Extracellular Vesicles as Stroke Biomarkers

The combination of rehabilitation protocols and regenerative therapies offers the outstanding opportunity to promote and enhance the endogenous regenerative and repair processes occurring in tissues damaged or lost due to injury, disease, or age. Still, one of the main hurdles in the clinical approach to regenerative rehabilitation is the lack of easily accessible and sensitive biomarkers for the evaluation of rehabilitation and therapy efficacy. Extracellular vesicles (EVs) are nanoscaled vesicles that mediate intercellular communication among organs. EVs were shown to be involved in the onset, progression and resolution of many disorders, being also used as valuable tool in the regenerative medicine field. However, the initial enthusiastic approach to EVs has been hindered in its transfer to clinics because of technological obstacles related to their dimensions and to their limited amount. The present project proposes the application of a Surface Plasmon Resonance imaging (SPRi)-based biosensor for the detection and characterization of blood EVs from stroke patients, before and after rehabilitation. After the successful SPRi detection of EVs of different cellular origin (brain and non-brain cells), the quantification of specific surface molecules related to pathological or regeneration processes will be accomplished. If successful, the project will 1) demonstrate the ability of the SPRi biosensor to reveal differences in the relative amount of specific cell-derived EV subpopulations and in their cargo during disease progression and rehabilitation induced recovery, 2) provide support for using the proposed SPRi-based biosensor for the detection and characterization of circulating EVs in order to evaluate the efficacy of rehabilitation protocols and regenerative therapies, 3) identify new biomarkers for the profiling of stroke patients to personalize the rehabilitation therapies.

OBJECTIVE: The aim of the project will be the evaluation of the prognostic power of circulating extracellular vesicles (EVs) in the serum of stroke patients. The characterization by SPRi of different families of EVs will allow to evaluate the activation status of the processes of neuroinflammation, neuronal regeneration, angiogenesis and cellular damage to provide a picture of the mechanisms of response to the damage taking place in the patient. These data will be correlated with emerging prognostic markers derived from the literature (Gandolfi et al, 2017) and with the outcome of the rehabilitation evaluated with specific functional and neurological scales that allow accurate profiling of the patient and the evaluation of functional recovery, according to what recently validated in the "Minimum rehabilitation evaluation protocol of the person with Cerebral Stroke Version 2020 - PMIC 2020" by the Italian Society of Physical and Rehabilitative Medicine (SIMFER) (https://springerhealthcare.it/mr/numero/volume-34-n- 2-June-2020 /).

The PMIC 2020 aims to provide each physiatrist / Rehabilitation Team with a uniform tool for the assessment of the person with stroke in the different phases of the disease, from acute hospital to territorial rehabilitation.

Specific objectives will be:

i) optimization of the SPRi method for the identification of different families of EVs in the serum of healthy subjects; ii) evaluation of the feasibility of the SPRi based method for the characterization of EVs from the serum of stroke patients; iii) preliminary study of the correlation between SPRi data, soluble markers of inflammation and regeneration, patient profiling and rehabilitation outcome at discharge.

IMPACT: The personalization of the rehabilitation plan in relation to the injury suffered and the patient's active response would lead to an increased probability of functional recovery, with benefits in terms of quality of life for the patient and family members, and a reduction in the management costs of the for the National Health System.

SAMPLE COLLECTION: 80 post-stroke patients will be asked to undergo three samples of biological material (10 ml of blood, two 5 ml tubes suitable for serum isolation): the first collection on the second day of hospitalization (t0) at IRCCS S. Maria Nascente (Mialno) or IRCCS Don Gnocchi (Florence) of Fondazioen Don Gnocchi; a second withdrawal at discharge (t1), or approximately 2 months after the first withdrawal. Where possible, a third sampling (t2 - follow up) will be performed 6 months after the event at the IRCCS S. Maria Nascente or at the IRCCS in Florence. 20 healthy subjects will be asked to undergo a single sample of biological material (10 ml of blood, two 5 ml tubes suitable for serum isolation).

EV ISOLATION: The blood sample will be processed for serum separation. The samples will be coded, sterile aliquoted and stored at -80°C at the Laboratory of Nanomedicine and Clinical Biophotonics of IRCCS S. Maria Nascente (Milan) until used.

EVs will be isolated from serum by size exclusion chromatography (qEV; Izon). The actual isolation will be verified with standard techniques, including for example western blot for specific protein markers (CD63, Flotillin 1), Nanoparticle Tracking Analysis or Transmission Electron Microscopy.

SPRi BIOSENSOR: The functionalization of the SPRi chip will be optimized for known markers of apoptotic bodies (Annexin V; index of extent of brain damage) and of EVs from neurons (Ephrin), microglia (IB4 or CD11b), astrocytes (GLAST) and endothelial cells ( CD31). The chip will be also functionalized for the recognition of the Klotho protein (Sahu et al, 2018).

Immobilized EVs will be tested also for the presence on the surface of receptors related to neuronal regeneration (TGFbR1 / 2), microglial activation (TSPO) and angiogenesis (VEGFR-2).

RAMAN ANALYSIS: EVs isolated from patient serum will also be analyzed by Raman spectroscopy according to a protocol already optimized for patients with Parkinson's disease(Gualerzi et al., 2019). Raman analysis will provide information on the biochemical characteristics of the EVs, which can be correlated with the molecular data obtained in SPRi.

ELISA: known markers of inflammation and neuronal activation will be quantified in serum by ELISA assays: inflammatory cytokines (IL6 and TNFa), adipokines (leptin), soluble adhesion molecules (sICAM, sFAS) and growth factors (BDNF).

Correlation between SPRi data, soluble inflammatory and neurotrophic factors will be performed to identify new rehabilitation markers for stroke patients

Eligibility Criteria:
Inclusion Criteria:

* Diagnosis of ischemic or haemorrhagic stroke related to intensive post-stroke rehabilitation hospitalization
* within 15 days of the ictal event

Exclusion Criteria:

* Previous head injuries/traumas
* Oncological diseases
* Diseases of the immune and haematological system
* Neurodegenerative diseases