Super-Resolution Ultrasound Imaging Reveals Spatiotemporal Vascular and Glial Remodelling Following Spinal Cord Injury

Super-Resolution Ultrasound Imaging Reveals Spatiotemporal Vascular and Glial Remodelling Following Spinal Cord 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 Article Super-Resolution Ultrasound Imaging Reveals Spatiotemporal Vascular and Glial Remodelling Following Spinal Cord Injury Abdul Karim Ghaith, F M Moinuddin, Ashis Dhar, Jingke Zhang, Yongbin Chen, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9151086/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background Spinal cord injury (SCI) induces complex secondary pathophysiological cascades, including microvascular disruption, glial activation, and chronic neuroinflammation. These vascular changes are dynamic and spatially heterogeneous across acute, subacute, and chronic phases. However, limitations in in vivo imaging resolution have impeded precise, longitudinal evaluation of spinal microcirculation. Objective To quantify post-SCI alterations in spinal cord vessel density, flow speed, and morphology over time using ultrasound localization microscopy (ULM), and to correlate these changes with chronic astrocytic reactivity assessed by GFAP expression. Methods Eleven Sprague-Dawley rats underwent T9–T11 laminectomy; nine received a standardized 200 kdyn contusion using the Infinite Horizons Impactor and were assigned to acute (24h), subacute (7d), and chronic (21d) groups (n = 3/group), while two served as uninjured controls. ULM was performed using a Vevo 3100 high-frequency ultrasound system with a 40 MHz transducer following intravenous Definity® microbubble injection. Vessel density (VD) and flow speed (FS) were quantified at the lesion epicenter and adjacent rostral/caudal regions. Arterial and venous compartments were distinguished by flow direction. Histological sections were stained for glial fibrillary acidic protein (GFAP), and %Area was quantified across phases. One-way ANOVA and Tukey’s test evaluated between-group differences; Pearson correlation identified phase-specific relationships between vascular metrics and chronic GFAP expression. Results ULM provided super-resolved imaging of spinal microvasculature at 5–10 µm resolution. Venous VD significantly declined from 0.92 ± 0.18 (acute) to 0.42 ± 0.23 (subacute) and 0.11 ± 0.07 (chronic, p 0.05), while FS demonstrated transient reductions in both arterial and venous systems during the subacute phase. Chronic venous FS partially recovered by Day 21. GFAP-positive area increased significantly in the chronic group (3.3 ± 0.87%) compared to acute (1.52 ± 0.42%) and subacute (1.40 ± 0.05%) (p < 0.05), indicating delayed astrocyte reactivity. Correlation analysis revealed significant inverse associations between chronic GFAP %Area and early ventral venous density (r = − 1.0, p < 0.05), as well as positive correlations with chronic ventral arterial FS (r = 1.0, p < 0.01), suggesting early microvascular deficits may predict long-term gliosis. Conclusion This study demonstrates the feasibility and power of ULM to quantify dynamic microvascular remodeling in SCI with unprecedented resolution. Venous rarefaction precedes chronic astrocytosis, while arterial flow speed partially recovers over time. These findings highlight key spatiotemporal biomarkers of injury progression and support the use of ULM as a noninvasive platform for monitoring SCI pathophysiology and guiding therapeutic timing. Biological sciences/Neuroscience Biological sciences/Neuroscience/Neurogenesis/Adult neurogenesis Spinal cord injury ultrasound localization microscopy microvascular density venous remodelling blood flow velocity glial fibrillary acidic protein astrocytosis super-resolution imaging neurovascular coupling secondary injury. Full Text Additional Declarations There is no duality of interest Cite Share Download PDF Status: Under Review Version 1 posted Review # 1 received at journal 24 Apr, 2026 Reviewer # 1 agreed at journal 11 Apr, 2026 Reviewers invited by journal 02 Apr, 2026 Editor assigned by journal 23 Mar, 2026 Submission checks completed at journal 23 Mar, 2026 First submitted to journal 21 Mar, 2026 Unknown event 20 Mar, 2026 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. 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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-9151086","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":616813476,"identity":"9855578e-8f6c-46dd-8de1-ba25d406692c","order_by":0,"name":"Abdul Karim 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16:35:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9151086/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9151086/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106724216,"identity":"461552f2-5a6e-4553-af0d-2245c6277f3c","added_by":"auto","created_at":"2026-04-12 18:26:45","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1140388,"visible":true,"origin":"","legend":"","description":"","filename":"MANUSCRIPTfinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9151086/v1_covered_324cd498-aafc-466d-9e61-f01f78a2e428.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"\u003cp\u003eSuper-Resolution Ultrasound Imaging Reveals Spatiotemporal Vascular and Glial Remodelling Following Spinal Cord Injury\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"spinal-cord","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"sc","sideBox":"Learn more about [Spinal Cord](http://www.nature.com/sc/)","snPcode":"41393","submissionUrl":"https://mts-sc.nature.com/cgi-bin/main.plex","title":"Spinal Cord","twitterHandle":"@journalsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Spinal cord injury, ultrasound localization microscopy, microvascular density, venous remodelling, blood flow velocity, glial fibrillary acidic protein, astrocytosis, super-resolution imaging, neurovascular coupling, secondary injury.","lastPublishedDoi":"10.21203/rs.3.rs-9151086/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9151086/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSpinal cord injury (SCI) induces complex secondary pathophysiological cascades, including microvascular disruption, glial activation, and chronic neuroinflammation. These vascular changes are dynamic and spatially heterogeneous across acute, subacute, and chronic phases. However, limitations in in vivo imaging resolution have impeded precise, longitudinal evaluation of spinal microcirculation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eObjective\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo quantify post-SCI alterations in spinal cord vessel density, flow speed, and morphology over time using ultrasound localization microscopy (ULM), and to correlate these changes with chronic astrocytic reactivity assessed by GFAP expression.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEleven Sprague-Dawley rats underwent T9\u0026ndash;T11 laminectomy; nine received a standardized 200 kdyn contusion using the Infinite Horizons Impactor and were assigned to acute (24h), subacute (7d), and chronic (21d) groups (n\u0026thinsp;=\u0026thinsp;3/group), while two served as uninjured controls. ULM was performed using a Vevo 3100 high-frequency ultrasound system with a 40 MHz transducer following intravenous Definity\u0026reg; microbubble injection. Vessel density (VD) and flow speed (FS) were quantified at the lesion epicenter and adjacent rostral/caudal regions. Arterial and venous compartments were distinguished by flow direction. Histological sections were stained for glial fibrillary acidic protein (GFAP), and %Area was quantified across phases. One-way ANOVA and Tukey\u0026rsquo;s test evaluated between-group differences; Pearson correlation identified phase-specific relationships between vascular metrics and chronic GFAP expression.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eULM provided super-resolved imaging of spinal microvasculature at 5\u0026ndash;10 \u0026micro;m resolution. Venous VD significantly declined from 0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 (acute) to 0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 (subacute) and 0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 (chronic, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), particularly in dorsal regions. Arterial VD remained stable across timepoints (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), while FS demonstrated transient reductions in both arterial and venous systems during the subacute phase. Chronic venous FS partially recovered by Day 21. GFAP-positive area increased significantly in the chronic group (3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87%) compared to acute (1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42%) and subacute (1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating delayed astrocyte reactivity. Correlation analysis revealed significant inverse associations between chronic GFAP %Area and early ventral venous density (r = \u0026minus;\u0026thinsp;1.0, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as well as positive correlations with chronic ventral arterial FS (r\u0026thinsp;=\u0026thinsp;1.0, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting early microvascular deficits may predict long-term gliosis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis study demonstrates the feasibility and power of ULM to quantify dynamic microvascular remodeling in SCI with unprecedented resolution. Venous rarefaction precedes chronic astrocytosis, while arterial flow speed partially recovers over time. These findings highlight key spatiotemporal biomarkers of injury progression and support the use of ULM as a noninvasive platform for monitoring SCI pathophysiology and guiding therapeutic timing.\u003c/p\u003e","manuscriptTitle":"Super-Resolution Ultrasound Imaging Reveals Spatiotemporal Vascular and Glial Remodelling Following Spinal Cord Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-08 19:50:09","doi":"10.21203/rs.3.rs-9151086/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-04-24T12:29:54+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-11T09:47:41+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-04-02T19:47:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-23T16:01:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-23T15:52:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Spinal Cord","date":"2026-03-21T05:02:26+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-03-20T14:21:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"spinal-cord","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"sc","sideBox":"Learn more about [Spinal Cord](http://www.nature.com/sc/)","snPcode":"41393","submissionUrl":"https://mts-sc.nature.com/cgi-bin/main.plex","title":"Spinal Cord","twitterHandle":"@journalsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"704d389a-9ee0-4385-9c3d-0528cbde43ae","owner":[],"postedDate":"April 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":65967492,"name":"Biological sciences/Neuroscience"},{"id":65967493,"name":"Biological sciences/Neuroscience/Neurogenesis/Adult neurogenesis"}],"tags":[],"updatedAt":"2026-04-08T19:50:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-08 19:50:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9151086","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9151086","identity":"rs-9151086","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}