Rheo-Erythrocrine Dysfunction as a Biomarker for Remote Ischemic Conditioning Treatment in Acute Ischemic Stroke: A Pilot Randomized Controlled Trial

HomeJournal of the American Heart AssociationAhead of PrintRheo‐Erythrocrine Dysfunction as a Biomarker for Remote Ischemic Conditioning Treatment in Acute Ischemic Stroke: A Pilot Randomized Controlled Trial Open AccessRapid CommunicationPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citations ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toOpen AccessRapid CommunicationPDF/EPUBRheo‐Erythrocrine Dysfunction as a Biomarker for Remote Ischemic Conditioning Treatment in Acute Ischemic Stroke: A Pilot Randomized Controlled Trial Maria Kjølhede, Grethe Andersen, Jan Brink Valentin, Marlene Christina Nielsen, Morten Nørgaard Andersen, Mohammad Badruzzaman Khan, Jesper Nørgaard Bech, David C. Hess and Rolf Ankerlund Blauenfeldt Maria KjølhedeMaria Kjølhede * Correspondence to: Maria Kjølhede, MD, Department of Neurology, Aarhus University Hospital, Palle Juul‐Jensens Boulevard 99, DK‐8200 Aarhus N, Denmark. Email: E-mail Address: [email protected] https://orcid.org/0000-0003-1544-6104 , Department of Neurology, , Aarhus University Hospital, , Aarhus, , Denmark, Search for more papers by this author , Grethe AndersenGrethe Andersen https://orcid.org/0000-0001-6807-2500 , Department of Neurology, , Aarhus University Hospital, , Aarhus, , Denmark, , Department of Clinical Medicine, , Aarhus University, , Aarhus, , Denmark, Search for more papers by this author , Jan Brink ValentinJan Brink Valentin https://orcid.org/0000-0002-8205-7179 , Danish Center for Clinical Health Services Research, Department of Clinical Medicine, , Aalborg University, , Aalborg, , Denmark, Search for more papers by this author , Marlene Christina NielsenMarlene Christina Nielsen https://orcid.org/0000-0003-2008-4224 , Department of Clinical Biochemistry, , Aarhus University Hospital, , Aarhus, , Denmark, Search for more papers by this author , Morten Nørgaard AndersenMorten Nørgaard Andersen https://orcid.org/0000-0003-0820-7789 , Department of Clinical Medicine, , Aarhus University, , Aarhus, , Denmark, , Department of Hematology, , Aarhus University Hospital, , Aarhus, , Denmark, , Department of Biomedicine, , Aarhus University, , Aarhus, , Denmark, Search for more papers by this author , Mohammad Badruzzaman KhanMohammad Badruzzaman Khan https://orcid.org/0000-0001-8231-1256 , Department of Neurology, Medical College of Georgia, , Augusta University, , Augusta, , GA, Search for more papers by this author , Jesper Nørgaard BechJesper Nørgaard Bech https://orcid.org/0000-0002-0605-1277 , University Clinic in Nephrology and Hypertension, Gødstrup Regional Hospital, , Herning, , Denmark, Search for more papers by this author , David C. HessDavid C. Hess https://orcid.org/0000-0002-8345-9153 , Department of Neurology, Medical College of Georgia, , Augusta University, , Augusta, , GA, Search for more papers by this author and Rolf Ankerlund BlauenfeldtRolf Ankerlund Blauenfeldt https://orcid.org/0000-0002-4846-9516 , Department of Neurology, , Aarhus University Hospital, , Aarhus, , Denmark, , Department of Clinical Medicine, , Aarhus University, , Aarhus, , Denmark, Search for more papers by this author Originally published10 Nov 2023https://doi.org/10.1161/JAHA.123.031466Journal of the American Heart Association. 2023;0:e031466Remote ischemic conditioning (RIC) is a simple and low‐cost intervention in which transient ischemia is induced in an extremity by repetitive inflation‐deflation of a blood pressure cuff. It has proven to be safe and feasible in clinical applications.1 RIC enhances collateral blood flow in ischemic areas.1 Cerebral blood flow depends on vasodilatory effects of NO generated by endothelial type NO synthetase 3, which has been discovered in red blood cells (RBCs), indicating that RBCs may synthesize, transport, and release NO, and be directly involved in the signaling for hypoxic vasodilation (erythrocrine function).2 In normal healthy states, RBCs are highly deformable, allowing for passage through microvessels half their size. During stroke, RBCs undergo severe morphologic changes, likely contributing to poor deformability and impaired microcirculatory flow.3 In this pilot study, we assessed whether rheo‐erythrocrine dysfunction in RBCs is a biomarker for RIC in patients with acute ischemic stroke (AIS).Data are available on reasonable request from the corresponding author, makjse@rm.dk.We performed a pilot, single‐center, randomized, patient‐assessor blinded, sham‐controlled study on 30 patients with AIS at Aarhus University Hospital, Denmark. Eligible patients met the following criteria: aged 18 to 80 years, modified Rankin Scale score 0 to 2, magnetic resonance imaging–documented ischemic stroke, and inclusion and randomization within 48 hours of symptom onset. A control group (n=15) consisted of patients with a nonstroke diagnosis. All patients signed the informed consent form before enrolling in the study. Race and ethnicity are not registered as routine in Denmark. The study was approved by the Central Denmark Region Committees on Health Research Ethics (identifier: 1‐10‐72‐184‐19) and the Danish Medicines Agency (identifier: 2019081802, EUDAMED CIV‐ID No. 19‐08‐029484) and was monitored by the regional Good Clinical Practice unit, Denmark.Patients were randomly assigned to RIC/sham (1:1) twice daily for 7 days. Automatic RIC/sham devices were preprogrammed to 5 cycles of unilateral cuff inflation followed by 5 minutes of cuff deflation, for a total treatment time of 50 minutes. The cuff was placed on the nonparetic limb. Blood samples were collected before RIC, 2 hours after initial treatment, and after 7 days of treatment.The rheological properties of RBCs were analyzed by ektacytometry (RheoScan‐AnD300; RheoMeditech, Seoul, South Korea), intracellular NO levels were analyzed by flow cytometry using the 4‐amino‐5‐methylamino‐2′, 7’‐difluorofluorescein diacetate (catalog number D23844; ThermoFisher Scientific, Waltham, MA), and whole blood nitrite levels were analyzed using ozone chemiluminescence technique (NOA280i; Zysense, CO).The sample size was based on unpublished experimental data with a difference in maximum elongation index between stroke and controls of 0 and 15, and we planned to include 30 patients with AIS (15 RIC and 15 sham) and 15 controls.Data managing and statistical analyses were performed using Stata 15 software (StataCorp; Stata Statistical Software: Release 15; College Station, TX: StataCorp LLC.). Differences in RBC deformability, NO content, plasma nitrite, and other continuous biomarkers over time (baseline, 2 hours, and 7 days) between RIC (yes/no) were examined using mixed effects linear regression models with a random intercept on subjects.Between July 28, 2020, and July 5, 2021, a total of 317 patients were screened, and 45 patients were included in the study. Of these patients, 30 (15 RIC and 15 sham) had AIS and 15 were controls. A total of 6 patients withdrew consent: 2 controls and 4 in the intervention group (3 RIC and 1 sham). The median (interquartile range) age was 67 (52–73) years for patients assigned to RIC and 60 (58–72) years for the sham group (P=0.85), and the mean National Institutes of Health Stroke Scale score was 2 in both groups. We found no significant differences in RBC deformability, whole‐blood nitrite levels, or NO content between RIC and sham (Figure). For AIS versus controls, baseline RBC deformability was significantly reduced between 5 and 10 Pa, and the critical shear stress required to disperse RBC aggregation significantly increased from baseline to day 7: 131.7 (95% CI, 70.5–192.8) mPa (P<0.001). RIC was well tolerated, and compliance was high.Download figureDownload PowerPointFigure . Plots on red blood cell (RBC) deformability, NO content, and whole‐blood nitrite in remote ischemic conditioning (RIC)– and sham‐treated patients.A, Profile plots of RBC deformability (elongation Index [EI]) for RIC and sham‐RIC at different shear stress (Pa) over time. B, Bar plots illustrate NO levels in RBCs for all groups over time analyzed using flow cytometry. 4‐Amino‐5‐methylamino‐2′, 7’‐difluorofluorescein diacetate (DAF) mean fluorescent intensity (MFI) fluorescence is a direct measurement of intracellular NO levels. C, Bar plots of whole‐blood nitrite levels for all groups over time. D, Box plots illustrating EI maximum (max) for RIC vs sham over time.In this exploratory pilot study, we could not demonstrate any effect of RIC on the rheo‐erythrocrine biomarkers. However, RBC deformability (elongation index) was reduced for AIS versus controls at baseline. The study has limitations related to its setting as a single center, the small sample size, low stroke severity, and a suboptimal treatment regimen (unilateral RIC for 1 week compared with 2 weeks of bilateral RIC).4 The requirement for patients to continue RIC treatment independently at home meant that only minor strokes were included, which could limit a demonstratable treatment effect. Furthermore, venous blood sampling may underestimate the arterial supply of NO to the brain, and dynamic changes of NO may not be captured at the specific sampling times used in study. Inevitable delays until analysis may have resulted in NO metabolization or reaction with other molecules. Further studies on RIC and rheo‐erythrocrine dysfunction in patients with stroke are ongoing and will indicate whether the effect of RIC is partly mediated by improvement of rheo‐erythrocrine dysfunction.5Sources of FundingThe trial received funding from the Novo Nordisk Foundation and the Lundbeck Foundation.DisclosuresNone.Footnotes* Correspondence to: Maria Kjølhede, MD, Department of Neurology, Aarhus University Hospital, Palle Juul‐Jensens Boulevard 99, DK‐8200 Aarhus N, Denmark. Email: makjse@rm.dkThis article was sent to Kori S. Zachrison, MD, MSc, Associate Editor, for review by expert referees, editorial decision, and final disposition.Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04266639.For Sources of Funding and Disclosures, see page 3.References1 Hess DC, Blauenfeldt RA, Andersen G, Hougaard KD, Hoda MN, Ding Y, Ji X. Remote ischaemic conditioning‐a new paradigm of self‐protection in the brain. Nat Rev Neurol. 2015; 11:698–710. doi: 10.1038/nrneurol.2015.223CrossrefMedlineGoogle Scholar2 Cortese‐Krott MM, Rodriguez‐Mateos A, Sansone R, Kuhnle GGC, Thasian‐Sivarajah S, Krenz T, Horn P, Krisp C, Wolters D, Heiß C, et al. Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease. Blood. 2012; 120:4229–4237. doi: 10.1182/blood-2012-07-442277CrossrefMedlineGoogle Scholar3 Pretorius E, Lipinski B. Thromboembolic ischemic stroke changes red blood cell morphology. Cardiovasc Pathol. 2013; 22:241–242. doi: 10.1016/j.carpath.2012.11.005CrossrefMedlineGoogle Scholar4 Chen H‐S, Cui Y, Li X‐Q, Wang X‐H, Ma Y‐T, Zhao Y, Jing Han J, Deng C‐Q, Hong M, Ying Bao Y, et al. Effect of remote ischemic conditioning vs usual care on neurologic function in patients with acute moderate ischemic stroke. JAMA. 2022; 328:627–636. doi: 10.1001/jama.2022.13123CrossrefMedlineGoogle Scholar5 Blauenfeldt RA, Hjort N, Gude MF, Behrndtz AB, Fisher M, Valentin JB, Kirkegaard H, Johnsen SP, Hess DC, Andersen G. A multicentre, randomised, sham‐controlled trial on REmote iSchemic conditioning In patients with acute STroke (RESIST)–rationale and study design. Eur Stroke J. 2020; 5:94–101. doi: 10.1177/2396987319884408CrossrefMedlineGoogle Scholar eLetters(0)eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. 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Published on behalf of the American Heart Association, Inc., by Wiley BlackwellThis is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.https://doi.org/10.1161/JAHA.123.031466PMID: 37947084 Manuscript receivedJune 23, 2023Manuscript acceptedSeptember 14, 2023Originally publishedNovember 10, 2023 Keywordsremote ischemic conditioningacute ischemic strokerheo‐erythrocrine dysfunctionPDF download SubjectsCerebrovascular Disease/Stroke