Circulating microglia-derived extracellular vesicles predict recovery after rehabilitation in stroke survivors

Background Timely intensive rehabilitation is crucial to contrast the negative escalation of events that follow a stroke injury, to promote tissue regeneration, and to restore the physiological function. This study aimed to identify measurable blood biomarkers that could be predictive of functional recovery in stroke survivors.

30 sub-acute stroke subjects were enrolled at Fondazione Don Carlo Gnocchi Onlus (Italy) during the observation prospective cohort study EXO4STROKE (NCT05370105). Among the clinical scales used for patient profiling, modified Barthel Index (MBI) was considered as primary outcome for the evaluation of rehabilitation recovery. Cytokines, Neurofilaments and circulating Extracellular Vesicles (EVs) were evaluated in the serum of patients at admission (T0) and at discharge (T1) in intensive rehabilitation unit. The Surface Plasmon Resonance imaging (SPRi) technique was exploited to obtain a multiplexed analysis of circulating EVs from brain (neurons, astrocytes, microglia) and non-brain (endothelium, skeletal muscle and platelets) cells, and for the relative quantification of markers of pathological or regenerative processes. Machine learning-based analysis complemented the study by integrating statistically significant features in a cross-validated prediction model targeting functional recovery at T1 from T0 data.

High serum levels of IL-6 and Neurofilament Light Chain at T0 were associated with higher residual disability (lower outcome) at T1. In contrast, elevated circulating levels of EVs from neurons (CD171+) and microglia (CD11b+) were associated with better recovery after rehabilitaion. Moreover, the overexpression of the VEGF receptor on microglial EVs (IB4+ and CD11b+), was associated with higher functional improvement. Alternatively, the overexpression of TGF-β receptor on IB4+ EVs during the subacute phase post-stroke was found associated with increased stroke severity. The MBI at T1 was predicted with a median [iqr] absolute error of 5.7[8.7] points.

This study identifies a panel of few circulating biomarkers associated to Evs able to objectively assess post-stroke status and predict functional recovery after rehabilitation. Competing Interest Statement The authors have declared no competing interest. Clinical Trial NCT05370105 Funding Statement This project was supported by the Italian Ministry of Health, Ricerca Corrente and Conto Capitale 2020-2022 to Fondazione Don Carlo Gnocchi. This study was funded by ?5xMille funds-2022-Italian Ministry of Health? through the competitive grant ?Bando per la Valorizzazione della Ricerca Interna? of Fondazione Don Carlo Gnocchi ONLUS. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The present study was approved by the Ethical Committee of Fondazione Don Carlo Gnocchi Onlus ((protocol number: 20/2018/CE_FdG/FC/SA).), and carried out according to the Declaration of Helsinki. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.