ischemic brain was assessed by measuring the expression of MPO and CD11b in the hippocampus. As shown in Fig. 2A–B, both markers were significantly upregulated in the UCAO‑alone group compared with sham controls, confirming robust neutrophil recruitment after ischemia. BJVL alone induced a moderate but detectable increase in MPO and CD11b expression, suggesting that venous reflux itself may provoke a low‑grade inflammatory response. Importantly, the BJVL+UCAO group exhibited the highest levels of MPO and CD11b among all groups, indicating that jugular venous reflux synergistically enhances neutrophil infiltration following cerebral ischemia. To determine whether enhanced neutrophil infiltration was accompanied by NET formation, we examined the expression of H3Cit, a specific marker of NETosis. H3Cit levels were markedly elevated in the hippocampus of UCAO‑alone rats (Fig. 2C–D). While BJVL alone induced only minimal H3Cit expression, the combination of BJVL and UCAO resulted in a pronounced increase in H3Cit signal, suggesting that venous reflux facilitates NET formation in the ischemic brain. Consistent with the local inflammatory changes, systemic inflammation was evaluated by measuring plasma cytokines. As depicted in Fig. 2E, UCAO alone significantly elevated the levels of IL‑1β, IL‑6, IL‑10, and TNF‑α. BJVL alone also increased most of these cytokines, except IL‑6. Notably, the BJVL+UCAO group displayed further elevation in IL‑6 and IL‑10 compared with UCAO alone, indicating that venous reflux exacerbates the systemic inflammatory response post‑ischemia. Taken together, these results demonstrate that jugular venous reflux amplifies both local neutrophil‑driven NET formation and systemic inflammation following cerebral ischemia–reperfusion injury. DNase I Treatment Attenuates Jugular Venous Reflux‑Aggravated Cerebral Ischemia–Reperfusion Injury by Disrupting NETs To examine the therapeutic potential of NET disruption, DNase I was administered intravenously starting 30 min after reperfusion and continued daily. DNase I treatment did not significantly alter body weight or Longa scores across groups (Fig. 3B–C), suggesting that its protective effect is not mediated through general physiological or behavioral modulation. However, TTC staining revealed that DNase I significantly reduced infarct volume in both the UCAO‑alone and BJVL+UCAO groups, while having no effect in sham or BJVL‑alone rats (Fig. 3D–E). These results indicate that DNase I specifically mitigates ischemia‑induced structural damage, particularly under conditions of venous reflux. We next investigated whether DNase I influences neutrophil recruitment. Western blot analysis showed that DNase I treatment did not significantly affect the elevated levels of MPO and CD11b in the hippocampus of UCAO or BJVL+UCAO rats (Fig. 3F–G), indicating that its protective mechanism is independent of neutrophil infiltration. In contrast, DNase I administration markedly reduced H3Cit expression in both the UCAO‑alone and BJVL+UCAO groups, as well as in the BJVL‑alone group (Fig. 3H–I). This demonstrates that DNase I effectively degrades pre‑formed NETs regardless of the underlying pathological trigger (ischemia, venous reflux, or both). Consistent with reduced NET burden, DNase I treatment significantly lowered plasma levels of IL‑1β, IL‑6, IL‑10, and TNF‑α in the UCAO‑alone and BJVL+UCAO groups, with a modest reduction also observed in the BJVL‑alone group (Fig. 3J). Collectively, these findings indicate that DNase I exerts neuroprotection primarily through the enzymatic disruption of NETs rather than by modulating neutrophil recruitment, and effectively attenuates both local NET‑associated damage and systemic inflammation exacerbated by jugular venous reflux. NETs Induced by PMA Exert Direct Neurotoxic Effects on Hippocampal Neurons Peripheral blood neutrophils were successfully isolated and showed high expression of MPO and CD11b, in contrast to PBMCs which served as a negative control (Fig. 4A–B). Stimulation with 100 nM PMA for 4 h robustly induced NET formation, reflected by increased H3Cit expression (Fig. 4C–D). Subsequent incubation with DNase I effectively reduced H3Cit levels, confirming NET disruption. To assess the direct neurotoxic potential of NETs, hippocampal neurons were co‑cultured with treated neutrophils using a Transwell system. Neurons exposed to PMA‑treated neutrophils displayed significantly reduced viability compared to vehicle‑treated controls (Fig. 4E). This reduction was partially mitigated when neutrophils were pre‑treated with DNase I. Apoptosis analysis corroborated these results, showing that co‑culture with PMA‑treated neutrophils significantly increased neuronal apoptosis, an effect attenuated by DNase I treatment (Fig. 4F–G). These findings collectively demonstrate that PMA‑induced NETs directly compromise hippocampal neuron viability and promote apoptosis in vitro, providing cellular‑level evidence that NETs contribute to neuronal injury. The reversibility of these effects upon NET disruption highlights the potential therapeutic relevance of targeting NETs in cerebral ischemia. Discussion The present study provides the first evidence that jugular venous reflux (JVR) exacerbates cerebral ischemia–reperfusion injury by promoting neutrophil extracellular trap (NET) formation, and that DNase I‑mediated NET disruption attenuates this pathological process. Our findings establish a novel link between cerebral venous drainage impairment, NET‑driven neuroinflammation, and ischemic brain damage, extending the current understanding of stroke pathophysiology beyond arterial occlusion‑centered mechanisms. Previous studies have established that impaired venous drainage contributes to intracranial hypertension and exacerbates brain edema in various neurological disorders 30-32 . However, whether JVR directly modulates the inflammatory cascade in ischemic stroke remained unknown. Our data demonstrate that BJVL alone induced mild but detectable upregulation of neutrophil markers and inflammatory cytokines, suggesting that venous stasis itself can provoke a low‑grade pro‑inflammatory state. Importantly, when combined with transient middle cerebral artery occlusion, JVR markedly amplified neutrophil infiltration, NET formation, and systemic inflammation, ultimately leading to larger infarct volumes and worse neurological outcomes. These results highlight the synergistic interaction between venous hemodynamic disturbance and arterial ischemic injury, supporting the concept that venous pathology is not merely a bystander but an active contributor to stroke progression. A key mechanistic insight from our study is the central role of NETs in mediating the detrimental effects of JVR. NETs have emerged as critical effectors of immunothrombosis and inflammatory damage in ischemic stroke 33 , 34 . Our experiments revealed that JVR substantially enhanced H3Cit expression in the ischemic hippocampus, indicating augmented NETosis. Notably, DNase I treatment significantly reduced H3Cit levels, infarct volume, and inflammatory cytokine release without affecting neutrophil recruitment, suggesting that NET degradation rather than inhibition of neutrophil infiltration underlies its protective effect. This observation is consistent with studies showing that DNase I protects against thrombosis and tissue injury in models of deep vein thrombosis 35 . Furthermore, DNase I has been proposed as a therapeutic strategy for immunothrombosis in various cardiovascular contexts 36 . Our findings extend its therapeutic potential to cerebral ischemia complicated by venous insufficiency. Our in vitro co‑culture experiments further elucidated the direct neurotoxicity of NETs. PMA‑induced NETs reduced hippocampal neuron viability and increased apoptosis, effects that were reversed by DNase I pretreatment. These data provide cellular‑level evidence that NETs are not only markers of inflammation but also active contributors to neuronal death, likely through the release of histones, granular enzymes, and other cytotoxic components 37 . Importantly, the neurotoxicity was attenuated by DNase I, reinforcing the therapeutic relevance of targeting NETs in ischemic stroke. Several limitations of this study warrant consideration. First, the BJVL model, while widely used to study JVR, does not fully replicate the dynamic and often intermittent nature of clinical venous reflux. Second, our study focused on acute outcomes; the long‑term effects of JVR and NET‑targeted therapy on functional recovery and neurogenesis remain to be investigated. Third, although DNase I effectively degraded NETs and reduced injury, it’s optimal dosing, timing, and potential side effects in the context of stroke require further optimization. From a translational perspective, our findings suggest that patients with impaired jugular venous drainage—such as those with internal jugular vein stenosis, heart failure, or increased central venous pressure—may be particularly vulnerable to NET‑driven ischemic brain injury. Detecting and therapeutically addressing venous dysfunction could therefore represent a novel adjunctive strategy in stroke management. Furthermore, DNase I, already FDA‑approved for cystic fibrosis, offers a clinically feasible approach to mitigate NET‑associated damage in stroke. Future studies should explore whether combining DNase I with reperfusion therapies (e.g., thrombolysis or thrombectomy) enhances neuroprotection in preclinical models and ultimately in patients. Conclusion In conclusion, our study demonstrates that jugular venous reflux aggravates cerebral ischemia–reperfusion injury by enhancing NET formation and neuroinflammation, and that DNase I‑mediated NET degradation confers significant neuroprotection. These results uncover a previously unrecognized role of venous hemodynamics in modulating immune responses in stroke and highlight NETs as a promising therapeutic target for patients with concomitant venous insufficiency. Abbreviations BJVL: Bilateral Jugular Vein Ligation; CCK-8: Cell Counting Kit‑8; CD11b: Cluster of Differentiation 11b; DMSO: Dimethyl Sulfoxide; DNase I: Deoxyribonuclease I; Elisa: Enzyme‑Linked Immunosorbent Assay; GAPDH: Glyceraldehyde‑3‑Phosphate Dehydrogenase; H3: Histone H3; H3Cit: Citrullinated Histone H3; IL-1β: Interleukin‑1 Beta; IL-6: Interleukin‑6; IL-10: Interleukin‑10; JVR: Jugular Venous Reflux; MPO: Myeloperoxidase; NET(s): Neutrophil Extracellular Trap(s); PBS: Phosphate‑Buffered Saline; PMA: Phorbol 12‑Myristate 13‑Acetate; SD: Standard Deviation; TNF-α: Tumor Necrosis Factor‑Alpha; TTC: 2,3,5‑Triphenyltetrazolium Chloride; UCAO: Unilateral Cerebral Artery Occlusion (in this context, transient Middle Cerebral Artery Occlusion). Declarations Ethics approval and consent to participate The study was performed in accordance with the guidelines for the ethical review of laboratory animal welfare in the People's Republic of China National Standard (GB/T 35892–2018) and approved by the Experimental Animal Management and Use Committee of Shenzhen People's Hospital (Approval No: AUP-230725-HYT-0334-01). All procedures involving animals were conducted in compliance with the ARRIVE guidelines. Consent for publication Not applicable. This manuscript does not contain data from any individual person. Availability of data and materials The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Shenzhen Science and Technology Program (Grant No: JCYJ20230807112400001). Authors' contributions Yitao He and Hui Zhang involved in the conception, design and implementation of experiments in Figure 1, Figure 2 and part of Figure 3; Guogao Zhang was responsible for the conception, design and carried out Figure 4; Zhili Cai was responsible for the design and conception of part of experiments in Figure 3; Yitao He participated in the framework, scheme and design of the overall scheme and guides the implementation of each figure and he final approval of the version to be published. Jian Deng was responsible for the analysis and interpretation of all the data. All authors completed the the drafting of the paper, revising it critically for intellectual content and agreed to be accountable for all aspects of the work. Acknowledgements Not applicable. References Young MJ, Regenhardt RW, Leslie-Mazwi TM, Stein MA. Disabling stroke in persons already with a disability: Ethical dimensions and directives. Neurology. 2020;94(7):306-310. 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Neutrophil Extracellular Traps: Villains and Targets in Arterial, Venous, and Cancer-Associated Thrombosis. Arterioscler Thromb Vasc Biol. 2019;39(9):1724-1738. Sollberger G, Tilley DO, Zychlinsky A. Neutrophil Extracellular Traps: The Biology of Chromatin Externalization. Dev Cell. 2018;44(5):542-553. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.zip 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. 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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-8877455","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":592150582,"identity":"f9896b98-6d7a-4fa6-97e9-3f0997827b7c","order_by":0,"name":"Yitao He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYBACAyA+AGaxNzY++EC0FrAensPNhjOI1QKxRiK9TZqDGC3m7D2Ghz/U2CRuuPmwQZqBwU5Ot4GAFsueMwYHDhxLS9xwO7HBuIAh2djsACGH3cgBamE7bCw5O7EheQbDgcRtBLXcfwPU8g+oZebBhsM8RGm5wWNw4GDbYTl+CcbGZuK0nEkrOHC2L02OnyexmXGGATF+OX5484eKbzY8bOzHn//4UGEnR1ALAwOHAbIJBJWDAPsDopSNglEwCkbBCAYAb3RKuUrf1EoAAAAASUVORK5CYII=","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yitao","middleName":"","lastName":"He","suffix":""},{"id":592150583,"identity":"2a8d90c1-8124-40f1-af19-8dfaf0a27bc6","order_by":1,"name":"Hui Zhang","email":"","orcid":"","institution":"Shenzhen People’s Hospital (The Second Clinical Medical College of Jinan University, The First Affiliated Hospital of Southern University of Science and Technology)","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Zhang","suffix":""},{"id":592150584,"identity":"a6aa28ca-f977-4092-a0d0-8c04a79dc4a2","order_by":2,"name":"Guogao Zhang","email":"","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Guogao","middleName":"","lastName":"Zhang","suffix":""},{"id":592150585,"identity":"fccee352-3289-4973-b049-a17c14aba027","order_by":3,"name":"Zhili Cai","email":"","orcid":"","institution":"Shenzhen People’s Hospital (The Second Clinical Medical College of Jinan University, The First Affiliated Hospital of Southern University of Science and Technology)","correspondingAuthor":false,"prefix":"","firstName":"Zhili","middleName":"","lastName":"Cai","suffix":""},{"id":592150586,"identity":"54afa8e1-650a-4aa3-9d0b-b42cb46f19c7","order_by":4,"name":"Jian Deng","email":"","orcid":"","institution":"Shenzhen People’s Hospital (The Second Clinical Medical College of Jinan University, The First Affiliated Hospital of Southern University of Science and Technology)","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Deng","suffix":""}],"badges":[],"createdAt":"2026-02-14 06:23:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8877455/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8877455/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103803255,"identity":"f2c31cb9-20c2-40b0-93b6-a81aa337ebc3","added_by":"auto","created_at":"2026-03-03 06:33:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9646598,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJugular venous reflux aggravates post‑ischemic neurological deficits and cerebral infarction.\u003c/strong\u003e\u003cbr\u003e\n (A) Experimental timeline illustrating the sequence of BJVL surgery, UCAO induction, and endpoint assessments. (B) Percentage change in body weight across experimental groups. (C) Longa neurological scores evaluated 24 h after UCAO. (D) Representative TTC‑stained coronal brain sections. (E) Quantitative analysis of infarct area expressed as percentage of total slice area.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8877455/v1/291224a647ed19cc1864c0a2.png"},{"id":103803252,"identity":"08053d31-9ce5-4528-8f63-7ef8bed5c156","added_by":"auto","created_at":"2026-03-03 06:33:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2708142,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJugular venous reflux promotes neutrophil infiltration, NET formation, and systemic inflammation following cerebral ischemia–reperfusion injury.\u003c/strong\u003e\u003cbr\u003e\n (A) Western blot analysis of neutrophil markers MPO and CD11b in hippocampal tissues. (B) Densitometric quantification of MPO and CD11b protein levels. (C) Western blot detection of NET formation marker histone H3 citrullination (H3Cit). (D) Quantification of H3Cit expression. (E) Plasma levels of inflammatory cytokines IL‑1β, IL‑6, IL‑10, and TNF‑α measured by ELISA.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8877455/v1/d5c6e629c334c0ce2dbfc1cd.png"},{"id":103803256,"identity":"fe187251-27df-4a1b-a8ff-6e8ae3e6ec66","added_by":"auto","created_at":"2026-03-03 06:33:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18680556,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNase I reduces infarct volume, NET formation, and systemic inflammation without affecting neutrophil infiltration.\u003c/strong\u003e\u003cbr\u003e\n (A) Schematic timeline of DNase I administration following reperfusion. (B) Body weight changes across groups after DNase I treatment. (C) Longa scores 24 h post‑UCAO. (D) Representative TTC‑stained brain sections. (E) Quantitative infarct area analysis. (F) Western blot of MPO and CD11b in hippocampal tissue after DNase I treatment. (G) Quantification of MPO and CD11b levels. (H) Western blot analysis of H3Cit expression. (I) Quantification of H3Cit levels. (J) Plasma cytokine levels measured by ELISA.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8877455/v1/0bb30c512706f36cd60e8102.png"},{"id":103803253,"identity":"02717afd-e5b9-42a1-85cc-b5e482a47211","added_by":"auto","created_at":"2026-03-03 06:33:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2753233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNETs induced by PMA reduce hippocampal neuron viability and promote apoptosis in vitro.\u003c/strong\u003e\u003cbr\u003e\n (A) Western blot analysis confirms expression of neutrophil markers MPO and CD11b in isolated rat peripheral blood neutrophils, with PBMCs serving as a negative control. (B) Densitometric quantification of MPO and CD11b. (C) Western blot detection of NET formation marker H3Cit in neutrophils following stimulation with 100 nM PMA with or without subsequent DNase I treatment. (D) Quantification of H3Cit levels. (E) CCK‑8 assay assessing hippocampal neuron viability after indirect co‑culture with treated neutrophils. (F) Flow cytometric analysis of neuronal apoptosis via Annexin V/7‑AAD staining. (G) Quantitative analysis of apoptotic rates.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8877455/v1/95646d633d4eaef592f6dba7.png"},{"id":104400982,"identity":"ae486376-4749-4515-9bd6-112e979fefc9","added_by":"auto","created_at":"2026-03-11 12:11:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":28697491,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8877455/v1/eeacbac7-7c62-4729-9ed4-991bf353cd89.pdf"},{"id":103803257,"identity":"1925ad14-c62e-4af0-9bfd-2e99b9310c01","added_by":"auto","created_at":"2026-03-03 06:33:14","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":13357167,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.zip","url":"https://assets-eu.researchsquare.com/files/rs-8877455/v1/493a1fda45ee9151b6a67d98.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Neutrophil Extracellular Traps Mediate the Detrimental Effects of Jugular Venous Reflux on Cerebral Ischemia–Reperfusion Injury","fulltext":[{"header":"Background","content":"\u003cp\u003eStroke is a leading cause of death and long-term disability worldwide, with ischemic stroke constituting the majority of cases\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e2\u003c/sup\u003e. Despite advances in acute revascularization therapies, many patients still suffer from severe neurological deficits and poor functional outcomes, particularly in cases of large hemispheric infarction\u003csup\u003e3-5\u003c/sup\u003e. The underlying pathological mechanisms are multifactorial and extend beyond the initial arterial occlusion to include secondary inflammatory responses\u003csup\u003e6-8\u003c/sup\u003e, blood-brain barrier disruption\u003csup\u003e9-11\u003c/sup\u003e, and impaired cerebral hemodynamics\u003csup\u003e12-14\u003c/sup\u003e. Notably, the role of venous drainage dysfunction in exacerbating ischemic brain injury has gained increasing attention, yet its molecular and cellular mechanisms remain poorly defined.\u003c/p\u003e\n\u003cp\u003eJugular venous reflux (JVR), characterized by retrograde flow in the internal jugular veins, is known to compromise cerebral venous outflow, elevate intracranial venous pressure, and reduce cerebral perfusion. Clinically, JVR has been associated with various neurological disorders, including idiopathic intracranial hypertension\u003csup\u003e15\u003c/sup\u003e and transient global amnesia\u003csup\u003e16\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e17\u003c/sup\u003e. In the context of cerebral ischemia, impaired venous clearance may theoretically aggravate edema, impede the removal of toxic metabolites and inflammatory mediators, and thus worsen ischemic injury. However, whether and how JVR interacts with arterial ischemic events at the cellular and immune levels has not been systematically investigated, representing a significant gap in stroke pathophysiology.\u003c/p\u003e\n\u003cp\u003eEmerging evidence highlights the critical role of neutrophil extracellular traps (NETs) in propagating inflammation and tissue damage in cerebral ischemia\u003csup\u003e18-21\u003c/sup\u003e. NETs are web-like structures composed of decondensed chromatin decorated with histones and granular proteins, released by activated neutrophils. They contribute to microvascular occlusion\u003csup\u003e22\u003c/sup\u003e, amplify inflammatory signaling\u003csup\u003e23\u003c/sup\u003e, and directly induce neuronal death\u003csup\u003e24\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e25\u003c/sup\u003e. NETs formation has been detected in both experimental stroke models and human stroke patients, and its inhibition has shown neuroprotective effects\u003csup\u003e18\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e20\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e26\u003c/sup\u003e. Nevertheless, whether venous hemodynamic disturbances such as JVR can modulate NETs dynamics in the ischemic brain remains entirely unexplored.\u003c/p\u003e\n\u003cp\u003eGiven that DNase I enzymatically degrades the DNA backbone of NETs and has shown efficacy in reducing injury in NETs-dependent diseases\u003csup\u003e27\u003c/sup\u003e, we hypothesized that JVR exacerbates cerebral ischemia\u0026ndash;reperfusion injury by enhancing NETs formation and that DNase I could attenuate this process. To test this, we established a rat model combining chronic bilateral jugular vein ligation (BJVL) with transient middle cerebral artery occlusion (UCAO). We evaluated functional outcomes, infarct volume, NETs markers, systemic inflammation, and the therapeutic potential of DNase I. Furthermore, we validated the neurotoxic role of NETs and the protective effect of DNase I in an in vitro co-culture system. This study aims to elucidate a novel mechanism linking venous drainage impairment to immune-mediated brain injury and to propose a translational therapeutic strategy targeting NETs in stroke.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal Experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdult male Sprague-Dawley rats (weight 280\u0026ndash;320 g) were housed under standard laboratory conditions (12 h light/dark cycle, with a temperature range of 21\u0026deg;C-24\u0026deg;C and humidity maintained at 50%-70%) with free access to food and water. After one week of acclimatization, rats were randomly divided into four groups: Sham, BJVL alone, UCAO alone, and BJVL + UCAO.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurgical Procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBilateral Jugular Vein Ligation (BJVL):\u003c/p\u003e\n\u003cp\u003eRats were anesthetized with 2% isoflurane in oxygen (flow rate 2 L/min) and placed in dorsal recumbency. A midline cervical incision (approximately 2-2.5 cm in length) was made. The sternocleidomastoid muscle was retracted laterally to expose the carotid sheath. The internal jugular vein (IJV) was isolated, doubly ligated with 6‑0 silk, and transected between ligatures. The same procedure was performed contralaterally. Sham animals underwent identical dissection without ligation.\u003c/p\u003e\n\u003cp\u003eTransient Middle Cerebral Artery Occlusion (UCAO):\u003c/p\u003e\n\u003cp\u003eOne week after BJVL (or sham), focal cerebral ischemia was induced using the intraluminal filament method as previously established in our laboratory\u003csup\u003e28\u003c/sup\u003e. Rats were anesthetized as above and placed supine. A midline incision exposed the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). After placing loose sutures around the CCA and ECA, the proximal CCA was clamped. A silicon-coated monofilament was introduced into the ECA and advanced into the ICA until mild resistance indicated occlusion of the middle cerebral artery origin (approximately 18\u0026ndash;20 mm from bifurcation). The filament was secured, and ischemia was maintained for 2 h. During ischemia, isoflurane was reduced to 1% to maintain light anesthesia. Body temperature was maintained with a heating pad. After 2 h, the filament was withdrawn to allow reperfusion. The ECA stump was sealed with gelatin sponge, and wounds were closed in layers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrug Administration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNase I (11284932001, Roche) was administered at a dose of 5 mg/kg via tail vein injection 30 minutes after reperfusion. Subsequent daily injections were continued until the experimental endpoint. The vehicle control group received an equivalent volume of phosphate-buffered saline (PBS) following the same schedule.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePostoperative Care\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter surgery, rats were placed in a warm, clean cage and monitored until fully recovered from anesthesia. Buprenorphine (0.05 mg/kg) was administered subcutaneously every 8\u0026ndash;12 h for postoperative analgesia for 48 h. Animals were inspected daily for signs of pain, distress, or infection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeurological function score\u003c/strong\u003e \u003cstrong\u003e(Longa Score)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNeurological function was evaluated at 24 hours after UCAO surgery using the standardized Longa scoring system \u003csup\u003e29\u003c/sup\u003e.Rats were placed in an open field and observed for 5 minutes by two investigators blinded to group assignment. Neurological deficits were scored as follows: 0 - normal, no neurological impairment. 1 - inability to fully extend the left front paw. 2 - turning to the left when walking. 3 - leaning to the left when walking. 4 - inability to walk on its own and showing loss of consciousness. The higher the score, the more severe the neurological function deficiency. Rats with a Longa score of 2 or higher were considered to have undergone successful ischemia induction and were included in further analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2,3,5-Triphenyltetrazolium chloride (TTC) staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the area of cerebral infarction, TTC staining was conducted on the brain sections obtained from each group of rats. Brains were frozen briefly and sectioned into 2 mm slices. Sections were incubated in 2% TTC solution (17779, Sigma) at 37\u0026deg;C for 20 min in the dark, then fixed in 4% PFA. Infarct areas (unstained) and total areas were measured using ImageJ, and infarct size was expressed as percentage of total slice area.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Culture and Treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. Isolation and Treatment of Rat Peripheral Blood Neutrophils\u003c/p\u003e\n\u003cp\u003eRat peripheral blood neutrophils were isolated using a commercial neutrophil extraction kit (Solarbio, P9200) according to the manufacturer\u0026rsquo;s instructions. Isolated neutrophils were divided into three groups and treated as follows:\u003c/p\u003e\n\u003cp\u003eVehicle group: Neutrophils were treated with DMSO at a volume equivalent to that of PMA for 4 h.\u003c/p\u003e\n\u003cp\u003ePMA group: Neutrophils were stimulated with 100 nM phorbol 12‑myristate 13‑acetate (PMA) (MCE, HY-18739) for 4 h to induce neutrophil extracellular trap (NET) formation.\u003c/p\u003e\n\u003cp\u003ePMA + DNase I group: Neutrophils were first treated with 100 nM PMA for 4 h, followed by incubation with 100 U/mL DNase I for 15 min to enzymatically disrupt NET structure.\u003c/p\u003e\n\u003cp\u003e2. Culture of Primary Rat Hippocampal Neurons\u003c/p\u003e\n\u003cp\u003ePrimary rat hippocampal neurons (iCell, RAT‑iCell‑n006) were cultured in iCell Primary Neuronal Cell Culture Medium (iCell, PriMed‑iCell‑005) and maintained at 37 \u0026deg;C under a humidified atmosphere of 5% CO₂, according to the supplier\u0026rsquo;s protocol.\u003c/p\u003e\n\u003cp\u003e3. Indirect Co‑culture Using Transwell System\u003c/p\u003e\n\u003cp\u003eNeurons were seeded in the lower chamber of a 24‑well Transwell plate (pore size 0.4 \u0026mu;m; Corning). Treated neutrophils from each group were added to the corresponding upper chambers. The 0.4 \u0026mu;m pore size allowed diffusion of soluble factors but prevented direct cell‑cell contact. Co‑culture was maintained for 24 h before subsequent analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCCK‑8 Assay for Cell Viability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 24 h of co‑culture in the Transwell system, the upper chamber containing neutrophils was removed. The hippocampal neurons remaining in the lower chamber were washed with PBS and incubated with 10% CCK‑8 reagent (CellCook, T01C) in culture medium for 1.5 h at 37 \u0026deg;C in a 5% CO₂ incubator. Absorbance was measured at 450 nm using a Bio‑Tek microplate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 24 h of co‑culture, hippocampal neurons in the lower chamber of the Transwell system were collected and washed with PBS. Cells were resuspended in binding buffer at a density of 1\u0026times;10⁶ cells/mL and stained with Annexin V‑FITC (KeyGen, KGA1022) and 7-Aminoactinomycin D (7-AAD) (Invitrogen, 00‑6993‑50) for 15 min at room temperature in the dark. Apoptotic cells were analyzed using a flow cytometer within 1 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins were extracted from hippocampal tissue or primary rat hippocampal neurons cells with RIPA buffer (P0013B, Beyotime) and quantified using BCA Protein Assay Kit (BL521A, Biosharp). Equal amounts of protein were separated by SDS‑PAGE, transferred to PVDF membranes, and blocked with 5% milk. Membranes were incubated overnight at 4 \u0026deg;C with primary antibodies against MPO (proteintech, 22225-1-AP), CD11b (abcam, ab133357), H3Cit (abcam, ab281584), GAPDH (60004-1-lg, Proteintech) and H3 (proteintech, 17168-1-AP), followed by HRP‑conjugated secondary antibodies for 1 h. Signals were detected by ECL and quantified using ImageJ, normalized to GAPDH or H3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme\u003c/strong\u003e\u003cstrong\u003e-linked immunosorbent assay\u003c/strong\u003e\u003cstrong\u003e (ELISA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasma levels of TNF‑\u0026alpha;, IL‑6, IL-10 and IL‑1\u0026beta; were measured using commercial rat ELISA kits (4A Biotech: TNF‑\u0026alpha; CRE0003, IL‑6 CRE0005, IL‑10 CRE0007, IL‑1\u0026beta; CRE0006) according to the manufacturer\u0026rsquo;s instructions. Briefly, samples and standards were added to pre‑coated plates and incubated for 90 min at 37 \u0026deg;C. After washing, biotin‑labeled detection antibodies were added and incubated for 60 min. Following another wash, streptavidin‑HRP was added for 30 min. Color was developed with TMB substrate for 15 min in the dark, and the reaction was stopped with stop solution. Absorbance was measured at 450 nm using a microplate reader. Cytokine concentrations were calculated from standard curves. All samples were run in duplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as mean \u0026plusmn; standard deviation (SD). Statistical analyses were performed using GraphPad Prism version 8.0. Comparisons between two groups were conducted using Student\u0026rsquo;s t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by Dunnett\u0026rsquo;s post hoc test. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eJugular venous reflux exacerbates neurological deficits and infarct volume after cerebral ischemia-reperfusion injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 1B, body weight was significantly reduced in rats subjected to UCAO alone compared with sham‑operated animals. Although BJVL alone did not cause significant weight loss, it markedly potentiated the weight reduction observed after UCAO when the two procedures were combined. Neurological function was assessed using the Longa scoring system at 24 h post‑reperfusion. Rats in the UCAO‑alone group exhibited clear neurological deficits, whereas BJVL‑alone rats showed no apparent impairment. Notably, the combination of BJVL and UCAO resulted in significantly more severe neurological dysfunction than UCAO alone (Fig. 1C), indicating that chronic jugular venous reflux exacerbates ischemia‑induced functional disability.\u003c/p\u003e\n\u003cp\u003eTo evaluate structural brain damage, TTC staining was performed. While no infarct was detected in the BJVL‑alone group, UCAO alone induced a well‑defined cortical‑subcortical infarction. Importantly, the infarct volume was significantly larger in the BJVL+UCAO group compared with the UCAO‑alone group (Fig. 1D, E). These results collectively demonstrate that jugular venous reflux not only worsens post‑ischemic neurological performance but also augments the extent of cerebral infarction, suggesting a synergistic detrimental effect of venous drainage impairment on ischemic brain injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJugular venous reflux enhances neutrophil infiltration and net formation in the ischemic hippocampus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe infiltration of neutrophils into the ischemic brain was assessed by measuring the expression of MPO and CD11b in the hippocampus. As shown in Fig. 2A\u0026ndash;B, both markers were significantly upregulated in the UCAO‑alone group compared with sham controls, confirming robust neutrophil recruitment after ischemia. BJVL alone induced a moderate but detectable increase in MPO and CD11b expression, suggesting that venous reflux itself may provoke a low‑grade inflammatory response. Importantly, the BJVL+UCAO group exhibited the highest levels of MPO and CD11b among all groups, indicating that jugular venous reflux synergistically enhances neutrophil infiltration following cerebral ischemia.\u003c/p\u003e\n\u003cp\u003eTo determine whether enhanced neutrophil infiltration was accompanied by NET formation, we examined the expression of H3Cit, a specific marker of NETosis. H3Cit levels were markedly elevated in the hippocampus of UCAO‑alone rats (Fig. 2C\u0026ndash;D). While BJVL alone induced only minimal H3Cit expression, the combination of BJVL and UCAO resulted in a pronounced increase in H3Cit signal, suggesting that venous reflux facilitates NET formation in the ischemic brain.\u003c/p\u003e\n\n\u003cp\u003eConsistent with the local inflammatory changes, systemic inflammation was evaluated by measuring plasma cytokines. As depicted in Fig. 2E, UCAO alone significantly elevated the levels of IL‑1\u0026beta;, IL‑6, IL‑10, and TNF‑\u0026alpha;. BJVL alone also increased most of these cytokines, except IL‑6. Notably, the BJVL+UCAO group displayed further elevation in IL‑6 and IL‑10 compared with UCAO alone, indicating that venous reflux exacerbates the systemic inflammatory response post‑ischemia. Taken together, these results demonstrate that jugular venous reflux amplifies both local neutrophil‑driven NET formation and systemic inflammation following cerebral ischemia\u0026ndash;reperfusion injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNase I Treatment Attenuates Jugular Venous Reflux‑Aggravated Cerebral Ischemia\u0026ndash;Reperfusion Injury by Disrupting NETs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine the therapeutic potential of NET disruption, DNase I was administered intravenously starting 30 min after reperfusion and continued daily. DNase I treatment did not significantly alter body weight or Longa scores across groups (Fig. 3B\u0026ndash;C), suggesting that its protective effect is not mediated through general physiological or behavioral modulation. However, TTC staining revealed that DNase I significantly reduced infarct volume in both the UCAO‑alone and BJVL+UCAO groups, while having no effect in sham or BJVL‑alone rats (Fig. 3D\u0026ndash;E). These results indicate that DNase I specifically mitigates ischemia‑induced structural damage, particularly under conditions of venous reflux.\u003c/p\u003e\n\u003cp\u003eWe next investigated whether DNase I influences neutrophil recruitment. Western blot analysis showed that DNase I treatment did not significantly affect the elevated levels of MPO and CD11b in the hippocampus of UCAO or BJVL+UCAO rats (Fig. 3F\u0026ndash;G), indicating that its protective mechanism is independent of neutrophil infiltration.\u003c/p\u003e\n\u003cp\u003eIn contrast, DNase I administration markedly reduced H3Cit expression in both the UCAO‑alone and BJVL+UCAO groups, as well as in the BJVL‑alone group (Fig. 3H\u0026ndash;I). This demonstrates that DNase I effectively degrades pre‑formed NETs regardless of the underlying pathological trigger (ischemia, venous reflux, or both). Consistent with reduced NET burden, DNase I treatment significantly lowered plasma levels of IL‑1\u0026beta;, IL‑6, IL‑10, and TNF‑\u0026alpha; in the UCAO‑alone and BJVL+UCAO groups, with a modest reduction also observed in the BJVL‑alone group (Fig. 3J).\u003c/p\u003e\n\u003cp\u003eCollectively, these findings indicate that DNase I exerts neuroprotection primarily through the enzymatic disruption of NETs rather than by modulating neutrophil recruitment, and effectively attenuates both local NET‑associated damage and systemic inflammation exacerbated by jugular venous reflux.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNETs Induced by PMA Exert Direct Neurotoxic Effects on Hippocampal Neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeripheral blood neutrophils were successfully isolated and showed high expression of MPO and CD11b, in contrast to PBMCs which served as a negative control (Fig. 4A\u0026ndash;B). Stimulation with 100 nM PMA for 4 h robustly induced NET formation, reflected by increased H3Cit expression (Fig. 4C\u0026ndash;D). Subsequent incubation with DNase I effectively reduced H3Cit levels, confirming NET disruption.\u003c/p\u003e\n\u003cp\u003eTo assess the direct neurotoxic potential of NETs, hippocampal neurons were co‑cultured with treated neutrophils using a Transwell system. Neurons exposed to PMA‑treated neutrophils displayed significantly reduced viability compared to vehicle‑treated controls (Fig. 4E). This reduction was partially mitigated when neutrophils were pre‑treated with DNase I.\u003c/p\u003e\n\u003cp\u003eApoptosis analysis corroborated these results, showing that co‑culture with PMA‑treated neutrophils significantly increased neuronal apoptosis, an effect attenuated by DNase I treatment (Fig. 4F\u0026ndash;G).\u003c/p\u003e\n\u003cp\u003eThese findings collectively demonstrate that PMA‑induced NETs directly compromise hippocampal neuron viability and promote apoptosis in vitro, providing cellular‑level evidence that NETs contribute to neuronal injury. The reversibility of these effects upon NET disruption highlights the potential therapeutic relevance of targeting NETs in cerebral ischemia.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study provides the first evidence that jugular venous reflux (JVR) exacerbates cerebral ischemia\u0026ndash;reperfusion injury by promoting neutrophil extracellular trap (NET) formation, and that DNase I‑mediated NET disruption attenuates this pathological process. Our findings establish a novel link between cerebral venous drainage impairment, NET‑driven neuroinflammation, and ischemic brain damage, extending the current understanding of stroke pathophysiology beyond arterial occlusion‑centered mechanisms.\u003c/p\u003e\n\u003cp\u003ePrevious studies have established that impaired venous drainage contributes to intracranial hypertension and exacerbates brain edema in various neurological disorders\u003csup\u003e30-32\u003c/sup\u003e. However, whether JVR directly modulates the inflammatory cascade in ischemic stroke remained unknown. Our data demonstrate that BJVL alone induced mild but detectable upregulation of neutrophil markers and inflammatory cytokines, suggesting that venous stasis itself can provoke a low‑grade pro‑inflammatory state. Importantly, when combined with transient middle cerebral artery occlusion, JVR markedly amplified neutrophil infiltration, NET formation, and systemic inflammation, ultimately leading to larger infarct volumes and worse neurological outcomes. These results highlight the synergistic interaction between venous hemodynamic disturbance and arterial ischemic injury, supporting the concept that venous pathology is not merely a bystander but an active contributor to stroke progression.\u003c/p\u003e\n\u003cp\u003eA key mechanistic insight from our study is the central role of NETs in mediating the detrimental effects of JVR. NETs have emerged as critical effectors of immunothrombosis and inflammatory damage in ischemic stroke\u003csup\u003e33\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e34\u003c/sup\u003e. Our experiments revealed that JVR substantially enhanced H3Cit expression in the ischemic hippocampus, indicating augmented NETosis. Notably, DNase I treatment significantly reduced H3Cit levels, infarct volume, and inflammatory cytokine release without affecting neutrophil recruitment, suggesting that NET degradation rather than inhibition of neutrophil infiltration underlies its protective effect. This observation is consistent with studies showing that DNase I protects against thrombosis and tissue injury in models of deep vein thrombosis\u003csup\u003e35\u003c/sup\u003e. Furthermore, DNase I has been proposed as a therapeutic strategy for immunothrombosis in various cardiovascular contexts\u003csup\u003e36\u003c/sup\u003e. Our findings extend its therapeutic potential to cerebral ischemia complicated by venous insufficiency.\u003c/p\u003e\n\u003cp\u003eOur in vitro co‑culture experiments further elucidated the direct neurotoxicity of NETs. PMA‑induced NETs reduced hippocampal neuron viability and increased apoptosis, effects that were reversed by DNase I pretreatment. These data provide cellular‑level evidence that NETs are not only markers of inflammation but also active contributors to neuronal death, likely through the release of histones, granular enzymes, and other cytotoxic components\u003csup\u003e37\u003c/sup\u003e. Importantly, the neurotoxicity was attenuated by DNase I, reinforcing the therapeutic relevance of targeting NETs in ischemic stroke.\u003c/p\u003e\n\u003cp\u003eSeveral limitations of this study warrant consideration. First, the BJVL model, while widely used to study JVR, does not fully replicate the dynamic and often intermittent nature of clinical venous reflux. Second, our study focused on acute outcomes; the long‑term effects of JVR and NET‑targeted therapy on functional recovery and neurogenesis remain to be investigated. Third, although DNase I effectively degraded NETs and reduced injury, it\u0026rsquo;s optimal dosing, timing, and potential side effects in the context of stroke require further optimization.\u003c/p\u003e\n\u003cp\u003eFrom a translational perspective, our findings suggest that patients with impaired jugular venous drainage\u0026mdash;such as those with internal jugular vein stenosis, heart failure, or increased central venous pressure\u0026mdash;may be particularly vulnerable to NET‑driven ischemic brain injury. Detecting and therapeutically addressing venous dysfunction could therefore represent a novel adjunctive strategy in stroke management. Furthermore, DNase I, already FDA‑approved for cystic fibrosis, offers a clinically feasible approach to mitigate NET‑associated damage in stroke. Future studies should explore whether combining DNase I with reperfusion therapies (e.g., thrombolysis or thrombectomy) enhances neuroprotection in preclinical models and ultimately in patients.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study demonstrates that jugular venous reflux aggravates cerebral ischemia\u0026ndash;reperfusion injury by enhancing NET formation and neuroinflammation, and that DNase I‑mediated NET degradation confers significant neuroprotection. These results uncover a previously unrecognized role of venous hemodynamics in modulating immune responses in stroke and highlight NETs as a promising therapeutic target for patients with concomitant venous insufficiency.\u003c/p\u003e\n"},{"header":"Abbreviations","content":"\u003cp\u003eBJVL: Bilateral Jugular Vein Ligation; CCK-8: Cell Counting Kit‑8; CD11b: Cluster of Differentiation 11b; DMSO: Dimethyl Sulfoxide; DNase I: Deoxyribonuclease I; Elisa: Enzyme‑Linked Immunosorbent Assay; GAPDH: Glyceraldehyde‑3‑Phosphate Dehydrogenase; H3: Histone H3; H3Cit: Citrullinated Histone H3; IL-1\u0026beta;: Interleukin‑1 Beta; IL-6: Interleukin‑6; IL-10: Interleukin‑10; JVR: Jugular Venous Reflux; MPO: Myeloperoxidase; NET(s): Neutrophil Extracellular Trap(s); PBS: Phosphate‑Buffered Saline; PMA: Phorbol 12‑Myristate 13‑Acetate; SD: Standard Deviation; TNF-\u0026alpha;: Tumor Necrosis Factor‑Alpha; TTC: 2,3,5‑Triphenyltetrazolium Chloride; UCAO: Unilateral Cerebral Artery Occlusion \u0026nbsp;(in this context, transient Middle Cerebral Artery Occlusion).\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was performed in accordance with the guidelines for the ethical review of laboratory animal welfare in the People\u0026apos;s Republic of China National Standard (GB/T 35892\u0026ndash;2018) and approved by the Experimental Animal Management and Use Committee of Shenzhen People\u0026apos;s Hospital (Approval No: AUP-230725-HYT-0334-01). All procedures involving animals were conducted in compliance with the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. This manuscript does not contain data from any individual person.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Shenzhen Science and Technology Program (Grant No: JCYJ20230807112400001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYitao He and Hui Zhang involved in the conception, design and implementation of experiments in Figure 1, Figure 2 and part of Figure 3; Guogao Zhang was responsible for the conception, design and carried out Figure 4; Zhili Cai was responsible for the design and conception of part of experiments in Figure 3; Yitao He participated in the framework, scheme and design of the overall scheme and guides the implementation of each figure and he final approval of the version to be published. Jian Deng was responsible for the analysis and interpretation of all the data. All authors completed the the drafting of the paper, revising it critically for intellectual content and agreed to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYoung MJ, Regenhardt RW, Leslie-Mazwi TM, Stein MA. Disabling stroke in persons already with a disability: Ethical dimensions and directives. Neurology.\u003cem\u003e \u003c/em\u003e2020;94(7):306-310.\u003c/li\u003e\n\u003cli\u003eJurjāns K, Cērpa M, Baborikina A, Kalējs O. Impact of Anticoagulants in Reducing Mortality and Disability in Cardioembolic Stroke Patients. 2022;58(10).\u003c/li\u003e\n\u003cli\u003eCappellari M, Bonetti B, Baracchini C, et al. 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Stroke.\u003cem\u003e \u003c/em\u003e2018;49(3):754-757.\u003c/li\u003e\n\u003cli\u003eLi C, Xing Y, Zhang Y, Hua Y, Hu J, Bai Y. Neutrophil Extracellular Traps Exacerbate Ischemic Brain Damage. 2022;59(1):643-656.\u003c/li\u003e\n\u003cli\u003eEtulain J, Martinod K, Wong SL, Cifuni SM, Schattner M, Wagner DD. P-selectin promotes neutrophil extracellular trap formation in mice. Blood.\u003cem\u003e \u003c/em\u003e2015;126(2):242-246.\u003c/li\u003e\n\u003cli\u003eTh\u0026aring;lin C, Hisada Y, Lundstr\u0026ouml;m S, Mackman N, Wall\u0026eacute;n H. Neutrophil Extracellular Traps: Villains and Targets in Arterial, Venous, and Cancer-Associated Thrombosis. Arterioscler Thromb Vasc Biol.\u003cem\u003e \u003c/em\u003e2019;39(9):1724-1738.\u003c/li\u003e\n\u003cli\u003eSollberger G, Tilley DO, Zychlinsky A. Neutrophil Extracellular Traps: The Biology of Chromatin Externalization. Dev Cell.\u003cem\u003e \u003c/em\u003e2018;44(5):542-553.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Jugular venous reflux, Cerebral ischemia–reperfusion injury, Neutrophil extracellular traps, Middle cerebral artery occlusion, Neutrophil infiltration, Neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-8877455/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8877455/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eJugular venous reflux (JVR) impairs cerebral venous drainage and may aggravate ischemic brain injury; however, its underlying mechanisms are unclear. Neutrophil extracellular traps (NETs) are key mediators of inflammatory damage in stroke. This study investigated whether JVR exacerbates cerebral ischemia-reperfusion injury by promoting NET formation and evaluated the therapeutic potential of DNase I.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eRats were allocated into Sham, BJVL alone, UCAO alone, and BJVL+UCAO groups. Chronic JVR was induced by bilateral jugular vein ligation (BJVL). One week later, transient middle cerebral artery occlusion (UCAO) was performed. Neurological deficits (Longa score), infarct volume (TTC staining), NET formation (H3Cit, MPO, CD11b), and plasma cytokines (IL-1β, IL-6, IL-10, TNF-α) were assessed. DNase I was administered post-reperfusion. In vitro, PMA-induced NETs were co-cultured with hippocampal neurons to assess cytotoxicity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eBJVL alone did not cause infarction but worsened UCAO-induced deficits, infarct volume, and weight loss. BJVL+UCAO increased MPO, CD11b, H3Cit, and pro-inflammatory cytokines. DNase I reduced infarct size, H3Cit, and cytokines without affecting neutrophil recruitment. In vitro, PMA-induced NETs decreased neuronal viability and increased apoptosis, effects reversed by DNase I.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e JVR aggravates cerebral ischemia-reperfusion injury by enhancing neutrophil infiltration and NET formation. DNase I mitigates injury by degrading NETs, highlighting its therapeutic potential in stroke with venous impairment.\u003c/p\u003e","manuscriptTitle":"Neutrophil Extracellular Traps Mediate the Detrimental Effects of Jugular Venous Reflux on Cerebral Ischemia–Reperfusion Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-03 06:33:04","doi":"10.21203/rs.3.rs-8877455/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":"af71c448-78f4-4105-aac1-23c87a9e9a1e","owner":[],"postedDate":"March 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-03T06:33:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-03 06:33:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8877455","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8877455","identity":"rs-8877455","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}